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Clinical pharmacology of ertapenem in the treatment of multidrug-resistant tuberculosisvan Rijn, Sander Pascal

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Clinicalpharmacologyof

ertapeneminthetreatmentof

Multidrug-resistantTuberculosis

SanderPascalvanRijn

VanRijnS.P.ClinicalpharmacologyofertapeneminthetreatmentofMultidrug-resistantTuberculosis

Thesis,UniversityofGroningen,TheNetherlandsPublicationof this thesiswas financially supportedbyUniversity ofGroningen,UniversityMedical Center Groningen, Graduate School of Medical Sciences, KNCV tuberculosisFoundation, Royal Dutch Pharmacists Association (KNMP), Stichting Beatrixoord Noord-Nederland,PharmIntelandTerWelle&Associés(TW&A).

Cover MaartenKarremans–MardoniCreativeProductionLay-out SandervanRijnPrintedby GVOdrukkers&vormgeversB.V.ISBN 978-94-6332-473-1©Copyright2018.S.P.vanRijn,Groningen,TheNetherlandsAllrightsreserved.Nopartsofthisthesismaybereproduced,storedinaretrievalsystem,ortransmitted in any form or by any means, electronical, mechanical, by photocopying,recordingorotherwise,withoutthepriorwrittenpermissionoftheauthor.

Clinical pharmacology of ertapenem in the treatment of

Multidrug-resistant Tuberculosis

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de rector magnificus prof. dr. E. Sterken

en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

woensdag 10 april 2019 om 12.45 uur

door

Sander Pascal van Rijn

geboren op 12 februari 1988 te Bochum (BRD)

VanRijnS.P.ClinicalpharmacologyofertapeneminthetreatmentofMultidrug-resistantTuberculosis

Thesis,UniversityofGroningen,TheNetherlandsPublicationof this thesiswas financially supportedbyUniversity ofGroningen,UniversityMedical Center Groningen, Graduate School of Medical Sciences, KNCV tuberculosisFoundation, Royal Dutch Pharmacists Association (KNMP), Stichting Beatrixoord Noord-Nederland,PharmIntelandTerWelle&Associés(TW&A).

PharmIntel Cover MaartenKarremans–MardoniCreativeProductionLay-out SandervanRijnPrintedby GVOdrukkers&vormgeversB.V.ISBN 978-94-6332-473-1©Copyright2018.S.P.vanRijn,Groningen,TheNetherlandsAllrightsreserved.Nopartsofthisthesismaybereproduced,storedinaretrievalsystem,ortransmitted in any form or by any means, electronical, mechanical, by photocopying,recordingorotherwise,withoutthepriorwrittenpermissionoftheauthor.

Clinical pharmacology of ertapenem in the treatment of

Multidrug-resistant Tuberculosis

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de rector magnificus prof. dr. E. Sterken

en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

woensdag 10 april 2019 om 12.45 uur

door

Sander Pascal van Rijn

geboren op 12 februari 1988 te Bochum (BRD)

Promotores

Prof.dr.J.W.C.Alffenaar

Prof.dr.T.S.vanderWerf

Prof.dr.J.G.W.Kosterink

Beoordelingscommissie

Prof.dr.B.Wilffert

Prof.dr.R.vanCrevel

Prof.dr.J.W.A.Rossen

Tableofcontents

Chapter1 GeneralIntroduction

Chapter2 Evaluation of Carbapenems for Treatment of Multi- and Extensively Drug-

ResistantMycobacteriumTuberculosis

Chapter3 QuantificationandValidationofErtapenemUsingaLiquidChromatography-

TandemMassSpectrometryMethod

Chapter4 Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant

Tuberculosis

Chapter5 Susceptibility Testing of Antibiotics That Degrade Faster than the Doubling

Time of Slow-Growing Mycobacteria: Ertapenem Sterilizing Effect Versus

MycobacteriumTuberculosis

Chapter6 Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of

TuberculosisandImplicationsonClinicalDosing

Chapter7 PharmacokineticModellingandLimitedSamplingStrategiesBasedonHealthy

VolunteersforMonitoringofErtapeneminPatientswithMultidrug-resistant

Tuberculosis

Chapter8 GeneralDiscussionandFuturePerspectives

Chapter9 Summary

Chapter10 SamenvattingDankwoordAbouttheAuthorPublicationList

CHAPTER 1

General Introduction

8

Chapter 1

GeneralIntroduction

Tuberculosis (TB) is caused byMycobacterium tuberculosis. TB is the deadliest infectious

diseaseworldwide. Itmostlyaffects the lungs,butcanalsoattackotherorgans; together,

theseformsofTBarereferredtoasextrapulmonary.Lymphnodes,thepleuralandperitoneal

space;theaxialskeleton;thegut;theurogenitalsystem;andthemostlethalform:thecentral

nervous system can all be affected. In 2017, 10 million new cases were reported, and

approximately1.6millionpeoplediedofTB[1].Anestimated1millionchildrenfellillwith

TB,aquarterofwhomdied.TBhasaglobalimpact,howeverover95%ofTBdeathsoccurin

low-andmiddle-incomecountries,with61%ofallnewcasesreportedinAsiaand26%ofnew

casesinAfrica,withsixcountriesaccountingfor60%ofthistotal.Anincreaseinrefugeeflow,

increasedtravellingandglobalization,socialinequalityandpoverty,lackofsafewater,poor

sanitationandpoorhygieneservicesareimportantriskfactorsforTB.Asimmigrationand

internationaltraveliscommoninaffluentregions,TBoughttobeaconcernforhighincome

countries[1-3].

Tuberculosisistransmittedviarespiratorydroplets,microbescarriedindropletsoraerosols

loadedwithM.tuberculosis froman infectedpersonviaclosepersonalcontact,coughing,

sneezingandlaughing.Onlyasmallminority–anestimated5-10%ofallinfectedindividuals

-willeverdevelopactiveTBwithsymptomssuchasproductivecoughwithpurulentsputum

thatmaycontainblood,weightloss,fatigueandnightsweats;besides,dependingonthesite

of disease manifestation, people may experience chest pain, back pain, abdominal pain,

headache,andseizures.TheimmuneresponseofmostpeoplesuccessfullyfightsoffTBbacilli

thatmaybekilledbyactivatedmacrophageimmunecells,withTBbacilliinthephagosome-

lysosome.TBbacillimayhoweveralsosurvivewithinthephagosomeofthesemacrophages,

andinthelattercase,thesepeoplearesaidtobelatentlyinfected.Theirbacillisurviveina

hibernatingmode,controlledbyageneticsystemreferredtoasdosRregulon[4].Thisisa

geneticprogramcontrolledbyasetof48genes thatallows the tuberclebacilli tosurvive

understressconditions;duringactiveimmunesuppression,hibernatingorganismsarecalled

‘latent’whileduringdrugtreatment,theseorganismsarecalled‘persistent’.Latentlyinfected

individualstypicallyhaveonlylimitednumbersof livingbacterialcellsthatslowlyreplicate

and are hardly metabolically active. People with latent TB infection feel well, have no

symptomsandarenotcontagious[5].

M. tuberculosis belongs to a small group of highly pathogenic bacteria (M. tuberculosis

complex)intheverylargefamilyofmycobacteria,characterizedbyathickcellwall,consisting

ofseveraldifferentspecificlipidmoleculesincludinglipoarabinomannanandmycolicacids.

Themajorityofcrosslinksinthepeptidoglycanlayerareformeddifferentlycomparedtogram

positivebacteria,makingmycobacteriamoreresistanttochemicaldamageandhydrophilic

antibiotics. As the replication rate ofM. tuberculosis – even if actively replicating and

metabolicallyactive-isveryslow(≈20h)comparedtootherbacteria(≈20min),TBrequires

morespecificantibioticsandprolongedtreatment,asmostantibioticsonlyworkonactively

replicatingbacteria[5].Typically,rapidlydividingmetabolicallyactivebacillicanbereduced

rapidlywithinweeks; severalhighlyactiveagentshavebactericidalproperties.Dueto the

slowreplicationrateofM.tuberculosis,especiallyofdifficulttoeradicatepersisterphenotype

bacteria. TB treatment needs to last long to obtain a sterilising effect. TheWorld Health

Organisation (WHO) therefore advises to threat TB with a standard first-line treatment

consistingofisoniazid(H),Rifampicin(R),pyrazinamide(Z)andethambutol(E)–HRZE–And

therebyintensivelydecreasethebacterialload(intensivephase).Followedbytheintensive

phase, a four-month continuationwith isoniazid and rifampicin is needed to provide the

opportunity to eliminate the last TB bacteria which are in a persistent state of being

capsulatedbymacrophages.

MDRTB-AntimicrobialresistanceofTB

Unfortunately, our world is facing a public health crisis and security threat due to the

treatmentofTBbecomingincreasinglychallengingwiththeemergenceofresistancetofirst-

linedrugs.Multidrug resistant (MDR)-TB is definedas an infectiousdisease causedbyM.

tuberculosisthatisresistanttoatleastisoniazidandrifampicin,whicharethecornerstone

drugs of drug-susceptible TB treatment. Extensively drug resistant (XDR)-TB is defined as

MDR-TBwithadditionalresistancetoatleastoneofthefluoroquinolonesandtoatleastone

oftheinjectablesecondlinedrugs[6].WHOestimatesthattherewere558.000newcases

withresistancetorifampicin,themosteffectivefirstlinedrug[2].

Development of new drugs is slow and expensive due to the obligatory market access

regulationssuchasrandomizedclinicaltrials.BedaquilineandDelamanid,bothwithanovel

mechanismofaction,were included intheWHOguidelinesonMDR-TB,afterapprovalby

9

1


GeneralIntroduction

Tuberculosis (TB) is caused byMycobacterium tuberculosis. TB is the deadliest infectious

diseaseworldwide. Itmostlyaffects the lungs,butcanalsoattackotherorgans; together,

theseformsofTBarereferredtoasextrapulmonary.Lymphnodes,thepleuralandperitoneal

space;theaxialskeleton;thegut;theurogenitalsystem;andthemostlethalform:thecentral

nervous system can all be affected. In 2017, 10 million new cases were reported, and

approximately1.6millionpeoplediedofTB[1].Anestimated1millionchildrenfellillwith

TB,aquarterofwhomdied.TBhasaglobalimpact,howeverover95%ofTBdeathsoccurin

low-andmiddle-incomecountries,with61%ofallnewcasesreportedinAsiaand26%ofnew

casesinAfrica,withsixcountriesaccountingfor60%ofthistotal.Anincreaseinrefugeeflow,

increasedtravellingandglobalization,socialinequalityandpoverty,lackofsafewater,poor

sanitationandpoorhygieneservicesareimportantriskfactorsforTB.Asimmigrationand

internationaltraveliscommoninaffluentregions,TBoughttobeaconcernforhighincome

countries[1-3].

Tuberculosisistransmittedviarespiratorydroplets,microbescarriedindropletsoraerosols

loadedwithM.tuberculosis froman infectedpersonviaclosepersonalcontact,coughing,

sneezingandlaughing.Onlyasmallminority–anestimated5-10%ofallinfectedindividuals

-willeverdevelopactiveTBwithsymptomssuchasproductivecoughwithpurulentsputum

thatmaycontainblood,weightloss,fatigueandnightsweats;besides,dependingonthesite

of disease manifestation, people may experience chest pain, back pain, abdominal pain,

headache,andseizures.TheimmuneresponseofmostpeoplesuccessfullyfightsoffTBbacilli

thatmaybekilledbyactivatedmacrophageimmunecells,withTBbacilliinthephagosome-

lysosome.TBbacillimayhoweveralsosurvivewithinthephagosomeofthesemacrophages,

andinthelattercase,thesepeoplearesaidtobelatentlyinfected.Theirbacillisurviveina

hibernatingmode,controlledbyageneticsystemreferredtoasdosRregulon[4].Thisisa

geneticprogramcontrolledbyasetof48genes thatallows the tuberclebacilli tosurvive

understressconditions;duringactiveimmunesuppression,hibernatingorganismsarecalled

‘latent’whileduringdrugtreatment,theseorganismsarecalled‘persistent’.Latentlyinfected

individualstypicallyhaveonlylimitednumbersof livingbacterialcellsthatslowlyreplicate

and are hardly metabolically active. People with latent TB infection feel well, have no

symptomsandarenotcontagious[5].

M. tuberculosis belongs to a small group of highly pathogenic bacteria (M. tuberculosis

complex)intheverylargefamilyofmycobacteria,characterizedbyathickcellwall,consisting

ofseveraldifferentspecificlipidmoleculesincludinglipoarabinomannanandmycolicacids.

Themajorityofcrosslinksinthepeptidoglycanlayerareformeddifferentlycomparedtogram

positivebacteria,makingmycobacteriamoreresistanttochemicaldamageandhydrophilic

antibiotics. As the replication rate ofM. tuberculosis – even if actively replicating and

metabolicallyactive-isveryslow(≈20h)comparedtootherbacteria(≈20min),TBrequires

morespecificantibioticsandprolongedtreatment,asmostantibioticsonlyworkonactively

replicatingbacteria[5].Typically,rapidlydividingmetabolicallyactivebacillicanbereduced

rapidlywithinweeks; severalhighlyactiveagentshavebactericidalproperties.Dueto the

slowreplicationrateofM.tuberculosis,especiallyofdifficulttoeradicatepersisterphenotype

bacteria. TB treatment needs to last long to obtain a sterilising effect. TheWorld Health

Organisation (WHO) therefore advises to threat TB with a standard first-line treatment

consistingofisoniazid(H),Rifampicin(R),pyrazinamide(Z)andethambutol(E)–HRZE–And

therebyintensivelydecreasethebacterialload(intensivephase).Followedbytheintensive

phase, a four-month continuationwith isoniazid and rifampicin is needed to provide the

opportunity to eliminate the last TB bacteria which are in a persistent state of being

capsulatedbymacrophages.

MDRTB-AntimicrobialresistanceofTB

Unfortunately, our world is facing a public health crisis and security threat due to the

treatmentofTBbecomingincreasinglychallengingwiththeemergenceofresistancetofirst-

linedrugs.Multidrug resistant (MDR)-TB is definedas an infectiousdisease causedbyM.

tuberculosisthatisresistanttoatleastisoniazidandrifampicin,whicharethecornerstone

drugs of drug-susceptible TB treatment. Extensively drug resistant (XDR)-TB is defined as

MDR-TBwithadditionalresistancetoatleastoneofthefluoroquinolonesandtoatleastone

oftheinjectablesecondlinedrugs[6].WHOestimatesthattherewere558.000newcases

withresistancetorifampicin,themosteffectivefirstlinedrug[2].

Development of new drugs is slow and expensive due to the obligatory market access

regulationssuchasrandomizedclinicaltrials.BedaquilineandDelamanid,bothwithanovel

mechanismofaction,were included intheWHOguidelinesonMDR-TB,afterapprovalby

10

Chapter 1

FederalDrugAdministration(FDA)andEuropeanMedicinesAgency(EMA)almostfiveyears

ago.Anindividualpatientdatameta-analysisrevealedthatofalldrugsusedtotreatMDR-TB,

theaddedvalueoftheinjectableagents’kanamycinandcapreomycinwasactuallyassociated

with poor outcome. Bedaquiline; the fluoroquinolones levofloxacin, gatifloxacin and

moxifloxacin;andlinezolidwereassociatedwithbeneficialoutcome[7].Basedonthismeta-

analysis,WHOissuedarapidcommunicationupdatingtheprovisionalguidelinesforMDR-TB

treatment [8]. Bedaquiline has now obtained a position in Group A; together with

FluoroquinolonesandLinezolid,thisdrugisnowconsideredamongthemostpowerfulagents

tofightMDR-TB.Unfortunately,resistancetothesenovelagentshasalreadybeendetected

[9].Obviously,thecostsofthesehighlyeffectivenovelagentsareaconstraintforuseinlow-

resourcedsettings.

The challenges to eradicate TB by 2030 are vast;many of the second line drugs are also

associatedwith toxicityandadverseeffects;and there is thereforeadesire foradditional

drugs with low inherent toxicity. Apart from developing additional new drugs, the

repurposingofdrugsthatarealreadyavailable forother indicationswouldbeanasset to

improveandextendcurrenttreatmentoptions,bydevelopingmoreactive–sterilizing-anti

TBdrugs[10-11].Multiplepartnershipshavebeeninitiatedwiththejointgoaloferadicating

resistancebydevelopingandproducingnewdrugsandrediscoveringolddrugs.

Rediscoveryofolddrugs

Oneparticularlyeffectivestrategy isrediscoveryofolddrugsasnewagentsfortreatment

against multidrug resistant tuberculosis. Linezolid and moxifloxacin already have been

exploredasnewagentsagainstMDR-TB[12-15].Benefitsofrepurposingoldantibioticsisthat

thesedrugsare commonly cheapandclinicalexperience is substantial resulting inawell-

establisheddrugsafetyprofile.TounlocktheirpotentialasnewTBagentsandobtainmarket

approval, efficacy, safety and toxicity profile needs to be established. Therefore, a

pharmacokinetic andpharmacodynamicsprofileneeds tobeestablishedanddose-finding

studiesareneededtoestablishrequireddoseinTBpatients.

Nowadays, beta-lactam antimicrobial drugs arewidely used drugs for the treatment of a

rangeofinfections[16].BeginningwiththediscoveryofpenicillinbyAlexanderFleminginthe

late1920s,antibioticschangedtreatmentofbacterialinfections,savingmillionsoflives.By

mid-1940 itbecameclearthatpenicillinwasnoteffectiveatkillingM.tuberculosis. Inthe

1960s it became clear that M. tuberculosis produced an enzyme, called beta-lactamase

(BLaC),whichrapidlyhydrolysis’sthebeta-lactamring.Carbapenemactivityhavetherefore

longbeenconsideredtobeoflimiteduse.However,morethanadecadeago,researchers

showedthatclavulanateirreversiblyblockedbeta-lactamaseenzymeofM.tuberculosis[17].

Recent studies suggest that beta-lactams, using clavulanate/clavulanic acid, show more

activityagainstM.tuberculosisandcouldbebeneficialinthetreatmentofTB[18-22].

CarbapenemsareearmarkedaspotentiallyactivedrugsforthetreatmentofM.tuberculosis.

Imipenem-cilastatinandmeropenemhavebeenlistedasadd-ondrugsintheupdatedWHO

treatment guidelines. Ertapenem, approved in 2001 by the FDA, an old drugwidely used

againstgrampositiveandnegativebacteriahasshowntobeactive inMDR-TB [23-25]. In

general,ertapenemappearstobefavourableandahighlypromisingdrugforthetreatment

ofMDR-TBthatwarrantsfurtherinvestigation.

Aimofthethesis

TobetterunderstandthepotentialroleofertapenemforthetreatmentofM/XDR-TB,the

aimofthisthesiswastoevaluatecurrentliterature,invitroactivity,andpharmacokinetics

andsafetyinTBpatients.

11

1


FederalDrugAdministration(FDA)andEuropeanMedicinesAgency(EMA)almostfiveyears

ago.Anindividualpatientdatameta-analysisrevealedthatofalldrugsusedtotreatMDR-TB,

theaddedvalueoftheinjectableagents’kanamycinandcapreomycinwasactuallyassociated

with poor outcome. Bedaquiline; the fluoroquinolones levofloxacin, gatifloxacin and

moxifloxacin;andlinezolidwereassociatedwithbeneficialoutcome[7].Basedonthismeta-

analysis,WHOissuedarapidcommunicationupdatingtheprovisionalguidelinesforMDR-TB

treatment [8]. Bedaquiline has now obtained a position in Group A; together with

FluoroquinolonesandLinezolid,thisdrugisnowconsideredamongthemostpowerfulagents

tofightMDR-TB.Unfortunately,resistancetothesenovelagentshasalreadybeendetected

[9].Obviously,thecostsofthesehighlyeffectivenovelagentsareaconstraintforuseinlow-

resourcedsettings.

The challenges to eradicate TB by 2030 are vast;many of the second line drugs are also

associatedwith toxicityandadverseeffects;and there is thereforeadesire foradditional

drugs with low inherent toxicity. Apart from developing additional new drugs, the

repurposingofdrugsthatarealreadyavailable forother indicationswouldbeanasset to

improveandextendcurrenttreatmentoptions,bydevelopingmoreactive–sterilizing-anti

TBdrugs[10-11].Multiplepartnershipshavebeeninitiatedwiththejointgoaloferadicating

resistancebydevelopingandproducingnewdrugsandrediscoveringolddrugs.

Rediscoveryofolddrugs

Oneparticularlyeffectivestrategy isrediscoveryofolddrugsasnewagentsfortreatment

against multidrug resistant tuberculosis. Linezolid and moxifloxacin already have been

exploredasnewagentsagainstMDR-TB[12-15].Benefitsofrepurposingoldantibioticsisthat

thesedrugsare commonly cheapandclinicalexperience is substantial resulting inawell-

establisheddrugsafetyprofile.TounlocktheirpotentialasnewTBagentsandobtainmarket

approval, efficacy, safety and toxicity profile needs to be established. Therefore, a

pharmacokinetic andpharmacodynamicsprofileneeds tobeestablishedanddose-finding

studiesareneededtoestablishrequireddoseinTBpatients.

Nowadays, beta-lactam antimicrobial drugs arewidely used drugs for the treatment of a

rangeofinfections[16].BeginningwiththediscoveryofpenicillinbyAlexanderFleminginthe

late1920s,antibioticschangedtreatmentofbacterialinfections,savingmillionsoflives.By

mid-1940 itbecameclearthatpenicillinwasnoteffectiveatkillingM.tuberculosis. Inthe

1960s it became clear that M. tuberculosis produced an enzyme, called beta-lactamase

(BLaC),whichrapidlyhydrolysis’sthebeta-lactamring.Carbapenemactivityhavetherefore

longbeenconsideredtobeoflimiteduse.However,morethanadecadeago,researchers

showedthatclavulanateirreversiblyblockedbeta-lactamaseenzymeofM.tuberculosis[17].

Recent studies suggest that beta-lactams, using clavulanate/clavulanic acid, show more

activityagainstM.tuberculosisandcouldbebeneficialinthetreatmentofTB[18-22].


Imipenem-cilastatinandmeropenemhavebeenlistedasadd-ondrugsintheupdatedWHO

treatment guidelines. Ertapenem, approved in 2001 by the FDA, an old drugwidely used

againstgrampositiveandnegativebacteriahasshowntobeactive inMDR-TB [23-25]. In

general,ertapenemappearstobefavourableandahighlypromisingdrugforthetreatment

ofMDR-TBthatwarrantsfurtherinvestigation.

Aimofthethesis

TobetterunderstandthepotentialroleofertapenemforthetreatmentofM/XDR-TB,the

aimofthisthesiswastoevaluatecurrentliterature,invitroactivity,andpharmacokinetics

andsafetyinTBpatients.

12

Chapter 1

OUTLINEOFTHETHESIS

In this thesis, we plan to evaluate the pharmacology of ertapenem in the treatment of

multidrugresistanttuberculosis.

inchapter2,Weplantostudyliteraturetoevaluatecurrentknowledgeoninvitro,invivo

andhumanactivityofcarbapenems

In chapter 3 we aim to develop a simple validated LC-MS/MS for the validation and

quantificationofertapenemrequiredforfuturepharmacokineticstudies.

Inchapter4weplantoevaluatepharmacokineticsandsafetyofertapenemusedtocomplete

atreatmentregimenforMDRTBpatients

Inchapter5weaimtodevelopasuitableexperiment toevaluate thesusceptibilityofM.

tuberculosisforertapenemasthecurrentlyusedassaysarenotsuitablebecauseertapenem

degradesfastunderstandardconditions(37C)

Inchapter6,weintendtostudythesterilizingeffectofertapenem-clavulanateinahollow

fibermodeloftuberculosistoselectadoseforfutureclinicalstudies

Inchapter7,weproposetodevelopapharmacokineticmodelandalimitedsamplingstrategy

whichcouldbeusedforafuturephaseIIstudy

References

1. Tuberculosisfactsheet.2018;Availableat:http://www.who.int/en/news-room/fact-

sheets/detail/tuberculosis

2. WHOreport:GlobalTuberculosisreport2017.2017.http://www.who.int/tb/en/

3. National Institute for Public Health and the Environment (RIVM). 2017. State of

infectiousdiseasesinTheNetherlands,2016.NationalInstituteforPublicHealthand

theEnvironment,Bilthoven,TheNetherlands.

4. VanAltena,DuggiralaS,GröschelMI,vanderWerf.2011.Immunologyintuberculosis:

challengesinmonitoringofdiseaseactivityandidentifyingcorrelatesofprotection.

CurrPharmDes.2011;17(27):2853-62.

5. WorldHealthOrganization.GuidelinesfortreatmentofTuberculosis.Fourthedition

ed.Geneva,Switserland:WorldHealthOrganization;2010.

6. FalzonD, SchünemannHJ, Harausz E, González-Angulo L, Lienhardt C, Jaramillo E,

Weyer K. 2017World Health Organization treatment guidelines for drug-resistant

tuberculosis,2016update.EurRespirJ.Mar22;49(3).

7. Collaborative Group for the Meta-Analysis of Individual Patient Data in MDR-TB

treatment–2017, Ahmad N, Ahuja SD, Akkerman OW, Alffenaar JC, Anderson LF,

BaghaeiP,BangD,BarryPM,BastosML,BeheraD,BenedettiA,BissonGP,Boeree

MJ,BonnetM,BrodeSK,BrustJCM,CaiY,CaumesE,CegielskiJP,CentisR,ChanPC,

ChanED,ChangKC,CharlesM,CiruleA,DalcolmoMP,D'AmbrosioL,deVriesG,Dheda

K,EsmailA,FloodJ,FoxGJ,Fréchet-JachymM,FregonaG,GayosoR,GegiaM,Gler

MT,Gu S,Guglielmetti L,Holtz TH,Hughes J, Isaakidis P, Jarlsberg L, KempkerRR,

KeshavjeeS,KhanFA,KipianiM,KoenigSP,KohWJ,KritskiA,KuksaL,KvasnovskyCL,

KwakN,LanZ,LangeC,Laniado-LaborínR,LeeM,LeimaneV,LeungCC,LeungEC,Li

PZ,LowenthalP,MacielEL,MarksSM,MaseS,MbuagbawL,MiglioriGB,MilanovV,

MillerAC,MitnickCD,ModongoC,MohrE,MonederoI,NahidP,NdjekaN,O'Donnell

MR,PadayatchiN,PalmeroD,PapeJW,PodewilsLJ,ReynoldsI,RiekstinaV,RobertJ,

RodriguezM,SeaworthB,SeungKJ,SchnippelK,ShimTS,SinglaR,SmithSE,SotgiuG,

SukhbaatarG,TabarsiP,TiberiS,TrajmanA,TrieuL,UdwadiaZF,vanderWerfTS,

VezirisN,ViikleppP,VilbrunSC,WalshK,WestenhouseJ,YewWW,YimJJ,ZetolaNM,

Zignol M, Menzies D. Treatment correlates of successful outcomes in pulmonary

13

1


OUTLINEOFTHETHESIS

In this thesis, we plan to evaluate the pharmacology of ertapenem in the treatment of

multidrugresistanttuberculosis.

inchapter2,Weplantostudyliteraturetoevaluatecurrentknowledgeoninvitro,invivo

andhumanactivityofcarbapenems

In chapter 3 we aim to develop a simple validated LC-MS/MS for the validation and

quantificationofertapenemrequiredforfuturepharmacokineticstudies.

Inchapter4weplantoevaluatepharmacokineticsandsafetyofertapenemusedtocomplete

atreatmentregimenforMDRTBpatients

Inchapter5weaimtodevelopasuitableexperiment toevaluate thesusceptibilityofM.

tuberculosisforertapenemasthecurrentlyusedassaysarenotsuitablebecauseertapenem

degradesfastunderstandardconditions(37C)

Inchapter6,weintendtostudythesterilizingeffectofertapenem-clavulanateinahollow

fibermodeloftuberculosistoselectadoseforfutureclinicalstudies

Inchapter7,weproposetodevelopapharmacokineticmodelandalimitedsamplingstrategy

whichcouldbeusedforafuturephaseIIstudy

References

1. Tuberculosisfactsheet.2018;Availableat:http://www.who.int/en/news-room/fact-

sheets/detail/tuberculosis

2. WHOreport:GlobalTuberculosisreport2017.2017.http://www.who.int/tb/en/

3. National Institute for Public Health and the Environment (RIVM). 2017. State of

infectiousdiseasesinTheNetherlands,2016.NationalInstituteforPublicHealthand

theEnvironment,Bilthoven,TheNetherlands.

4. VanAltena,DuggiralaS,GröschelMI,vanderWerf.2011.Immunologyintuberculosis:

challengesinmonitoringofdiseaseactivityandidentifyingcorrelatesofprotection.

CurrPharmDes.2011;17(27):2853-62.

5. WorldHealthOrganization.GuidelinesfortreatmentofTuberculosis.Fourthedition

ed.Geneva,Switserland:WorldHealthOrganization;2010.

6. FalzonD, SchünemannHJ, Harausz E, González-Angulo L, Lienhardt C, Jaramillo E,

Weyer K. 2017World Health Organization treatment guidelines for drug-resistant

tuberculosis,2016update.EurRespirJ.Mar22;49(3).


treatment–2017, Ahmad N, Ahuja SD, Akkerman OW, Alffenaar JC, Anderson LF,

BaghaeiP,BangD,BarryPM,BastosML,BeheraD,BenedettiA,BissonGP,Boeree

MJ,BonnetM,BrodeSK,BrustJCM,CaiY,CaumesE,CegielskiJP,CentisR,ChanPC,

ChanED,ChangKC,CharlesM,CiruleA,DalcolmoMP,D'AmbrosioL,deVriesG,Dheda

K,EsmailA,FloodJ,FoxGJ,Fréchet-JachymM,FregonaG,GayosoR,GegiaM,Gler

MT,Gu S,Guglielmetti L,Holtz TH,Hughes J, Isaakidis P, Jarlsberg L, KempkerRR,

KeshavjeeS,KhanFA,KipianiM,KoenigSP,KohWJ,KritskiA,KuksaL,KvasnovskyCL,

KwakN,LanZ,LangeC,Laniado-LaborínR,LeeM,LeimaneV,LeungCC,LeungEC,Li

PZ,LowenthalP,MacielEL,MarksSM,MaseS,MbuagbawL,MiglioriGB,MilanovV,

MillerAC,MitnickCD,ModongoC,MohrE,MonederoI,NahidP,NdjekaN,O'Donnell

MR,PadayatchiN,PalmeroD,PapeJW,PodewilsLJ,ReynoldsI,RiekstinaV,RobertJ,

RodriguezM,SeaworthB,SeungKJ,SchnippelK,ShimTS,SinglaR,SmithSE,SotgiuG,

SukhbaatarG,TabarsiP,TiberiS,TrajmanA,TrieuL,UdwadiaZF,vanderWerfTS,

VezirisN,ViikleppP,VilbrunSC,WalshK,WestenhouseJ,YewWW,YimJJ,ZetolaNM,

Zignol M, Menzies D. Treatment correlates of successful outcomes in pulmonary

14

Chapter 1

multidrug-resistant tuberculosis: an individual patient data meta-analysis. Lancet.

2018Sep8;392(10150):821-834.doi:10.1016/S0140-6736(18)31644-1.Review.

8. WHO report: Rapid Communication: Key changes to treatment of multidrug- and

rifampicin-resistant tuberculosis (MDR/RR-TB) 2018. Available at:

http://www.who.int/tb/publications/2018/rapid_communications_MDR/en/

9. NguyenTVA,AnthonyRM,BañulsAL,VuDH,AlffenaarJC.Bedaquilineresistance:Its

emergence, mechanism and prevention. Clin Infect Dis. 2017 Nov 8. doi:

10.1093/cid/cix992.

10. WorldHealthOrganization(WHO(ed.).2014.TheendTBstrategy:Globalstrategyand

targets for tuberculosis prevention, care and control after 2015. World Health

OrganizationGeneva,Switzerland.

11. United nations development Programme: Goal 3; Good-health and well-being:

http://www.undp.org/content/undp/en/home/sustainable-development-goals/goal-

3-good-health-and-well-being.html

12. MillardJ,PertinezH,BonnettL,HodelEM,DartoisV,JohnsonJL,CawsM,TiberiS,

Alffenaar JC, Davies G, Sloan DJ. 2018. Linezolid pharmacokinetics in MDR-TB: a

systematic review, meta-analysis and Monte Carlo simulation. J Antimicrob.

Chemother.

13. SotgiuG,CentisR,D’AmbrosioL,SpanevelloA,MiglioriGB;InternationalGroupfor

the study of Linezolid. 2013. Linezolid to treat extensively drug-resistant TB:

retrospective data are confirmed by experimental evidence. Eur Respir J.

Jul;42(1):288-90.

14. PrangerAD,AlffenaarJW,AarnoutseRE.2011.Fluoroquinolones,thecornerstoneof

treatmentofdrug-resistant tuberculosis:apharmacokineticandpharmacodynamic

approach.CurrPharmDes.2011;17027):2900-30

15. PrangerAD,vanAltenaR,AarnoutseRE,vanSoolingenD,UgesDR,KosterinkJG,van

der Werf TS, Alffenaar JW. Evaluation of moxifloxacin for the treatment of

tuberculosis:3yearsofexperience.EurRespirJ.201138(4):888-894

16. YatesTA,KhanPY,KnightGM,etal.ThetransmissionofMycobacteriumtuberculosis

inhighburdensettings.LancetInfectDis2016;16(2):227-38.

17. Hugonnet JE, Blanchard JS. 2007. Irreversible inhibition of the Mycobacterium

tuberculosis beta-lactamase by clavulanate. Biochemistry. 46:11998-12004. doi:

10.1021/bi701506h.

18. HugonnetJE,TremblayLW,BoshoffHI,Barry3rdCE,BlanchardJS.2009.Meropenem-

clavulanate is effective against extensively drug-resistant Mycobacterium

tuberculosis. Science. 323:1215-1218. doi: 10.1126/science.1167498;

10.1126/science.1167498.

19. DeshpandeD,SrivastavaS,ChapagainM,MagombedzeG,MartinKR,CirrincioneKN,

Lee PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactam has potent

sterilizing activity against highly drug-resistant tuberculosis. Sci Adv. Aug

30;3(8):e1701102

20. DeshpandeD,SrivastavaS,BendetP,MartinKR,CirrincioneKN,LeePS,Pasipanodya

JG,DhedaK,GumboT.2018.AntibacterialandSterilizingEffectofBenzylpenicillinin

Tuberculosis.AntimicrobAgentsChemother.Jan25;62(2).

21. Cynamon,M.H.,andG.S.Palmer.1983.Invitroactivityofamoxicillinincombination

with clavulanic acid against Mycobacterium tuberculosis. Antimicrob Agents

Chemother.24:429-431.




23. KaushikA,MakkerN,PandeyP,ParrishN,SinghU,LamichaneG.2015.Carbapenems

and Rifampicin exhibit synergy against Mycobacterium tuberculosis and

Mycobacterium abscessus. Antimicrob Agents Chemother 59:6561-6567.

Doi:10.1128/AAC.01158-15

24. Veziris, N., C. Truffot, J. L.Mainardi, and V. Jarlier. 2011. Activity of carbapenems

combined with clavulanate against murine tuberculosis. Antimicrob. Agents

Chemother.55:2597-2600.doi:10.1128/AAC.01824-10;10.1128/AAC.01824-10.

25. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,Alffenaar JWC,

MiglioriGB.2015. Ertapenem in the treatmentofmultidrug-resistant tuberculosis:

firstclinicalexperience.EurRespirJ47:333-336.Doi:10.1183/13993003.01278-2015

15

1


multidrug-resistant tuberculosis: an individual patient data meta-analysis. Lancet.

2018Sep8;392(10150):821-834.doi:10.1016/S0140-6736(18)31644-1.Review.

8. WHO report: Rapid Communication: Key changes to treatment of multidrug- and



9. NguyenTVA,AnthonyRM,BañulsAL,VuDH,AlffenaarJC.Bedaquilineresistance:Its


10.1093/cid/cix992.

10. WorldHealthOrganization(WHO(ed.).2014.TheendTBstrategy:Globalstrategyand

targets for tuberculosis prevention, care and control after 2015. World Health

OrganizationGeneva,Switzerland.

11. United nations development Programme: Goal 3; Good-health and well-being:

http://www.undp.org/content/undp/en/home/sustainable-development-goals/goal-

3-good-health-and-well-being.html

12. MillardJ,PertinezH,BonnettL,HodelEM,DartoisV,JohnsonJL,CawsM,TiberiS,

Alffenaar JC, Davies G, Sloan DJ. 2018. Linezolid pharmacokinetics in MDR-TB: a

systematic review, meta-analysis and Monte Carlo simulation. J Antimicrob.

Chemother.

13. SotgiuG,CentisR,D’AmbrosioL,SpanevelloA,MiglioriGB;InternationalGroupfor

the study of Linezolid. 2013. Linezolid to treat extensively drug-resistant TB:

retrospective data are confirmed by experimental evidence. Eur Respir J.

Jul;42(1):288-90.

14. PrangerAD,AlffenaarJW,AarnoutseRE.2011.Fluoroquinolones,thecornerstoneof

treatmentofdrug-resistant tuberculosis:apharmacokineticandpharmacodynamic

approach.CurrPharmDes.2011;17027):2900-30

15. PrangerAD,vanAltenaR,AarnoutseRE,vanSoolingenD,UgesDR,KosterinkJG,van

der Werf TS, Alffenaar JW. Evaluation of moxifloxacin for the treatment of

tuberculosis:3yearsofexperience.EurRespirJ.201138(4):888-894

16. YatesTA,KhanPY,KnightGM,etal.ThetransmissionofMycobacteriumtuberculosis

inhighburdensettings.LancetInfectDis2016;16(2):227-38.



10.1021/bi701506h.

18. HugonnetJE,TremblayLW,BoshoffHI,Barry3rdCE,BlanchardJS.2009.Meropenem-

clavulanate is effective against extensively drug-resistant Mycobacterium


10.1126/science.1167498.

19. DeshpandeD,SrivastavaS,ChapagainM,MagombedzeG,MartinKR,CirrincioneKN,

Lee PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactam has potent

sterilizing activity against highly drug-resistant tuberculosis. Sci Adv. Aug

30;3(8):e1701102




21. Cynamon,M.H.,andG.S.Palmer.1983.Invitroactivityofamoxicillinincombination

with clavulanic acid against Mycobacterium tuberculosis. Antimicrob Agents





23. KaushikA,MakkerN,PandeyP,ParrishN,SinghU,LamichaneG.2015.Carbapenems

and Rifampicin exhibit synergy against Mycobacterium tuberculosis and

Mycobacterium abscessus. Antimicrob Agents Chemother 59:6561-6567.

Doi:10.1128/AAC.01158-15

24. Veziris, N., C. Truffot, J. L.Mainardi, and V. Jarlier. 2011. Activity of carbapenems

combined with clavulanate against murine tuberculosis. Antimicrob. Agents


25. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,Alffenaar JWC,

MiglioriGB.2015. Ertapenem in the treatmentofmultidrug-resistant tuberculosis:

firstclinicalexperience.EurRespirJ47:333-336.Doi:10.1183/13993003.01278-2015

CHAPTER 2

Antimicrob. Agents Chemother. 2019 Jan 29; 63(2).PMID: 30455232.

*Both authors contributed equally

S.P. van Rijn*M.A. Zuur*R. AnthonyB. Wilffert

R. van AltenaO.W. Akkerman

W.C.M. de LangeT.S. van der WerfJ.G.W. KosterinkJ.W.C. Alffenaar

Evaluation of Carbapenems for Treatment of Multi- and Extensively

Drug-Resistant Mycobacterium Tuberculosis

18

Chapter 2

Abstract

M/XDR-TB has become an increasing threat in high burden countries but also in affluent

regionsduetoincreasedinternationaltravelandglobalization.Carbapenemsareearmarked

aspotentiallyactivedrugs for the treatmentofM.tuberculosis.Tobetterunderstand the

potential of carbapenems for the treatment ofM/XDR-TB, the aim of this reviewwas to

evaluatetheliteratureoncurrentlyavailableinvitro,invivoandclinicaldataoncarbapenems

inthetreatmentofM.tuberculosisanddetectionofknowledgegaps,inordertotargetfuture

research.InFebruary2018,asystematicliteraturesearchofPubMedandWebofSciencewas

performed.Overalltheresultsofthestudiesidentifiedinthisreview,whichusedavarietyof

carbapenemsusceptibilitytestsonclinicalandlabstrainsofM.tuberculosis,areconsistent.

In vitro the activity of carbapenems againstM. tuberculosis is increased when used in

combinationwith clavulanate, a BLaC inhibitor.However, clavulanate is not commercially

available alone, and therefore is it practically impossible to prescribe carbapenems in

combinationwith clavulanateat this time. Few in vivo studieshavebeenperformed,one

prospective, two observational and seven retrospective clinical studies to assess

effectiveness,safetyandtolerabilityofthreedifferentcarbapenems(imipenem,meropenem

andertapenem).Presentlywefoundnoclearevidencetoselectoneparticularcarbapenem

among the different candidate compounds, to design an effective M/XDR-TB regimen.

Therefore, more clinical evidence and dose optimization substantiated by hollow fiber

infection studies are needed to support repurposing carbapenems for the treatment of

M/XDR-TB.

Introduction

Treatmentoftuberculosis(TB),adiseasecausedbyMycobacteriumtuberculosis,hasbecome

morechallengingwiththeemergenceofmultidrugresistant(MDR)-TBandextensivelydrug

resistant(XDR)-TBamongpreviouslyandnewlydetectedcases(1).M/XDR-TBhasbecomean

increasing threat in high burden countries but also in affluent regions due to increased

internationaltravelandglobalization.

MDR-TBisdefinedasaninfectiousdiseasecausedbyM.tuberculosisthatisresistanttoat

leastisoniazidandrifampicin.XDR-TBisdefinedasMDR-TBwithadditionalresistancetoat

least one of the fluoroquinolones and to at least one of the injectable second line drugs

(amikacin, capreomycinor kanamycin).NewTBdrugs,with anovelmechanismof action,

includebedaquilineanddelamanid thathave recentlybeenapprovedand included in the

World Health Organization guidelines on MDR-TB as add-on agents (2). Unfortunately,

resistancetotheseagentshasalreadybeendetected(3).Explorationofcurrentlyavailable

drugs for their potential effect against TB, may be an additional source for potential

candidatestobeusedincaseofextensiveresistancetotrytocomposeatreatmentregimen

(4-5).

Beta-lactam antimicrobial drugs are widely used drugs for the treatment of a range of

infections.Also,imipenem-cilastatinandmeropenemhavebeenlistedasadd-ondrugsinthe

updatedWHOtreatmentguidelines(6).Carbapenemactivityhaslongbeenconsideredtobe

of limited use, due to rapid hydrolysis of the beta -lactam ring by broad-spectrum

mycobacterialclassAbeta-lactamases(BLaC).TheadditionoftheBLaCinhibitorclavulanate

suggests that beta-lactams combined with BLaC inhibitors could be beneficial in the

treatmentofTB(7).Recentstudiessuggestthatbeta-lactams,usingclavulanate/clavulanic

acid,showmoreactivityagainstM.tuberculosis(7-14).

Thebacterialactivityofbeta-lactamsisduetotheinactivationofbacterialtranspeptidases,

commonlyknownaspenicillinbindingproteins(PBP),whichinhibitthebiosynthesisofthe

peptidoglycanlayerofthecellwallofbacteria(8,15).Polymerizationsofthepeptidoglycan

layer in most bacteria are predominantly cross-linked by D,D-transpeptidases (DDT), the

19

2

Evaluation of Carbapenems for Treatment of M/XDR Mycobacterium Tuberculosis

Abstract

M/XDR-TB has become an increasing threat in high burden countries but also in affluent

regionsduetoincreasedinternationaltravelandglobalization.Carbapenemsareearmarked

aspotentiallyactivedrugs for the treatmentofM.tuberculosis.Tobetterunderstand the

potential of carbapenems for the treatment ofM/XDR-TB, the aim of this reviewwas to

evaluatetheliteratureoncurrentlyavailableinvitro,invivoandclinicaldataoncarbapenems

inthetreatmentofM.tuberculosisanddetectionofknowledgegaps,inordertotargetfuture

research.InFebruary2018,asystematicliteraturesearchofPubMedandWebofSciencewas

performed.Overalltheresultsofthestudiesidentifiedinthisreview,whichusedavarietyof

carbapenemsusceptibilitytestsonclinicalandlabstrainsofM.tuberculosis,areconsistent.

In vitro the activity of carbapenems againstM. tuberculosis is increased when used in

combinationwith clavulanate, a BLaC inhibitor.However, clavulanate is not commercially

available alone, and therefore is it practically impossible to prescribe carbapenems in

combinationwith clavulanateat this time. Few in vivo studieshavebeenperformed,one

prospective, two observational and seven retrospective clinical studies to assess


andertapenem).Presentlywefoundnoclearevidencetoselectoneparticularcarbapenem

among the different candidate compounds, to design an effective M/XDR-TB regimen.

Therefore, more clinical evidence and dose optimization substantiated by hollow fiber

infection studies are needed to support repurposing carbapenems for the treatment of

M/XDR-TB.

Introduction

Treatmentoftuberculosis(TB),adiseasecausedbyMycobacteriumtuberculosis,hasbecome

morechallengingwiththeemergenceofmultidrugresistant(MDR)-TBandextensivelydrug

resistant(XDR)-TBamongpreviouslyandnewlydetectedcases(1).M/XDR-TBhasbecomean

increasing threat in high burden countries but also in affluent regions due to increased

internationaltravelandglobalization.

MDR-TBisdefinedasaninfectiousdiseasecausedbyM.tuberculosisthatisresistanttoat

leastisoniazidandrifampicin.XDR-TBisdefinedasMDR-TBwithadditionalresistancetoat

least one of the fluoroquinolones and to at least one of the injectable second line drugs

(amikacin, capreomycinor kanamycin).NewTBdrugs,with anovelmechanismof action,

includebedaquilineanddelamanid thathave recentlybeenapprovedand included in the

World Health Organization guidelines on MDR-TB as add-on agents (2). Unfortunately,

resistancetotheseagentshasalreadybeendetected(3).Explorationofcurrentlyavailable

drugs for their potential effect against TB, may be an additional source for potential

candidatestobeusedincaseofextensiveresistancetotrytocomposeatreatmentregimen

(4-5).

Beta-lactam antimicrobial drugs are widely used drugs for the treatment of a range of

infections.Also,imipenem-cilastatinandmeropenemhavebeenlistedasadd-ondrugsinthe

updatedWHOtreatmentguidelines(6).Carbapenemactivityhaslongbeenconsideredtobe

of limited use, due to rapid hydrolysis of the beta -lactam ring by broad-spectrum

mycobacterialclassAbeta-lactamases(BLaC).TheadditionoftheBLaCinhibitorclavulanate

suggests that beta-lactams combined with BLaC inhibitors could be beneficial in the

treatmentofTB(7).Recentstudiessuggestthatbeta-lactams,usingclavulanate/clavulanic

acid,showmoreactivityagainstM.tuberculosis(7-14).

Thebacterialactivityofbeta-lactamsisduetotheinactivationofbacterialtranspeptidases,

commonlyknownaspenicillinbindingproteins(PBP),whichinhibitthebiosynthesisofthe

peptidoglycanlayerofthecellwallofbacteria(8,15).Polymerizationsofthepeptidoglycan

layer in most bacteria are predominantly cross-linked by D,D-transpeptidases (DDT), the

20

Chapter 2

enzymesinhibitedbybeta-lactams(8,16).Themajorityofcrosslinksinpeptidoglycanappear

tobeformedbythenon-classicalL,D-transpeptidases(LDT)inM.tuberculosis(17-23).Several

studiesrevealedthestructuralbasisandtheinactivationmechanismofLDTandtheactive

roleofcarbapenems,providingabasisforthepotentialuseofcarbapenemsininhibitingM.

tuberculosis(24-28).

Beta-lactams show time-dependent activity, carbapenems have been shown to have

bactericidalactivitywhenthefreedrugplasmaconcentrationexceedstheMICforatleast40

%ofthetimeinnon-TBbacterialspecies(29-30).


TobetterunderstandthepotentialofcarbapenemsforthetreatmentofM/XDR-TB,theaim

ofthisreviewwastoevaluatetheliteratureoncurrentlyavailableinvitro,invivoandclinical

dataoncarbapenemsinthetreatmentofM.tuberculosisanddetectionofknowledgegaps,

inordertotargetfutureresearch.

Methods

Prisma

ThissystematicreviewwasconductedinaccordancewiththePreferredReportingItemsfor

SystematicReviewsandMeta-Analyses(PRISMA)statement.

Search

In February2018, a systematic literature searchof PubMedandWebof Science,without

restrictions with respect to publication date was employed using the key words

(´Carbapenem’ OR ‘Carbapenems’ OR ‘Imipenem’ OR ‘Meropenem’ OR ‘Ertapenem’ OR

‘Doripenem’ OR ‘Faropenem’ OR ‘Biapenem’ OR ‘Panipenem’ OR ‘Tebipenem’) AND

(‘Tuberculosis’ORTBORMycobacteriumtuberculosis)asMeShTerms.Retrievedstudiesand

abstractsfrombothPubMedandWebofSciencewerepooledandduplicateswereremoved.

Titlesandabstractsofretrievedarticleswerescreened.Reviews,case-reportsorstudieson

otherspeciesthanTBorstudiesonotherdrugsthancarbapenemswereexcluded.Studies

werescreenedforeligibility.Ifeligible,thefull-textwasreadbyaresearcher(SvR).Asecond

researcher(MZ)independentlyrepeatedthearticlesearchandselection.Discrepancieswere

resolved by discussion, or a third researcher was consulted (JWA). Full text papers were

subdividedintothreesections;invitro,invivoandclinicaldata.Fulltextpapersforinvitro

data were eligible for inclusion if an M. tuberculosis strain was studied and minimum

inhibitoryconcentrationswere reported.Full textpapers for invivodatawereeligible for

inclusioniftreatmentofM.tuberculosisinfectionswithcarbapenemswerestudiedinanimal

models,andifcolonyformingunitsand/orsurvivaldatawerereported.Fulltextpapersfor

clinical data were eligible for inclusion if pharmacokinetics of carbapenems or safety or

responsetotreatmentmeasuredassurrogateendpoints(sputumconversion)orclinicalend

pointswerestudiedandreported.Referencesofallincludedarticleswerescreenedbyhand.

Thesamesystematic searchwasperformedusingclinicaltrials.gov to findongoing studies

investigatingcarbapenemsinTBpatients(Feb2018).

Dataextraction

Aresearcher(SvR)performeddataextractionfirstbyusingastructureddatacollectionform.

Asecondresearcher(MZ)verifiedthedataextractionindependently.Dataweresubdivided

intothreesections;invitro,invivoandclinicaldata.Variablesinthesection‘invitro’included;

M. tuberculosis strain, experimental methods, drug of interest. Minimal inhibitory

concentration,minimal inhibitory concentrationwith clavulanic acid,minimal bactericidal

concentrationandcolonyformingunits(CFU)wereextractedfromtheincludedarticles.For

thesection‘invivo’thefollowingdatawereincluded;M.tuberculosisstrain,mice,routeof

infection, drug of interest with or without clavulanic acid, dose, and treatment, colony

formingunits and survival rate,were retrieved from the includedarticles. For the clinical

section,weextracteddatafromtheincludedarticlesontypeofstudypopulation,numberof

subjects, study design, drug of interest, and dosage. Sputum smear, sputum culture,

treatment success, adverseeventsand interruptiondue toadverseeventswerenotedas

outcomes.AUC,Peakdrugconcentration(Cmax),half-life(t1/2),Distributionvolume(Vd),and

clearancewereextracted.Possibilityofpoolingdatafromincludeddatawasassessedondata

presentation.

21

2


enzymesinhibitedbybeta-lactams(8,16).Themajorityofcrosslinksinpeptidoglycanappear

tobeformedbythenon-classicalL,D-transpeptidases(LDT)inM.tuberculosis(17-23).Several

studiesrevealedthestructuralbasisandtheinactivationmechanismofLDTandtheactive

roleofcarbapenems,providingabasisforthepotentialuseofcarbapenemsininhibitingM.

tuberculosis(24-28).

Beta-lactams show time-dependent activity, carbapenems have been shown to have

bactericidalactivitywhenthefreedrugplasmaconcentrationexceedstheMICforatleast40

%ofthetimeinnon-TBbacterialspecies(29-30).


TobetterunderstandthepotentialofcarbapenemsforthetreatmentofM/XDR-TB,theaim

ofthisreviewwastoevaluatetheliteratureoncurrentlyavailableinvitro,invivoandclinical

dataoncarbapenemsinthetreatmentofM.tuberculosisanddetectionofknowledgegaps,

inordertotargetfutureresearch.

Methods

Prisma

ThissystematicreviewwasconductedinaccordancewiththePreferredReportingItemsfor

SystematicReviewsandMeta-Analyses(PRISMA)statement.

Search

In February2018, a systematic literature searchof PubMedandWebof Science,without

restrictions with respect to publication date was employed using the key words

(´Carbapenem’ OR ‘Carbapenems’ OR ‘Imipenem’ OR ‘Meropenem’ OR ‘Ertapenem’ OR

‘Doripenem’ OR ‘Faropenem’ OR ‘Biapenem’ OR ‘Panipenem’ OR ‘Tebipenem’) AND

(‘Tuberculosis’ORTBORMycobacteriumtuberculosis)asMeShTerms.Retrievedstudiesand

abstractsfrombothPubMedandWebofSciencewerepooledandduplicateswereremoved.

Titlesandabstractsofretrievedarticleswerescreened.Reviews,case-reportsorstudieson

otherspeciesthanTBorstudiesonotherdrugsthancarbapenemswereexcluded.Studies

werescreenedforeligibility.Ifeligible,thefull-textwasreadbyaresearcher(SvR).Asecond

researcher(MZ)independentlyrepeatedthearticlesearchandselection.Discrepancieswere

resolved by discussion, or a third researcher was consulted (JWA). Full text papers were

subdividedintothreesections;invitro,invivoandclinicaldata.Fulltextpapersforinvitro

data were eligible for inclusion if an M. tuberculosis strain was studied and minimum

inhibitoryconcentrationswere reported.Full textpapers for invivodatawereeligible for

inclusioniftreatmentofM.tuberculosisinfectionswithcarbapenemswerestudiedinanimal

models,andifcolonyformingunitsand/orsurvivaldatawerereported.Fulltextpapersfor

clinical data were eligible for inclusion if pharmacokinetics of carbapenems or safety or

responsetotreatmentmeasuredassurrogateendpoints(sputumconversion)orclinicalend

pointswerestudiedandreported.Referencesofallincludedarticleswerescreenedbyhand.

Thesamesystematic searchwasperformedusingclinicaltrials.gov to findongoing studies

investigatingcarbapenemsinTBpatients(Feb2018).

Dataextraction

Aresearcher(SvR)performeddataextractionfirstbyusingastructureddatacollectionform.

Asecondresearcher(MZ)verifiedthedataextractionindependently.Dataweresubdivided

intothreesections;invitro,invivoandclinicaldata.Variablesinthesection‘invitro’included;

M. tuberculosis strain, experimental methods, drug of interest. Minimal inhibitory

concentration,minimal inhibitory concentrationwith clavulanic acid,minimal bactericidal

concentrationandcolonyformingunits(CFU)wereextractedfromtheincludedarticles.For

thesection‘invivo’thefollowingdatawereincluded;M.tuberculosisstrain,mice,routeof

infection, drug of interest with or without clavulanic acid, dose, and treatment, colony

formingunits and survival rate,were retrieved from the includedarticles. For the clinical

section,weextracteddatafromtheincludedarticlesontypeofstudypopulation,numberof

subjects, study design, drug of interest, and dosage. Sputum smear, sputum culture,

treatment success, adverseeventsand interruptiondue toadverseeventswerenotedas

outcomes.AUC,Peakdrugconcentration(Cmax),half-life(t1/2),Distributionvolume(Vd),and

clearancewereextracted.Possibilityofpoolingdatafromincludeddatawasassessedondata

presentation.

22

Chapter 2

Dataquality

No validated tool for risk of bias assessment for in vitro studies, in vivo studies and

pharmacokinetic studieswasavailable.Tobeable toassess thequalityofeachstudy,we

verifiedifeachstudyreportedonkey-elementsrequiredforadequatedatainterpretation.If

studiesreportedadequatelyonthekey-elements,riskofbiaswasconsideredtobelow.If

studieshadmissingdataorifprocedureswerenotclearornotmentioned,riskofbiaswas

considered to be high. The following key-elements were identified for in vitro studies;

descriptionof laborclinical strains,minimalsamplesizeof>10strains,>3concentrations

testedperdrug,MIC/CFUdeterminedusingtheproportionmethod,evaluationendpointof

minimal inhibitory concentration (MIC 50 orMIC 90), evaluation of endpoint of minimal

bactericidal concentration (MBC99) and CFU reduction, for in vivo studies; description of

laboratory or clinical strains, type ofmice, route of administration of the drug, dose and

treatment duration, MIC/CFU determined using the proportion method, evaluation of

endpointofCFUandsurvivalrateandforclinicalstudies;forhumanstudies;studydesign,

patientpopulation(TB/MDR-TB;HIVco-infection),numberofstudyparticipants,endpoints

tested,definedassputumsmearconversion,sputumcultureconversion,treatmentsuccess,

adverse events. The following components were checked for pharmacokinetic studies:

samplesize,typeofpatients,typeofassay,numberofplasmasamplesdrawnperpatient,

samplehandling,useofvalidatedanalyticalmethodsandmethodofAUCcalculation.

Results

Basedon theselectioncriteria,250articleswereretrieved inPubMedand260 inWebof

Science.Afterremovalof146duplicates,364articlesremainedforscreening.Afterscreening

ofthetitleandabstract,46articlesremainedforfulltextevaluation.Reasonsforexclusion

included;notavailable(n=6),otherdrugs(n=2),noMIC(n=1),case-report(n=1),other(n=1).

Afterthisprocess,35relevantarticleswereincludedinthisstudy(Flowchart;Fig1).Dueto

lownumberandhighdiversityofstrains,analyticalmethodsandstudydesigns,presenceof

biochemicalinstabilityofthedrugsofinterest,theshorthalf-lifeofdrugsofinterestinmice

andthediversity inMICdetermination,wedidnothaveenoughdata toperformameta-

analysis.RiskofbiasoftheincludedstudiesisshownintableS1.Studiesonclinicaltrials.gov

areshowninS2.

Figure1.Flowchart

Invitro

Resultsoftheinvitrostudiesreportingoncarbapenemsarepresentedintable1.

Imipenem

Susceptibility testing of imipenem, using various analyticalmethods against strainH37Rv,

H37Ra,ErdmanandclinicalisolatesofM.tuberculosisshowedarangeofMIC’sbetween2–

32mg/LwithoutclavulanicacidandarangeofMIC’sbetween0.16–32withclavulanicacid.

(8,32-37).WhenImipenemwascombinedwithclavulanateitshoweda4-16-foldlowerMIC

againsttheM.tuberculosisH37Rvreferencestrain(8,33-35).

23

2


Dataquality

No validated tool for risk of bias assessment for in vitro studies, in vivo studies and

pharmacokinetic studieswasavailable.Tobeable toassess thequalityofeachstudy,we

verifiedifeachstudyreportedonkey-elementsrequiredforadequatedatainterpretation.If

studiesreportedadequatelyonthekey-elements,riskofbiaswasconsideredtobelow.If

studieshadmissingdataorifprocedureswerenotclearornotmentioned,riskofbiaswas

considered to be high. The following key-elements were identified for in vitro studies;

descriptionof laborclinical strains,minimalsamplesizeof>10strains,>3concentrations

testedperdrug,MIC/CFUdeterminedusingtheproportionmethod,evaluationendpointof

minimal inhibitory concentration (MIC 50 orMIC 90), evaluation of endpoint of minimal

bactericidal concentration (MBC99) and CFU reduction, for in vivo studies; description of

laboratory or clinical strains, type ofmice, route of administration of the drug, dose and

treatment duration, MIC/CFU determined using the proportion method, evaluation of

endpointofCFUandsurvivalrateandforclinicalstudies;forhumanstudies;studydesign,

patientpopulation(TB/MDR-TB;HIVco-infection),numberofstudyparticipants,endpoints

tested,definedassputumsmearconversion,sputumcultureconversion,treatmentsuccess,

adverse events. The following components were checked for pharmacokinetic studies:

samplesize,typeofpatients,typeofassay,numberofplasmasamplesdrawnperpatient,

samplehandling,useofvalidatedanalyticalmethodsandmethodofAUCcalculation.

Results

Basedon theselectioncriteria,250articleswereretrieved inPubMedand260 inWebof

Science.Afterremovalof146duplicates,364articlesremainedforscreening.Afterscreening

ofthetitleandabstract,46articlesremainedforfulltextevaluation.Reasonsforexclusion

included;notavailable(n=6),otherdrugs(n=2),noMIC(n=1),case-report(n=1),other(n=1).

Afterthisprocess,35relevantarticleswereincludedinthisstudy(Flowchart;Fig1).Dueto

lownumberandhighdiversityofstrains,analyticalmethodsandstudydesigns,presenceof

biochemicalinstabilityofthedrugsofinterest,theshorthalf-lifeofdrugsofinterestinmice

andthediversity inMICdetermination,wedidnothaveenoughdata toperformameta-

analysis.RiskofbiasoftheincludedstudiesisshownintableS1.Studiesonclinicaltrials.gov

areshowninS2.

Figure1.Flowchart

Invitro

Resultsoftheinvitrostudiesreportingoncarbapenemsarepresentedintable1.

Imipenem

Susceptibility testing of imipenem, using various analyticalmethods against strainH37Rv,

H37Ra,ErdmanandclinicalisolatesofM.tuberculosisshowedarangeofMIC’sbetween2–

32mg/LwithoutclavulanicacidandarangeofMIC’sbetween0.16–32withclavulanicacid.

(8,32-37).WhenImipenemwascombinedwithclavulanateitshoweda4-16-foldlowerMIC

againsttheM.tuberculosisH37Rvreferencestrain(8,33-35).

24

Chapter 2

Tabl

e1.

Resultsoftheinvitrostudiesreportingoncarbapenems.

Firs

taut

hor

(ref

).St

rain

N

Met

hod

Carb

apen

em(s

)β-

Lact

amas

ein

hibi

tor(s)

Valu

e(s)

[mg/

liter

;med

ian

(ran

ge)]fo

r:

Carbapenem

CarbapenemwithCLV(2.5mg/L)

MIC

MIC

50

MIC

90

MIC

MIC

50

MIC

90

MBC

99

ΔlogCFU

reduction

Cham

bers

et

al(3

2)

H37Ra,

H37Rv,

clinical

isolates

7

BactecTB

system

Imipenem

None

(2–4)

Cohe

net

al

(38)

H37Rv,

Clinical

isolates

91

Microplate

alamarBlue

assay

Meropenem

Clavulanate

22

(2–

32)

5.4(0.5–32)

Cava

naug

het

al

(39)

Clinical

isolates

15

3

Resazurin

microdilution

assay

Meropenem

Clavulanate

(<0.12–>16)

1

8

Des

hpan

de

eta

l(47

) H37Ra,THP1

monocytes

1

Resazurin

microdilution

assay,CFU

counts

Faropenem

None

1

2.71log

Dha

reta

l(4

9)

H37Rv,

Erdman

2

96W

ellflat-

bottom

polystyrene

microtiter

plate

Faropenem,

meropenem,

imipenem

Clavulanate

1.3;

2.5;

2.5

1.3;

0.3;

0.5

Engl

and

eta

l(4

0)

H37Rv,

macrophages

1

CFUcounts

Meropenem

Clavulanate

2log

Fors

man

eta

l(4

1)

H37Rv,

Clinical

isolates

69

Broth

microdilution

Meropenem

Clavulanate

(0.125–32)

1

2log

Gon

zalo

eta

l(4

2)

H37Rv,

Clinical

isolates

28

960M

GIT

system

Meropenem

None

Resistan

t at

5

mg/L

(1.28–2.56)

Gur

umur

thy

eta

l(48

) H37Rv

1

96W

ellsplate

Faropenem

Clavulanate,

avibactama

(5–10)

20

0log

Hor

itae

tal

(43)

H37Rv,

Clinical

isolates

42

Broth

microdilution

Meropenem

Biapenem

Tebipenem

Clavulanate

(1–32),

(1–32),

(0.25–8)

16,

16,

4

32,

32,

8

(0.063–8),

(0.25–8),

(0.063–8)

2,

2,

1

4,

4,

1

Hug

onne

tet

al(8

)Erdman,

H37Rv,

Clinical

isolates

15

Broth

microdilution

Imipenem

Meropenem

Clavulanate

0.16,

(0.23–1.25)

Kaus

hik

eta

l(3

3)

H37Rv,

Clinical

isolates

1

Broth

microdilution

Imipenem,

meropenem,

ertapenem,

doripenem,

biapenem,

faropenem,

tebipenem,

panipenem

None

(40–80),

(5–10),

(10–20),

(2.5–5),

(2.5–5),

(2.5–5),

(1.25–2.5)

>80

(20–40),

(2.5–5),

(5–10),

(1.25–2.5),

(0.6–1.2),

(2.5–5),

(0.31–0.62),

ND

ND

80

ND

20

20

20

10

ND

Kaus

hik

eta

l(5

1)

H37Rv,strain

115R,

strain

124R

3

Broth

microdilution

Biapenem

Clavulanate

(2–16)

Sala

eta

l(44

)18bcells

1

Serial

dilutions,CFU

counts

Meropenem

Clavulanate

Sola

pure

et

al(3

4)

H37Rv,

18b

cells

1

Resazurin

microdilution

assay,CFU

counts

Imipenem,

meropenem,

faropenem

Clavulanate

4,8,4

0.5,1,2

4

2

4

2log

Sriv

asta

vae

tal

(45)

H37Ra

1

Resazurin

microdilution

assay

Ertapenem

Clavulanate

0.6

Veziris

eta

l(3

5)

H37Rv

1

Broth

microdilution

Imipenem,

meropenem,

ertapenem

16,8,

16

1,1,

4

2.38log10

a.MICvaluesforcarbapenemswithavibactamarenotshowninthistable.

N:numberofstrains,MIC:Minimalinhibitoryconcentration(mg/L),M

IC50:Minimalinhibitoryconcentrationrequiredtoinhibitgrowthof50%

oftheorganisms,MIC90:Minimalinhibitoryconcentrationrequiredtoinhibitgrowthof90%oftheorganisms,CLV:clavulanate(mg/L),M

BC99:

minimalbactericidalconcentrationthatkills99%ofreplicationculture(mg/L),CFU:colonyform

ingunits(Log/(CFU/ml))

25

2


Tabl

e1.

Resultsoftheinvitrostudiesreportingoncarbapenems.

Firs

taut

hor

(ref

).St

rain

N

Met

hod

Carb

apen

em(s

)β-

Lact

amas

ein

hibi

tor(s)

Valu

e(s)

[mg/

liter

;med

ian

(ran

ge)]fo

r:

Carbapenem

CarbapenemwithCLV(2.5mg/L)

MIC

MIC

50

MIC

90

MIC

MIC

50

MIC

90

MBC

99

ΔlogCFU

reduction

Cham

bers

et

al(3

2)

H37Ra,

H37Rv,

clinical

isolates

7

BactecTB

system

Imipenem

None

(2–4)

Cohe

net

al

(38)

H37Rv,

Clinical

isolates

91

Microplate

alamarBlue

assay

Meropenem

Clavulanate

22

(2–

32)

5.4(0.5–32)

Cava

naug

het

al

(39)

Clinical

isolates

15

3

Resazurin

microdilution

assay

Meropenem

Clavulanate

(<0.12–>16)

1

8

Des

hpan

de

eta

l(47

)H37Ra,THP1

monocytes

1

Resazurin

microdilution

assay,CFU

counts

Faropenem

None

1

2.71log

Dha

reta

l(4

9)

H37Rv,

Erdman

2

96W

ellflat-

bottom

polystyrene

microtiter

plate

Faropenem,

meropenem,

imipenem

Clavulanate

1.3;

2.5;

2.5

1.3;

0.3;

0.5

Engl

and

eta

l(4

0)

H37Rv,

macrophages

1

CFUcounts

Meropenem

Clavulanate

2log

Fors

man

eta

l(4

1)

H37Rv,

Clinical

isolates

69

Broth

microdilution

Meropenem

Clavulanate

(0.125–32)

1

2log

Gon

zalo

eta

l(4

2)

H37Rv,

Clinical

isolates

28

960M

GIT

system

Meropenem

None

Resistan

t at

5

mg/L

(1.28–2.56)

Gur

umur

thy

eta

l(48

)H37Rv

1

96W

ellsplate

Faropenem

Clavulanate,

avibactama

(5–10)

20

0log

Hor

itae

tal

(43)

H37Rv,

Clinical

isolates

42

Broth

microdilution

Meropenem

Biapenem

Tebipenem

Clavulanate

(1–32),

(1–32),

(0.25–8)

16,

16,

4

32,

32,

8

(0.063–8),

(0.25–8),

(0.063–8)

2,

2,

1

4,

4,

1

Hug

onne

tet

al(8

) Erdman,

H37Rv,

Clinical

isolates

15

Broth

microdilution

Imipenem

Meropenem

Clavulanate

0.16,

(0.23–1.25)

Kaus

hik

eta

l(3

3)

H37Rv,

Clinical

isolates

1

Broth

microdilution

Imipenem,

meropenem,

ertapenem,

doripenem,

biapenem,

faropenem,

tebipenem,

panipenem

None

(40–80),

(5–10),

(10–20),

(2.5–5),

(2.5–5),

(2.5–5),

(1.25–2.5)

>80

(20–40),

(2.5–5),

(5–10),

(1.25–2.5),

(0.6–1.2),

(2.5–5),

(0.31–0.62),

ND

ND

80

ND

20

20

20

10

ND

Kaus

hik

eta

l(5

1)

H37Rv,strain

115R,

strain

124R

3

Broth

microdilution

Biapenem

Clavulanate

(2–16)

Sala

eta

l(44

)18bcells

1

Serial

dilutions,CFU

counts

Meropenem

Clavulanate

Sola

pure

et

al(3

4)

H37Rv,

18b

cells

1

Resazurin

microdilution

assay,CFU

counts

Imipenem,

meropenem,

faropenem

Clavulanate

4,8,4

0.5,1,2

4

2

4

2log

Sriv

asta

vae

tal

(45)

H37Ra

1

Resazurin

microdilution

assay

Ertapenem

Clavulanate

0.6

Veziris

eta

l(3

5)

H37Rv

1

Broth

microdilution

Imipenem,

meropenem,

ertapenem

16,8,

16

1,1,

4

2.38log10

a.MICvaluesforcarbapenemswithavibactamarenotshowninthistable.

N:numberofstrains,MIC:Minimalinhibitoryconcentration(mg/L),M

IC50:Minimalinhibitoryconcentrationrequiredtoinhibitgrowthof50%

oftheorganisms,MIC90:Minimalinhibitoryconcentrationrequiredtoinhibitgrowthof90%oftheorganisms,CLV:clavulanate(mg/L),M

BC99:

minimalbactericidalconcentrationthatkills99%ofreplicationculture(mg/L),CFU:colonyform

ingunits(Log/(CFU/ml))

26

Chapter 2

Meropenem

Multiplestudiesreportedthatmeropeneminpresenceofclavulanateisactiveinvitroagainst

clinicalandlabstrains,H37RvandH37Ra,ofM.tuberculosis,showingMIC’s≤1mg/L.Invitro

studiesreportingsusceptibilityofmeropenemofM.tuberculosisreferencestrainandclinical

isolatesshowedMICvaluesbetween1-32mg/L(8,33-44).Meropenemincombinationwith

clavulanicacidwasshowntohaveaMICbetween0.063–32mg/L(33-35,38,43)Meropenem

in combinationwith clavulanate killed the non-replicating ss18b strain ofM. tuberculosis

moderatelyandwasshowntohaveaMICof0.125–2.56mg/LagainstM.tuberculosisH37Rv

strains(8,34-35,40).Adecreaseof2log10CFUsoversixdayswasreportedinM.tuberculosis-

infectedmurinemacrophages(40).

Ertapenem

InclinicalstrainsofM.tuberculosistheMICofertapenem,assingleagent,was16mg/Land

when combinedwith clavulanate 4mg/L (33,35). Another study showed ertapenemwas

unstabledegradingfasterthanthedoublingtimeofM.tuberculosisinthegrowthmediaused,

suggestingpreviouspublishedMICsofertapenemarelikelytobefalselyhigh(45).Inahollow

fibermodelwith supplementationofertapenem inabrothmicrodilution test,ertapenem

showedaMICof0.6ml/L(46).A28-dayexposure-responsehollowfibermodelofTBstudy

tested8differentdosesofertapenem in combinationwith clavulanateand identified the

ertapenemexposureassociatedwithoptimalsterilizingeffectforclinicaluse.MonteCarlo

simulationwith10,000MDR-TBpatientsidentifiedasusceptibilitybreakpointMICof2mg/L

foranintravenousdoseof2gonceadaythatachievedorexceeded40%T>MIC(46)

Faropenem

Faropenemshoweda4-foldreductionwhencombinedwithclavulanicacid(33,34),resulting

inaMICrangebetween1-5mg/L(33-34,47-49)Inahollowfibermodel,theoptimaltarget

exposurewasidentifiedtobeassociatedwithoptimalefficacyinchildrenwithdisseminated

TB using Monte Carlo simulations; the predicted optimal oral dose was 30 mg/kg of

faropenem-medoxomil3-4timesdaily.Theexposuretargetforfaropenem-medoxomilwas

60%Tfree>MIC(50).

Othercarbapenems

Othercarbapenems,suchasdoripenem,biapenemandtebipenemshowedatleasta2-fold

reductioninMICwhencombinedwithclavulanicacid(33,37,43,51).

Invivo

Resultsoftheinvivostudiesreportingoncarbapenemsarepresentedintable2.

Imipenem

Thebacterialburdeninimipenem-treatedCD-1femalemice(twicedaily(BID)100mg/kg),

infectedwithM.tuberculosisstrainH37Rv,wasreducedby1.8log10insplenictissueand1.2

log10inlungtissueafter28days,showingananti-mycobacterialeffectaswellasimproved

survivalinthismousemodel(52).InanotherstudySwissmice,infectedwithM.tuberculosis

strainH37Rv,weretreatedwithasubcutaneousadministrationof100-mg/kgimipenemin

combinationwithclavulanateonceadaytosimulateahumanequivalentdose.TheCFUcount

after28daysoftreatmentincreasedcomparedtotheCFUcountatstartoftreatment.There

onlywasasignificantdifferenceintheimipenem-clavulanatetreatedmice(35).

Meropenem

It has been reported that 300mg/kgBIDmeropenemalone, and in combinationwith 50

mg/kgclavulanatebothresultedinasignificant,thoughmodestreduction, inCFUsin lung

andspleentissuesinC57BL/6mice(40).Vezirisetal.reportedaCFUincreasecomparedto

startofthetreatmentofmeropenemwhengivenasmono-therapyorincombinationwith

clavulanateinadoseof100mg/kg,onCFUs,spleenweights,orlunglesionsinSwissmice

(35).Meropeneminadoseof300mg/kgincombinationwithclavulanate,75mg/kgthrice-

dailygiventoBALB/cmiceshowedmarginalreductioninCFUcountsintheacutemodeland

noreductioninthechronicmodel(34).Meropenem,givensubcutaneously300mg/kgthree

timesaday,showedaCFUcountreductionof1.7loginthelungsofTF3157DHP-1deficient

mice(53).

27

2


Meropenem

Multiplestudiesreportedthatmeropeneminpresenceofclavulanateisactiveinvitroagainst

clinicalandlabstrains,H37RvandH37Ra,ofM.tuberculosis,showingMIC’s≤1mg/L.Invitro

studiesreportingsusceptibilityofmeropenemofM.tuberculosisreferencestrainandclinical

isolatesshowedMICvaluesbetween1-32mg/L(8,33-44).Meropenemincombinationwith

clavulanicacidwasshowntohaveaMICbetween0.063–32mg/L(33-35,38,43)Meropenem

in combinationwith clavulanate killed the non-replicating ss18b strain ofM. tuberculosis

moderatelyandwasshowntohaveaMICof0.125–2.56mg/LagainstM.tuberculosisH37Rv

strains(8,34-35,40).Adecreaseof2log10CFUsoversixdayswasreportedinM.tuberculosis-

infectedmurinemacrophages(40).

Ertapenem

InclinicalstrainsofM.tuberculosistheMICofertapenem,assingleagent,was16mg/Land

when combinedwith clavulanate 4mg/L (33,35). Another study showed ertapenemwas

unstabledegradingfasterthanthedoublingtimeofM.tuberculosisinthegrowthmediaused,

suggestingpreviouspublishedMICsofertapenemarelikelytobefalselyhigh(45).Inahollow

fibermodelwith supplementationofertapenem inabrothmicrodilution test,ertapenem

showedaMICof0.6ml/L(46).A28-dayexposure-responsehollowfibermodelofTBstudy

tested8differentdosesofertapenem in combinationwith clavulanateand identified the

ertapenemexposureassociatedwithoptimalsterilizingeffectforclinicaluse.MonteCarlo

simulationwith10,000MDR-TBpatientsidentifiedasusceptibilitybreakpointMICof2mg/L

foranintravenousdoseof2gonceadaythatachievedorexceeded40%T>MIC(46)

Faropenem

Faropenemshoweda4-foldreductionwhencombinedwithclavulanicacid(33,34),resulting

inaMICrangebetween1-5mg/L(33-34,47-49)Inahollowfibermodel,theoptimaltarget

exposurewasidentifiedtobeassociatedwithoptimalefficacyinchildrenwithdisseminated

TB using Monte Carlo simulations; the predicted optimal oral dose was 30 mg/kg of

faropenem-medoxomil3-4timesdaily.Theexposuretargetforfaropenem-medoxomilwas

60%Tfree>MIC(50).

Othercarbapenems

Othercarbapenems,suchasdoripenem,biapenemandtebipenemshowedatleasta2-fold

reductioninMICwhencombinedwithclavulanicacid(33,37,43,51).

Invivo

Resultsoftheinvivostudiesreportingoncarbapenemsarepresentedintable2.

Imipenem

Thebacterialburdeninimipenem-treatedCD-1femalemice(twicedaily(BID)100mg/kg),

infectedwithM.tuberculosisstrainH37Rv,wasreducedby1.8log10insplenictissueand1.2

log10inlungtissueafter28days,showingananti-mycobacterialeffectaswellasimproved

survivalinthismousemodel(52).InanotherstudySwissmice,infectedwithM.tuberculosis

strainH37Rv,weretreatedwithasubcutaneousadministrationof100-mg/kgimipenemin

combinationwithclavulanateonceadaytosimulateahumanequivalentdose.TheCFUcount

after28daysoftreatmentincreasedcomparedtotheCFUcountatstartoftreatment.There

onlywasasignificantdifferenceintheimipenem-clavulanatetreatedmice(35).

Meropenem

It has been reported that 300mg/kgBIDmeropenemalone, and in combinationwith 50

mg/kgclavulanatebothresultedinasignificant,thoughmodestreduction, inCFUsin lung

andspleentissuesinC57BL/6mice(40).Vezirisetal.reportedaCFUincreasecomparedto

startofthetreatmentofmeropenemwhengivenasmono-therapyorincombinationwith

clavulanateinadoseof100mg/kg,onCFUs,spleenweights,orlunglesionsinSwissmice

(35).Meropeneminadoseof300mg/kgincombinationwithclavulanate,75mg/kgthrice-

dailygiventoBALB/cmiceshowedmarginalreductioninCFUcountsintheacutemodeland

noreductioninthechronicmodel(34).Meropenem,givensubcutaneously300mg/kgthree

timesaday,showedaCFUcountreductionof1.7loginthelungsofTF3157DHP-1deficient

mice(53).

28

Chapter 2

Table2.Resultsofthe

invivostud

iesr

eportin

gon

carbap

enem

s.

Firsta

utho

r(ref).

Stra

in

Miceof

miceof

ot

hera

nimal

mod

el

Infection

Drug

Do

se

Infection

mod

el

Trea

tmen

tEn

d-po

int

Organ

sCF

Ure

duction

Surviva

lrate

CFU/

clv

Cham

bers

etal(52

) H3

7Rv

CD-1Fem

ale

mice

su

bcutan

eous

ly

Imipen

em

Bid10

0mg/kg

ND

28

day

sCF

Uco

unt,

Survivalra

te

Spleen

,lung

s 1.8log

65%

ND

Dhareta

l(49)

H37R

vad

ultC

57BL

/6J

mice

intratrach

eal

Farope

nem

500mg/kg

ac

uteTB

8da

ys

CFUco

unt

Lung

sredu

ctionof

CFU:

10^

5-

10^6

ND

ND

Englan

det

al(4

0)

H37R

vC5

7BL/6Mice

su

bcutan

eous

ly

Merop

enem

bid30

0mg/kg

Ch

ronic

stag

e 2wee

ks

CFUco

unt

Spleen

,lung

s 1log

ND

1log

NewZea

land

whitera

bbits

intrav

enou

sbo

lus

Merop

enem

75

mg/kg

12

5mg/kg

ND

ND

PK

data

ND

ND

ND

ND

Kaus

hiket

al(5

1)

H37R

vBA

LB/cm

ice

Aeroso

lBiap

enem

20

0mg/kg

BID

300mg/kg

BID

Latepha

se

acuteTB

rifam

picin

resis

tantTB

8wee

ks

4wee

ks

CFUco

unt

Lung

s1log

ND

ND

ND

Rulla

seta

l(53)

H37R

vTF

3157

DHP

-IKO

su

bcutan

eous

ly

Merop

enem

Fa

rope

nem

TID30

0mg/kg

TID50

0mg/kg

AcuteTB

mod

el

21day

sCF

Uco

unt

Lung

s1.7log

2log

ND

ND

ND

ND

Solapu

reet

al(3

4)

H37R

vBA

LB/cm

ice

Aeroso

lMerop

enem

TID30

0mg/kg

Ac

utean

dch

ronic

mod

el

4wee

ks

CFUco

unt

lung

sno

redu

ction

ND

no

redu

ction

Vezir

isetal

(35)

H37R

vFe

maleSw

iss

mice

Intrav

enou

sly

Imipen

em

Merop

enem

Ertape

nem

100mg/kg

10

0mg/kg

10

0mg/kg

prev

entiv

emod

el

28day

sCF

Uco

unt,

Survivalra

te

Spleen

,lung

s >1

.2lo

g*

>1.8lo

g*

>1.7lo

g*

1de

ad

3de

ad

3de

ad

>0.9lo

g*

>1.4lo

g*

>1.6lo

g*

*Th

erewasaCFU

increa

sein

thegrou

psw

ithand

with

outc

lavu

lana

teco

mpa

redtoth

estarto

fthe

trea

tmen

t.

MIC:M

inim

alin

hibitorycon

centratio

n(m

g/L),C

LV:c

lavu

lana

te(m

g/L),MBC

:minim

albacteric

idalcon

centratio

n(m

g/L),C

FU:c

olon

yform

ing

units

,od:onc

eada

y,bid:twice

aday

,tid:three

timesaday

,qid:fou

rtim

esaday

,ND:notdescribed

.

Ertapenem

InamurineTBmodel infectedwithH37Rv,adoseof100mg/kgoncedailyertapenemas

monotherapyorincombinationwithclavulanatehadneitherbactericidalnorbacteriostatic

activityinlungsandspleensofTB-infectedmice.Spleenweightandlunglesionsremained

similarcomparedtotheuntreatedgroupofmice.TherewasanincreaseinCFUscompared

totheCFUsatthestartofthetreatment(35).

OtherCarbapenems

Anoraldoseof500mg/kgfaropenem-medoxomil,giventhreetimesdaily,gaveareduction

of2logCFUcountinthelungsofTF3157DHP-1deficientmice(53).Neitherinvivonorclinical

studiesforothercarbapenemsaspartofamulti-drugregimenagainstTBwereretrieved.

Clinicalstudies

Resultsoftheclinicalstudiesreportingoncarbapenemsarepresentedintable3.

Imipenem

Ten patients were treated with imipenem in combination with two or more other

antimicrobialagents.Itwasreportedthatitwaslikelythat1gofimipenem(BID)contributed

to sputum culture conversion in these patients (52). A prospective study evaluated 1000

mg/day imipenem/clavulanateatadoseofoncedaily in12patients,11of thesepatients

receivedlinezolid-containingregimens.Allpatientsshowedsputumandcultureconversion

within180days.Noadverseeventswerereportedforimipenem/clavulanate(54).Inalarge

observational study, the clinical outcomes of 84 patients, treated with 500 mg

imipenem/clavulanatefourtimesaday,werecomparedwithresultsfrom168controls.The

studyshowedthatimipenem-containingregimensachievedcomparableresultscomparedto

theimipenemsparingregimens,whilesuccessratesweresimilartomajorinternationalMDR-

TBcohorts(55).

Meropenem

A regimen includingmeropenem-clavulanate given to 18 patientswith severe pulmonary

XDR-TB led to sputum culture conversion in 15 patients, of which 10 has successfully

completedandfivepatientswereconsideredcuredaccordingtoWHOguidelines.Long-

29

2


Table2.Resultsofthe

invivostud

iesr

eportin

gon

carbap

enem

s.

Firsta

utho

r(ref).

Stra

in

Miceof

miceof

ot

hera

nimal

mod

el

Infection

Drug

Do

se

Infection

mod

el

Trea

tmen

tEn

d-po

int

Organ

sCF

Ure

duction

Surviva

lrate

CFU/

clv

Cham

bers

etal(52

)H3

7Rv

CD-1Fem

ale

mice

su

bcutan

eous

ly

Imipen

em

Bid10

0mg/kg

ND

28

day

sCF

Uco

unt,

Survivalra

te

Spleen

,lung

s1.8log

65%

ND

Dhareta

l(49)

H37R

vad

ultC

57BL

/6J

mice

intratrach

eal

Farope

nem

500mg/kg

ac

uteTB

8da

ys

CFUco

unt

Lung

sredu

ctionof

CFU:

10^

5-

10^6

ND

ND

Englan

det

al(4

0)

H37R

vC5

7BL/6Mice

su

bcutan

eous

ly

Merop

enem

bid30

0mg/kg

Ch

ronic

stag

e2wee

ks

CFUco

unt

Spleen

,lung

s1log

ND

1log

NewZea

land

whitera

bbits

intrav

enou

sbo

lus

Merop

enem

75

mg/kg

12

5mg/kg

ND

ND

PK

data

ND

ND

ND

ND

Kaus

hiket

al(5

1)

H37R

vBA

LB/cm

ice

Aeroso

lBiap

enem

20

0mg/kg

BID

300mg/kg

BID

Latepha

se

acuteTB

rifam

picin

resis

tantTB

8wee

ks

4wee

ks

CFUco

unt

Lung

s1log

ND

ND

ND

Rulla

seta

l(53)

H37R

vTF

3157

DHP

-IKO

su

bcutan

eous

ly

Merop

enem

Fa

rope

nem

TID30

0mg/kg

TID50

0mg/kg

AcuteTB

mod

el

21day

sCF

Uco

unt

Lung

s1.7log

2log

ND

ND

ND

ND

Solapu

reet

al(3

4)

H37R

vBA

LB/cm

ice

Aeroso

lMerop

enem

TID30

0mg/kg

Ac

utean

dch

ronic

mod

el

4wee

ks

CFUco

unt

lung

sno

redu

ction

ND

no

redu

ction

Vezir

isetal

(35)

H37R

vFe

maleSw

iss

mice

Intrav

enou

sly

Imipen

em

Merop

enem

Ertape

nem

100mg/kg

10

0mg/kg

10

0mg/kg

prev

entiv

emod

el

28day

sCF

Uco

unt,

Survivalra

te

Spleen

,lung

s>1

.2lo

g*

>1.8lo

g*

>1.7lo

g*

1de

ad

3de

ad

3de

ad

>0.9lo

g*

>1.4lo

g*

>1.6lo

g*

*Th

erewasaCFU

increa

sein

thegrou

psw

ithand

with

outc

lavu

lana

teco

mpa

redtoth

estarto

fthe

trea

tmen

t.

MIC:M

inim

alin

hibitorycon

centratio

n(m

g/L),C

LV:c

lavu

lana

te(m

g/L),MBC

:minim

albacteric

idalcon

centratio

n(m

g/L),C

FU:c

olon

yform

ing

units

,od:onc

eada

y,bid:twice

aday

,tid:three

timesaday

,qid:fou

rtim

esaday

,ND:notdescribed

.

Ertapenem

InamurineTBmodel infectedwithH37Rv,adoseof100mg/kgoncedailyertapenemas

monotherapyorincombinationwithclavulanatehadneitherbactericidalnorbacteriostatic

activityinlungsandspleensofTB-infectedmice.Spleenweightandlunglesionsremained

similarcomparedtotheuntreatedgroupofmice.TherewasanincreaseinCFUscompared

totheCFUsatthestartofthetreatment(35).

OtherCarbapenems

Anoraldoseof500mg/kgfaropenem-medoxomil,giventhreetimesdaily,gaveareduction

of2logCFUcountinthelungsofTF3157DHP-1deficientmice(53).Neitherinvivonorclinical

studiesforothercarbapenemsaspartofamulti-drugregimenagainstTBwereretrieved.

Clinicalstudies

Resultsoftheclinicalstudiesreportingoncarbapenemsarepresentedintable3.

Imipenem

Ten patients were treated with imipenem in combination with two or more other

antimicrobialagents.Itwasreportedthatitwaslikelythat1gofimipenem(BID)contributed

to sputum culture conversion in these patients (52). A prospective study evaluated 1000

mg/day imipenem/clavulanateatadoseofoncedaily in12patients,11of thesepatients

receivedlinezolid-containingregimens.Allpatientsshowedsputumandcultureconversion

within180days.Noadverseeventswerereportedforimipenem/clavulanate(54).Inalarge

observational study, the clinical outcomes of 84 patients, treated with 500 mg

imipenem/clavulanatefourtimesaday,werecomparedwithresultsfrom168controls.The

studyshowedthatimipenem-containingregimensachievedcomparableresultscomparedto

theimipenemsparingregimens,whilesuccessratesweresimilartomajorinternationalMDR-

TBcohorts(55).

Meropenem

A regimen includingmeropenem-clavulanate given to 18 patientswith severe pulmonary

XDR-TB led to sputum culture conversion in 15 patients, of which 10 has successfully

completedandfivepatientswereconsideredcuredaccordingtoWHOguidelines.Long-

30

Chapter 2

Tabl

e3.

Resultsoftheinhum

anstudiesreportin

gon

carbapenems.

Firs

taut

hor(

ref)

Year

of

publ

icat

ion

Coun

try

Stud

ypo

pula

tion

Stud

yde

sign

Drug

Do

sage

Pa

tient

sPa

edi

atric

Sp

utum

Sm

ear

Sput

um

cultu

re

Trea

tmen

tsuc

ces

Adve

rse

even

ts

inte

rrup

tion

due

AE

Arbexetal(54)

2016

Brazil

2013-2

015

Observatio

nal,

retrospective

Imipenem

1goc

12

No

12/12

12/12

7/12

0/12

0/12

Cham

bersetal

(52)

2005

USA

ND

Prospective

Imipenem

1gbid

10

No

ND

8/10

7/10

ND

ND

DeLorenzoetal

(58)

2014

Italy,

The

Netherla

nds

2001-2012

Observatio

nal

case-con

trol

Merop

ene

m

1gtid

37

No

28/32

31/37

ND

5/37

2/5

Payenetal(57)

2018

Belgium

2009-2016

Retrospective

casese

ries

Merop

ene

m

2g

tid

(thenbid)

18

No

16/18

16/18

15/18

0/18

0/18

Palmero

etal

(59)

2015

Argentina

2012-2013

Retrospective

Merop

ene

m

2g

tid

(then

1g

tid)

10

No

ND

8/10

3/6

0/10

ND

Van

Rijnetal

(10)

2016

The

Netherla

nds

2010-2013

Retrospective

Ertapenem

1goc

18

yes

ND

15/18

15/18

2/18

3/18

Tiberietal(61)

2016

Italy,

2008-2015

Retrospective,

coho

rt

Ertapenem

1goc

5No

3/5

3/5

4/5

0/5

0/5

Tiberietal(60)

2016

Multicentred

in3cou

ntrie

s 2003-2015

Observatio

nal,

retrospective,

coho

rt

Merop

ene

m

1gtid

(2g

tid)

96

No

55/58

55/58

55/96

6/93

8/94

Tiberi

etal(11,

55)

2016

Multicentred

in8cou

ntrie

s 2003-2015

Observatio

nal

retrospective

case-con

trol

Imipenem

500mgqid

84

No

51/64

51/64

34/57

3/56

4/55

od:onceaday,bid:twiceaday,tid:th

reetim

esadat,qid:fou

rtimesaday.PK:pharm

acokinetic,N

D:notdescribed

termsafetywasnotaprobleminthisstudyasnoadverseeventswerereported(56-57).The

first study, that evaluated efficacy, safety and tolerability,was a case-control study in 37

patients, who received meropenem/clavulanate as part of a linezolid based multi-drug

regimen.Thisisthefirststudythatshowedanaddedvalueofmeropenem/clavulanateina

multi-drug regimen. The meropenem/clavulanate containing regimen showed a sputum

microscopy conversion of 87.5 % and a sputum culture conversion of 83.8%, while the

meropenem/clavulanatesparingregimenshowedasputummicroscopyconversionof56.3%

andasputumcultureconversionof62.5%after90daysoftreatment(58).Inanotherstudy,

10XDRandpre-XDRfemalepatientswere treatedwithmulti-drugregimensandreceived

meropenem/clavulanatefor6monthsormore.Eightpatientsachievedsputumconversion

after 6 months, while two patients died. (59). Pharmacokinetic parameters of 1 g

meropenem/clavulanategiven intravenouslyover5minutes showedaserumpeakof112

mg/mlandaconcentrationof28.6mg*h/L (39). Inanobservational retrospectivecohort-

study,efficacyandsafetywereevaluatedin96patientstreatedwithmeropenem/clavulanate

containingregimensandcomparedwith168controls.Sputumsmearandcultureconversion

rates were found to be similar (60) In an observational study comparing therapeutic

contribution, such as sputum smear and culture conversion rates and success rates, of

imipenem/clavulanate andmeropenem/ clavulanate in a background regimen, suggested

thatmeropenem/clavulanatecancontributetotheefficacyofaregimenintreatingM/XDR-

TBpatients(11).

Ertapenem

Thefirstreportonclinicalexperiencewithertapenempresenteddatafromfivepatientswho

were treated with an intravenous injection of 1 g ertapenem once daily in a multi-drug

regimen.Threeofthesepatientsshowedsputumsmearandcultureconversion;fouroffive

patientshadasuccessfultreatmentoutcome.Twopatientsinterruptedtreatmentduetoan

adverseevent.Theseadverseeventswereconsideredunrelatedtothestudydrug(61).Inan

observationalstudy18patientsweretreatedwith1gertapenemoncedaily;fifteenofthese

patientshadasuccessfultreatmentoutcomewerecured.Threepatientswere lostdueto

follow-up.Threepatientsstoppedertapenemtreatmentduetoertapenemunrelatedadverse

events.Pharmacokineticparameterswereevaluated in12patients, showingameanpeak

plasmaof127.5(range73.9-277.9)mg/LandanAUCof544.9(range390-1130)mg*h/L.

31

2


Tabl

e3.

Resultsoftheinhum

anstudiesreportin

gon

carbapenems.

Firs

taut

hor(

ref)

Year

of

publ

icat

ion

Coun

try

Stud

ypo

pula

tion

Stud

yde

sign

Drug

Do

sage

Pa

tient

sPa

edi

atric

Sp

utum

Sm

ear

Sput

um

cultu

re

Trea

tmen

tsuc

ces

Adve

rse

even

ts

inte

rrup

tion

due

AE

Arbexetal(54)

2016

Brazil

2013-2

015

Observatio

nal,

retrospective

Imipenem

1goc

12

No

12/12

12/12

7/12

0/12

0/12

Cham

bersetal

(52)

2005

USA

ND

Prospective

Imipenem

1gbid

10

No

ND

8/10

7/10

ND

ND

DeLorenzoetal

(58)

2014

Italy,

The

Netherla

nds

2001-2012

Observatio

nal

case-con

trol

Merop

ene

m

1gtid

37

No

28/32

31/37

ND

5/37

2/5

Payenetal(57)

2018

Belgium

2009-2016

Retrospective

casese

ries

Merop

ene

m

2g

tid

(thenbid)

18

No

16/18

16/18

15/18

0/18

0/18

Palmero

etal

(59)

2015

Argentina

2012-2013

Retrospective

Merop

ene

m

2g

tid

(then

1g

tid)

10

No

ND

8/10

3/6

0/10

ND

Van

Rijnetal

(10)

2016

The

Netherla

nds

2010-2013

Retrospective

Ertapenem

1goc

18

yes

ND

15/18

15/18

2/18

3/18

Tiberietal(61)

2016

Italy,

2008-2015

Retrospective,

coho

rt

Ertapenem

1goc

5No

3/5

3/5

4/5

0/5

0/5

Tiberietal(60)

2016

Multicentred

in3cou

ntrie

s2003-2015

Observatio

nal,

retrospective,

coho

rt

Merop

ene

m

1gtid

(2g

tid)

96

No

55/58

55/58

55/96

6/93

8/94

Tiberi

etal(11,

55)

2016

Multicentred

in8cou

ntrie

s2003-2015

Observatio

nal

retrospective

case-con

trol

Imipenem

500mgqid

84

No

51/64

51/64

34/57

3/56

4/55

od:onceaday,bid:twiceaday,tid:th

reetim

esadat,qid:fou

rtimesaday.PK:pharm

acokinetic,N

D:notdescribed

termsafetywasnotaprobleminthisstudyasnoadverseeventswerereported(56-57).The

first study, that evaluated efficacy, safety and tolerability,was a case-control study in 37

patients, who received meropenem/clavulanate as part of a linezolid based multi-drug

regimen.Thisisthefirststudythatshowedanaddedvalueofmeropenem/clavulanateina

multi-drug regimen. The meropenem/clavulanate containing regimen showed a sputum

microscopy conversion of 87.5 % and a sputum culture conversion of 83.8%, while the

meropenem/clavulanatesparingregimenshowedasputummicroscopyconversionof56.3%

andasputumcultureconversionof62.5%after90daysoftreatment(58).Inanotherstudy,

10XDRandpre-XDRfemalepatientswere treatedwithmulti-drugregimensandreceived

meropenem/clavulanatefor6monthsormore.Eightpatientsachievedsputumconversion

after 6 months, while two patients died. (59). Pharmacokinetic parameters of 1 g

meropenem/clavulanategiven intravenouslyover5minutes showedaserumpeakof112

mg/mlandaconcentrationof28.6mg*h/L (39). Inanobservational retrospectivecohort-

study,efficacyandsafetywereevaluatedin96patientstreatedwithmeropenem/clavulanate

containingregimensandcomparedwith168controls.Sputumsmearandcultureconversion

rates were found to be similar (60) In an observational study comparing therapeutic

contribution, such as sputum smear and culture conversion rates and success rates, of

imipenem/clavulanate andmeropenem/ clavulanate in a background regimen, suggested

thatmeropenem/clavulanatecancontributetotheefficacyofaregimenintreatingM/XDR-

TBpatients(11).

Ertapenem

Thefirstreportonclinicalexperiencewithertapenempresenteddatafromfivepatientswho

were treated with an intravenous injection of 1 g ertapenem once daily in a multi-drug

regimen.Threeofthesepatientsshowedsputumsmearandcultureconversion;fouroffive

patientshadasuccessfultreatmentoutcome.Twopatientsinterruptedtreatmentduetoan

adverseevent.Theseadverseeventswereconsideredunrelatedtothestudydrug(61).Inan

observationalstudy18patientsweretreatedwith1gertapenemoncedaily;fifteenofthese

patientshadasuccessfultreatmentoutcomewerecured.Threepatientswere lostdueto

follow-up.Threepatientsstoppedertapenemtreatmentduetoertapenemunrelatedadverse

events.Pharmacokineticparameterswereevaluated in12patients, showingameanpeak

plasmaof127.5(range73.9-277.9)mg/LandanAUCof544.9(range390-1130)mg*h/L.

32

Chapter 2

BasedonaMICof0.25mg/ml11/12patientsreachedthetargetvalueof40%Tfree>MICwas

exceeded(10).Thepharmacokineticmodelcomposedinthisstudywasshowntoadequately

predictertapenemexposureinMDR-TBpatients.TheMonteCarlosimulation,whichhada

timerestrictionof0–6h,showedthatthebestperforminglimitedsamplingstrategywasat1

and 5 h after intravenous injection. (62). In another pharmacokinetic model study using

prospectivedatafrom12TBpatientsitwasobservedthat2gertapenemoncedailyresulted

inamorethanadose-proportionalincreaseinAUCcomparedtooncedaily1gertapenem.

BasedonaMICof1.0mg/L,11outof12patientsreachedthetargetvalueof40%Tfree>MIC

(63).

DiscussionHugonnetandcolleaguesfirststatedthatcarbapenemshaveantimycobacterialactivity(7).

Subsequently,studiesaddressingtheinactivationmechanismofLDTprovidedtheunderlying

evidencetosupportthehypothesisofactivityofcarbapenemsagainstM.tuberculosis(14-

28).Inspiteofthisaseriesofinvitrostudieshavebeencarriedout,someofwhichdetected

an effect and some of which did not (8,32-50). Only later, was it recognized that these

confusing results are probably explained by the chemical instability of carbapenems, in

culturemedia at the temperatures typically used in in vitro studies, andmanypreviously

publishedinvitrostudiesarelikelytohavereportedfalselyhighMICs(45).

Overall the results of the studies identified in this review, which used a variety of

experimental methods to test clinical and laboratory strains of M. tuberculosis for

susceptibility to carbapenems, are consistent. Carbapenems are more active against M.

tuberculosisifusedincombinationwithclavulanate,aBLaCinhibitor.(8,32-50).Inlinewith

theseinvitrostudiestheadditionofclavulanateimprovedthesurvivalrateinmice(35).As

theEuropeanMedicinesAgency(EMA)hasacceptedandqualifiedthe invitrohollowfiber

systemmodelsasamethodologytodefinepharmacokineticandpharmacodynamic(PK/PD)

parameters,thesemoderninvitrostudiescanbeusedtoavoidtheproblemsassociatedwith

thechemicalinstabilityoftheseagentsinstandardagar-basedMICtesting.Thus,hollowfiber

systems have the potential for dose finding and regimen selection studies on the use of

carbapenemsinthetreatmentofTB(64-65).

Few in vivo studies have been performed due to the short half-life and lower serum

concentrationsofcarbapenemsinmice(35).

One prospective, two observational and seven retrospective clinical studies to assess


and ertapenem) have been performed. Adverse events due to carbapenems were mild,

confirmingwhatweknowfromotherinfectiousdiseases;butincontrasttootherrepurposed

drugslikelinezolid(55,58,60).Todate,onlytwolargeretrospectivestudieswithM/XDR-TB

patientshavebeenperformedwithimipenem(84patients),andmeropenem(96patients)

(11).Meropenem/clavulanatewassuggestedtobemoreefficient inmanagingM/XDR-TB

(11),howeverinterpretationallimitationswerementioned.

We found no clear evidence to select one particular carbapenem among the different

candidatecompounds,whendesigninganeffectiveM/XDR-TBregimen.Botheconomicaland

clinicalfactorsplayarole.Whereas imipenemisthecheapercarbapenem,ertapenemhas

thepotentialadvantagethatitisonlygivenoncedaily;andmeropenemisbysomeauthors

believedtobethemosteffectiveinhumans,butnohead-to-headcomparisonstudieshave

confirmedthistodate.Therefore,moreclinicalevidenceanddoseoptimizationsubstantiated

for example by hollow fiber infection studies are needed to support the repurposing

carbapenemsforthetreatmentofM/XDR-TB.

Clinicalstudiesarehamperedbythefactthatcurrentlynocombinationofacarbapenemwith

clavulanateiscommerciallyavailable.Furthermore,clavulanateisnotavailablealonesoat

present it isnotpracticallypossible toprescribecarbapenemwithclavulanate.Therefore,

amoxicillin–clavulanateisoftenco-administeredalongwithacarbapenemincasethelatter

is preferred for treatment. Unfortunately, amoxicillin has gastrointestinal side effects

potentiallycomplicatingprolongedtreatment.Therefore,combinedtreatmentamoxicillin–

clavulanate with a carbapenem is only an option for TB treatment of complicated cases

showingmulti-orextensivedrugresistance(42).Although,Gonzaloetal.reportedapotential

benefitthatMICvaluesdropwhenamoxicillinisaddedtoacombinationofmeropenemand

clavulanate.

33

2


BasedonaMICof0.25mg/ml11/12patientsreachedthetargetvalueof40%Tfree>MICwas

exceeded(10).Thepharmacokineticmodelcomposedinthisstudywasshowntoadequately

predictertapenemexposureinMDR-TBpatients.TheMonteCarlosimulation,whichhada

timerestrictionof0–6h,showedthatthebestperforminglimitedsamplingstrategywasat1

and 5 h after intravenous injection. (62). In another pharmacokinetic model study using

prospectivedatafrom12TBpatientsitwasobservedthat2gertapenemoncedailyresulted

inamorethanadose-proportionalincreaseinAUCcomparedtooncedaily1gertapenem.

BasedonaMICof1.0mg/L,11outof12patientsreachedthetargetvalueof40%Tfree>MIC

(63).

DiscussionHugonnetandcolleaguesfirststatedthatcarbapenemshaveantimycobacterialactivity(7).

Subsequently,studiesaddressingtheinactivationmechanismofLDTprovidedtheunderlying

evidencetosupportthehypothesisofactivityofcarbapenemsagainstM.tuberculosis(14-

28).Inspiteofthisaseriesofinvitrostudieshavebeencarriedout,someofwhichdetected

an effect and some of which did not (8,32-50). Only later, was it recognized that these

confusing results are probably explained by the chemical instability of carbapenems, in

culturemedia at the temperatures typically used in in vitro studies, andmanypreviously

publishedinvitrostudiesarelikelytohavereportedfalselyhighMICs(45).

Overall the results of the studies identified in this review, which used a variety of

experimental methods to test clinical and laboratory strains of M. tuberculosis for

susceptibility to carbapenems, are consistent. Carbapenems are more active against M.

tuberculosisifusedincombinationwithclavulanate,aBLaCinhibitor.(8,32-50).Inlinewith

theseinvitrostudiestheadditionofclavulanateimprovedthesurvivalrateinmice(35).As

theEuropeanMedicinesAgency(EMA)hasacceptedandqualifiedthe invitrohollowfiber

systemmodelsasamethodologytodefinepharmacokineticandpharmacodynamic(PK/PD)

parameters,thesemoderninvitrostudiescanbeusedtoavoidtheproblemsassociatedwith

thechemicalinstabilityoftheseagentsinstandardagar-basedMICtesting.Thus,hollowfiber

systems have the potential for dose finding and regimen selection studies on the use of

carbapenemsinthetreatmentofTB(64-65).

Few in vivo studies have been performed due to the short half-life and lower serum

concentrationsofcarbapenemsinmice(35).

One prospective, two observational and seven retrospective clinical studies to assess


and ertapenem) have been performed. Adverse events due to carbapenems were mild,

confirmingwhatweknowfromotherinfectiousdiseases;butincontrasttootherrepurposed

drugslikelinezolid(55,58,60).Todate,onlytwolargeretrospectivestudieswithM/XDR-TB

patientshavebeenperformedwithimipenem(84patients),andmeropenem(96patients)

(11).Meropenem/clavulanatewassuggestedtobemoreefficient inmanagingM/XDR-TB

(11),howeverinterpretationallimitationswerementioned.

We found no clear evidence to select one particular carbapenem among the different

candidatecompounds,whendesigninganeffectiveM/XDR-TBregimen.Botheconomicaland

clinicalfactorsplayarole.Whereas imipenemisthecheapercarbapenem,ertapenemhas

thepotentialadvantagethatitisonlygivenoncedaily;andmeropenemisbysomeauthors

believedtobethemosteffectiveinhumans,butnohead-to-headcomparisonstudieshave

confirmedthistodate.Therefore,moreclinicalevidenceanddoseoptimizationsubstantiated

for example by hollow fiber infection studies are needed to support the repurposing

carbapenemsforthetreatmentofM/XDR-TB.

Clinicalstudiesarehamperedbythefactthatcurrentlynocombinationofacarbapenemwith

clavulanateiscommerciallyavailable.Furthermore,clavulanateisnotavailablealonesoat

present it isnotpracticallypossible toprescribecarbapenemwithclavulanate.Therefore,

amoxicillin–clavulanateisoftenco-administeredalongwithacarbapenemincasethelatter

is preferred for treatment. Unfortunately, amoxicillin has gastrointestinal side effects

potentiallycomplicatingprolongedtreatment.Therefore,combinedtreatmentamoxicillin–

clavulanate with a carbapenem is only an option for TB treatment of complicated cases

showingmulti-orextensivedrugresistance(42).Although,Gonzaloetal.reportedapotential

benefitthatMICvaluesdropwhenamoxicillinisaddedtoacombinationofmeropenemand

clavulanate.

34

Chapter 2

Duetodifferentprocedures,analyticalmethodsanddesign,thebiochemicalinstabilityofthe

drugsofinterest,theshorthalf-liveofdrugsofinterestinmice,diversityinMICdetermination

and intolerance in addition to resistance, itwas not possible to perform ameta-analysis.

Whiletheobservationaldataarepromising,carbapenemscanonlyrecommendedincaseof

resistancetogroupAandgroupBdrugsinM/XDR-TBtreatment.

Theidealcarbapenemwouldhavetheantimycobacterialactivityofimipenem,thehalf-lifeof

ertapenem and the oral bioavailability of tebipenem-pivoxil. Due to increasing resistance

observed in XDR-TB isolates (66-67) and in MDR-TB patients with resistance to an

aminoglycoside,carbapenemsmaybeavaluablealternativetothecurrentinjectablesecond

line drugs. Assessment of intracellular activity as well as activity against dormant M.

tuberculosis by carbapenems is a critical step to further explore the potential of these

repurposeddrugs.

As successful treatment outcome forM/XDR-TB is still poor, ranging from 25-50% (1) an

improvementofthecurrenttreatmentisurgentlyneeded.Anindividualdatameta-analysis

among12,030 individualpatients from50studies showedasignificantlybetter treatment

outcomeforpatientswhoreceivedcarbapenemscomparedtootherdrugstraditionallyused

fortreatmentofMDR-TB. (68).Sincethere isaneedforneworrepurposeddrugs forthe

treatmentofM/XDR-TB,phaseII/IIIclinicaltrialsareurgentlyneededforcarbapenemsto

further evaluate their potential. Long term safety and activity againstM. tuberculosis are

supported by observational data and several studies (41,50,69). A phase II prospective

randomized controlled study evaluating a carbapenemplus a BLaC inhibitor on top of an

optimizedbackgroundregimenversusstandardofcarewouldbeanappropriatestrategyto

testthepotentialbenefitsofcarbapenemsforM/XDR-TBtreatment.

Conclusion

Now the variable results of in vitro studies have been explained and the activity of

carbapenemsinthepresenceofaBLaCinhibitorisestablished,thesedrugsshouldbefurther

developed for the treatment of multi- and extensive drug resistant M. tuberculosis.

Ultimately,awell-designedphase2studyisneededtosubstantiatetheclaimedbenefitsof

carbapenemsinpatientswithdrug-resistantTB.

References

1. World Health Organization. Global Tuberculosis Report 2017.

http://www.who.int/tb/publications/global_report/en/accessed23march2018

2. FalzonD,SchünemannHJ,HarauszE,González-AnguloL,LienhardtC,JaramilloE,Weyer

K.2017WorldHealthOrganizationtreatmentguidelinesfordrug-resistanttuberculosis,

2016update.EurRespirJ.Mar22;49(3).

3. Nguyen TVA, Anthony RM, Bañuls AL, VuDH, Alffenaar JC. Bedaquiline resistance: Its


10.1093/cid/cix992.

4. Alsaad,N.,B.Wilffert,R.vanAltena,W.C.deLange,T.S.vanderWerf,J.G.Kosterink,

andJ.W.Alffenaar.2013.PotentialantimicrobialagentsforthetreatmentofMDR-TB.

Eur.Respir.J.doi:10.1183/09031936.00113713.

5. vanderPaardtAF,WilffertB,AkkermanOW,deLangeWC,vanSoolingenD,SinhaB,van

derWerfTS,KosterinkJG,AlffenaarJW.2015.Evaluationofmacrolidesforpossibleuse

againstmultidruf-resistantMycobacteriumtuberculosis.Eur.RespirJ.aug;46(2):444-55.

6. World Health Organization. WHO treatment guidelines for drug-resistant

tuberculosis.http://www.who.int/tb/areas-of-work/drug-resistant-tb/treatment/en/

accessed23march2018



10.1021/bi701506h.

8. Hugonnet JE,TremblayLW,BoshoffHI,Barry3rdCE,Blanchard JS.2009.Meropenem-

clavulanate is effective against extensively drug-resistantMycobacterium tuberculosis.

Science.323:1215-1218.doi:10.1126/science.1167498;10.1126/science.1167498.

9. Cynamon,M.H.,andG.S.Palmer.1983.Invitroactivityofamoxicillinincombinationwith

clavulanic acid against Mycobacterium tuberculosis. Antimicrob Agents Chemother.

24:429-431.

10. VanRijnSP,vanAltenaR,Akkerman,OW,vanSoolingenD,vanderLaanT,deLangeWCM,

KosterinkJGW,vanderWerfTS,AlffenaarJWC.2016.Pharmacokineticsofertapenemin

patients with multidrug-resistant tuberculosis. Eur Respir J. 47:1229-1234.

Doi:10.1183/13993003.01654-2015.

35

2


Duetodifferentprocedures,analyticalmethodsanddesign,thebiochemicalinstabilityofthe

drugsofinterest,theshorthalf-liveofdrugsofinterestinmice,diversityinMICdetermination

and intolerance in addition to resistance, itwas not possible to perform ameta-analysis.

Whiletheobservationaldataarepromising,carbapenemscanonlyrecommendedincaseof

resistancetogroupAandgroupBdrugsinM/XDR-TBtreatment.

Theidealcarbapenemwouldhavetheantimycobacterialactivityofimipenem,thehalf-lifeof

ertapenem and the oral bioavailability of tebipenem-pivoxil. Due to increasing resistance

observed in XDR-TB isolates (66-67) and in MDR-TB patients with resistance to an

aminoglycoside,carbapenemsmaybeavaluablealternativetothecurrentinjectablesecond

line drugs. Assessment of intracellular activity as well as activity against dormant M.

tuberculosis by carbapenems is a critical step to further explore the potential of these

repurposeddrugs.

As successful treatment outcome forM/XDR-TB is still poor, ranging from 25-50% (1) an

improvementofthecurrenttreatmentisurgentlyneeded.Anindividualdatameta-analysis

among12,030 individualpatients from50studies showedasignificantlybetter treatment

outcomeforpatientswhoreceivedcarbapenemscomparedtootherdrugstraditionallyused

fortreatmentofMDR-TB. (68).Sincethere isaneedforneworrepurposeddrugs forthe

treatmentofM/XDR-TB,phaseII/IIIclinicaltrialsareurgentlyneededforcarbapenemsto

further evaluate their potential. Long term safety and activity againstM. tuberculosis are

supported by observational data and several studies (41,50,69). A phase II prospective

randomized controlled study evaluating a carbapenemplus a BLaC inhibitor on top of an

optimizedbackgroundregimenversusstandardofcarewouldbeanappropriatestrategyto

testthepotentialbenefitsofcarbapenemsforM/XDR-TBtreatment.

Conclusion

Now the variable results of in vitro studies have been explained and the activity of

carbapenemsinthepresenceofaBLaCinhibitorisestablished,thesedrugsshouldbefurther

developed for the treatment of multi- and extensive drug resistant M. tuberculosis.

Ultimately,awell-designedphase2studyisneededtosubstantiatetheclaimedbenefitsof

carbapenemsinpatientswithdrug-resistantTB.

References

1. World Health Organization. Global Tuberculosis Report 2017.

http://www.who.int/tb/publications/global_report/en/accessed23march2018

2. FalzonD,SchünemannHJ,HarauszE,González-AnguloL,LienhardtC,JaramilloE,Weyer

K.2017WorldHealthOrganizationtreatmentguidelinesfordrug-resistanttuberculosis,

2016update.EurRespirJ.Mar22;49(3).

3. Nguyen TVA, Anthony RM, Bañuls AL, VuDH, Alffenaar JC. Bedaquiline resistance: Its


10.1093/cid/cix992.

4. Alsaad,N.,B.Wilffert,R.vanAltena,W.C.deLange,T.S.vanderWerf,J.G.Kosterink,

andJ.W.Alffenaar.2013.PotentialantimicrobialagentsforthetreatmentofMDR-TB.

Eur.Respir.J.doi:10.1183/09031936.00113713.

5. vanderPaardtAF,WilffertB,AkkermanOW,deLangeWC,vanSoolingenD,SinhaB,van

derWerfTS,KosterinkJG,AlffenaarJW.2015.Evaluationofmacrolidesforpossibleuse

againstmultidruf-resistantMycobacteriumtuberculosis.Eur.RespirJ.aug;46(2):444-55.

6. World Health Organization. WHO treatment guidelines for drug-resistant

tuberculosis.http://www.who.int/tb/areas-of-work/drug-resistant-tb/treatment/en/

accessed23march2018



10.1021/bi701506h.

8. Hugonnet JE,TremblayLW,BoshoffHI,Barry3rdCE,Blanchard JS.2009.Meropenem-


Science.323:1215-1218.doi:10.1126/science.1167498;10.1126/science.1167498.

9. Cynamon,M.H.,andG.S.Palmer.1983.Invitroactivityofamoxicillinincombinationwith

clavulanic acid against Mycobacterium tuberculosis. Antimicrob Agents Chemother.

24:429-431.




Doi:10.1183/13993003.01654-2015.

36

Chapter 2

11. TiberiS,SotgiuG,D’AmbrosioL,CentisR,ArbexMA,ArrascueEA,AlffenaarJW,Caminero

JA,GagaM,GualanoG,SkrahinaA,SolovicA,SulisG,TadoliniM,GuizadoVA,DeLorenzo

S,AriasAJR,ScardigliA,AkkermanOW,AleksaA,ArtsukevichJ,AvchinkoV,BoniniEH,

Marín FAC, Collahuazo López L, de Vries G, Dore S, Kunst H,Matteelli A,Moschos C,

PalmiereF,PapavasileiouA,PayenMC,PianaA, SpaneveloA,VasquezDV,ViggianiP,

White V, Zumla A, Migliori GB. 2016. Comparison of effectiveness and safety of

imipenem/clavulanate-versus meropenem/clavulanate- containing regiments in the

treatmentofMDR-andXDR-TB.EurRespirJ.47(6):1758-66

12. DeshpandeD,SrivastavaS,BendetP,MartinKR,CirrincioneKN,LeePS,PasipanodyaJG,

Dheda K, Gumbo T. 2018. Antibacterial and Sterilizing Effect of Benzylpenicillin in


13. DeshpandeD,SrivastavaS,ChapagainM,MagombedzeG,MartinKR,CirrincioneKN,Lee

PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactamhas potent sterilizing

activityagainsthighlydrug-resistanttuberculosis.SciAdv.Aug30;3(8):e1701102

14. Kurz, S. G., K. A.Wolff, S. Hazra, C. R. Bethel, A.M. Hujer, K.M. Smith, Y. Xu, L.W.

Tremblay, J. S. Blanchard, L. Nguyen, and R. A. Bonomo. 2013. Can inhibitor-resistant

substitutionsintheMycobacteriumtuberculosisbeta-LactamaseBLaCleadtoclavulanate

resistance?:abiochemicalrationalefortheuseofbeta-lactam-beta-lactamaseinhibitor

combinations.Antimicrob.AgentsChemother.57:6085-6096.doi:10.1128/AAC.01253-

13;10.1128/AAC.01253-13.

15. Lavollay, M., M. Arthur, M. Fourgeaud, L. Dubost, A. Marie, N. Veziris, D. Blanot, L.

Gutmann,andJ.L.Mainardi.2008.Thepeptidoglycanofstationary-phaseMycobacterium

tuberculosis predominantly contains cross-links generated by L,D-transpeptidation. J.

Bacteriol.190:4360-4366.doi:10.1128/JB.00239-08;10.1128/JB.00239-08.

16. Kumar,P.,K.Arora,J.R.Lloyd,I.Y.Lee,V.Nair,E.Fischer,H.I.Boshoff,andC.E.Barry

3rd. 2012. Meropenem inhibits D,D-carboxypeptidase activity in Mycobacterium

tuberculosis. Mol. Microbiol. 86:367-381. doi: 10.1111/j.1365-2958.2012.08199.x;

10.1111/j.1365-2958.2012.08199.x.

17. BrammerBastaLA,GhoshA,oanY,JakoncicJ,LloydEP,TownsendCA,LamichhaneG,

Bianchet MA 2015. Loss of functionally and structurally distinct LD-transpeptidase,

LDtmt5, compromises cell wall integrity of mycobacterium tuberculosis. J Biol Chem

290:25670-25685

18. Cordillot,M.,V.Dubee,S.Triboulet,L.Dubost,A.Marie,J.E.Hugonnet,M.Arthur,andJ.

L.Mainardi.2013.Invitrocross-linkingofMycobacteriumtuberculosispeptidoglycanby

L,D-transpeptidases and inactivation of these enzymes by carbapenems. Antimicrob.

AgentsChemother.57:5940-5945.doi:10.1128/AAC.01663-13;10.1128/AAC.01663-13.

19. Dubee,V.,S.Triboulet, J.L.Mainardi,M.Etheve-Quelquejeu,L.Gutmann,A.Marie,L.

Dubost,J.E.Hugonnet,andM.Arthur.2012.InactivationofMycobacteriumtuberculosis

l,d-transpeptidase LdtMt(1) by carbapenems and cephalosporins. Antimicrob. Agents


20. Erdemli,S.B.,R.Gupta,W.R.Bishai,G.Lamichhane,L.M.Amzel,andM.A.Bianchet.

2012.TargetingthecellwallofMycobacteriumtuberculosis:structureandmechanismof

L,D-transpeptidase 2. Structure. 20:2103-2115. doi: 10.1016/j.str.2012.09.016;

10.1016/j.str.2012.09.016.

21. KumarP,KaushikA,LloydE,LiSG,MattooR,AmmermanNC,BellDT,PerrymanAL,Zandi

TA, Ekins S,Ginell SL, TownsendCA, Freundlich JS, LamichhaneG. 2017.Non-classical

transpeptidases yield insight into new antibacterials. Nature Chemical Biology. Doi:

10.38/Nchemobio.2237

22. Lecoq,L.,V.Dubee,S.Triboulet,C.Bougault,J.E.Hugonnet,M.Arthur,andJ.P.Simorre.

2013. Structure of Enterococcus faeciuml, d-Transpeptidase Acylated by Ertapenem

Provides Insight into the Inactivation Mechanism. ACS Chem. Biol. doi:

10.1021/cb4001603.

23. Triboulet, S.,M. Arthur, J. L.Mainardi, C. Veckerle, V. Dubee, A. Nguekam-Moumi, L.

Gutmann,L.B.Rice,andJ.E.Hugonnet.2011.Inactivationkineticsofanewtargetofbeta-

lactam antibiotics. J. Biol. Chem. 286:22777-22784. doi: 10.1074/jbc.M111.239988;

10.1074/jbc.M111.239988.

24. DubéeV,ArthurM,FiefH,TribouletS,Mainardi JL,GutmannL,SollogoubM,RiceLB,

Ethève-Quelquejeu, Hugonnet JE. 2012. Kinetic Analysis of enterococcus Faecium L,D-

transpeptidaseinactivationofcarbapenems.AntimicrobAgentsChemother56(6)3409-

3412

25. Kim,H.S.,J.Kim,H.N.Im,J.Y.Yoon,D.R.An,H.J.Yoon,J.Y.Kim,H.K.Min,S.J.Kim,J.

Y.Lee,B.W.Han,andS.W.Suh.2013.StructuralbasisfortheinhibitionofMycobacterium

tuberculosisL,D-transpeptidasebymeropenem,adrugeffectiveagainstextensivelydrug-

resistant strains. Acta Crystallogr. D Biol. Crystallogr. 69:420-431. doi:

37

2







White V, Zumla A, Migliori GB. 2016. Comparison of effectiveness and safety of

imipenem/clavulanate-versus meropenem/clavulanate- containing regiments in the

treatmentofMDR-andXDR-TB.EurRespirJ.47(6):1758-66

12. DeshpandeD,SrivastavaS,BendetP,MartinKR,CirrincioneKN,LeePS,PasipanodyaJG,

Dheda K, Gumbo T. 2018. Antibacterial and Sterilizing Effect of Benzylpenicillin in



PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactamhas potent sterilizing

activityagainsthighlydrug-resistanttuberculosis.SciAdv.Aug30;3(8):e1701102

14. Kurz, S. G., K. A.Wolff, S. Hazra, C. R. Bethel, A.M. Hujer, K.M. Smith, Y. Xu, L.W.

Tremblay, J. S. Blanchard, L. Nguyen, and R. A. Bonomo. 2013. Can inhibitor-resistant

substitutionsintheMycobacteriumtuberculosisbeta-LactamaseBLaCleadtoclavulanate

resistance?:abiochemicalrationalefortheuseofbeta-lactam-beta-lactamaseinhibitor

combinations.Antimicrob.AgentsChemother.57:6085-6096.doi:10.1128/AAC.01253-

13;10.1128/AAC.01253-13.

15. Lavollay, M., M. Arthur, M. Fourgeaud, L. Dubost, A. Marie, N. Veziris, D. Blanot, L.

Gutmann,andJ.L.Mainardi.2008.Thepeptidoglycanofstationary-phaseMycobacterium

tuberculosis predominantly contains cross-links generated by L,D-transpeptidation. J.

Bacteriol.190:4360-4366.doi:10.1128/JB.00239-08;10.1128/JB.00239-08.

16. Kumar,P.,K.Arora,J.R.Lloyd,I.Y.Lee,V.Nair,E.Fischer,H.I.Boshoff,andC.E.Barry

3rd. 2012. Meropenem inhibits D,D-carboxypeptidase activity in Mycobacterium

tuberculosis. Mol. Microbiol. 86:367-381. doi: 10.1111/j.1365-2958.2012.08199.x;

10.1111/j.1365-2958.2012.08199.x.

17. BrammerBastaLA,GhoshA,oanY,JakoncicJ,LloydEP,TownsendCA,LamichhaneG,

Bianchet MA 2015. Loss of functionally and structurally distinct LD-transpeptidase,

LDtmt5, compromises cell wall integrity of mycobacterium tuberculosis. J Biol Chem

290:25670-25685

18. Cordillot,M.,V.Dubee,S.Triboulet,L.Dubost,A.Marie,J.E.Hugonnet,M.Arthur,andJ.

L.Mainardi.2013.Invitrocross-linkingofMycobacteriumtuberculosispeptidoglycanby

L,D-transpeptidases and inactivation of these enzymes by carbapenems. Antimicrob.

AgentsChemother.57:5940-5945.doi:10.1128/AAC.01663-13;10.1128/AAC.01663-13.

19. Dubee,V.,S.Triboulet, J.L.Mainardi,M.Etheve-Quelquejeu,L.Gutmann,A.Marie,L.

Dubost,J.E.Hugonnet,andM.Arthur.2012.InactivationofMycobacteriumtuberculosis

l,d-transpeptidase LdtMt(1) by carbapenems and cephalosporins. Antimicrob. Agents


20. Erdemli,S.B.,R.Gupta,W.R.Bishai,G.Lamichhane,L.M.Amzel,andM.A.Bianchet.

2012.TargetingthecellwallofMycobacteriumtuberculosis:structureandmechanismof

L,D-transpeptidase 2. Structure. 20:2103-2115. doi: 10.1016/j.str.2012.09.016;

10.1016/j.str.2012.09.016.

21. KumarP,KaushikA,LloydE,LiSG,MattooR,AmmermanNC,BellDT,PerrymanAL,Zandi

TA, Ekins S,Ginell SL, TownsendCA, Freundlich JS, LamichhaneG. 2017.Non-classical

transpeptidases yield insight into new antibacterials. Nature Chemical Biology. Doi:

10.38/Nchemobio.2237




10.1021/cb4001603.

23. Triboulet, S.,M. Arthur, J. L.Mainardi, C. Veckerle, V. Dubee, A. Nguekam-Moumi, L.

Gutmann,L.B.Rice,andJ.E.Hugonnet.2011.Inactivationkineticsofanewtargetofbeta-

lactam antibiotics. J. Biol. Chem. 286:22777-22784. doi: 10.1074/jbc.M111.239988;

10.1074/jbc.M111.239988.

24. DubéeV,ArthurM,FiefH,TribouletS,Mainardi JL,GutmannL,SollogoubM,RiceLB,

Ethève-Quelquejeu, Hugonnet JE. 2012. Kinetic Analysis of enterococcus Faecium L,D-

transpeptidaseinactivationofcarbapenems.AntimicrobAgentsChemother56(6)3409-

3412

25. Kim,H.S.,J.Kim,H.N.Im,J.Y.Yoon,D.R.An,H.J.Yoon,J.Y.Kim,H.K.Min,S.J.Kim,J.

Y.Lee,B.W.Han,andS.W.Suh.2013.StructuralbasisfortheinhibitionofMycobacterium

tuberculosisL,D-transpeptidasebymeropenem,adrugeffectiveagainstextensivelydrug-

resistant strains. Acta Crystallogr. D Biol. Crystallogr. 69:420-431. doi:

38

Chapter 2

10.1107/S0907444912048998;10.1107/S0907444912048998.




10.1021/cb4001603.

27. Schoonmaker MK, Bishai WR, Lamichhane G. 2014. Nonclassical transpeptidases of

mycobacterium tuberculosis alter cell size, morphology, the cytosolic matric, protein

localization,virulence,andresistance toβ-lactams.AmericanSociety formicrobiology.

Doi:10.1128/JB.01396-13

28. TribouletS,DubéeV, LecoqL,BougaultC,Mainardi JL,RiceLB,Ethève-quelquejeuM,

GutmannL,MarieA,DubostL,HugonnetJE,SImorreJP,ArthurM.2013.Kineticfeatures

of L,D-Transpeptidase inactivationcritical forβ-lactamantibacterialactivity.PLoSOne.

8(7):e67831

29. Nicolau,D.P.2008.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.

Clin.Infect.Dis.47Suppl1:S32-40.doi:10.1086/590064;10.1086/590064.

30. CraigWA.2003.Basicpharmacodynamicsofantibacterialswithclinicalapplicationstothe

useofbeta-lactams,glycopeptides,andlinezolid.Infect.Dis.Clin.NorthAm.17:479-501

31. MoherD,LiberatiA,TetzlaffJ,AltmanDG,PRISMAGroup.2009.Preferredreportingitems

forsystematicreviewsandmeta-analyses:thePRISMAstatement.JClinEpidemiol;62:

1006-1012.

32. Chambers,H.F.,D.Moreau,D.Yajko,C.Miick,C.Wagner,C.Hackbarth,S.Kocagoz,E.

Rosenberg,W. K.Hadley, andH.Nikaido. 1995. Canpenicillins andother beta-lactam

antibioticsbeusedtotreattuberculosis?Antimicrob.AgentsChemother.39:2620-2624.

33. KaushikA,MakkerN,PandeyP,ParrishN,SinghU,LamichaneG.2015.Carbapenemsand

Rifampicin exhibit synergy against Mycobacterium tuberculosis and Mycobacterium

abscessus.AntimicrobAgentsChemother59:6561-6567.Doi:10.1128/AAC.01158-15

34. SolapureS,DineshN,ShandilR,RamachandranV,SharmaS,BhattacharjeeD,GangulyS,

Reddy J, Ahuja V, Panduga V, Parab M, Vishwas KG, Kumar N, Balganesh M,

BalasubramanianV.2013.Invitroandinvivoefficacyofbeta-lactamsagainstreplicating

andslowlygrowing/nonreplicatingM.tuberculosis.AntimicrobAgentsChemother.doi:

10.1128/AAC.00023-13.

35. Veziris, N., C. Truffot, J. L. Mainardi, and V. Jarlier. 2011. Activity of carbapenems

combinedwithclavulanateagainstmurinetuberculosis.Antimicrob.AgentsChemother.

55:2597-2600.doi:10.1128/AAC.01824-10;10.1128/AAC.01824-10.

36. Watt,B.,J.R.Edwards,A.Rayner,A.J.Grindey,andG.Harris.1992.Invitroactivityof

meropenem and imipenem against mycobacteria: development of a daily antibiotic

dosingschedule.Tuber.LungDis.73:134-136.doi:10.1016/0962-8479(92)90145-A.

37. ZhangD,WangY,LuJ,PangY.2016.Invitroactivityofβ-lactamsincombinationwithβ-

lactamase inhibitors against multidrug-resistant Mycobacterium tuberculosis isolates.

AntimicrobAgentsChemother60:393-399.

38. CohenKA,El-HayT,WyresKL,WeissbrodO,MunsamyV,YanoverC,AharonovR,Shaham

O,ConwayTC,GoldschmidtY,BishaiWR,PymA.2016.Paradoxicalhypersusceptibilityof

drug-resistantMycobacteriumtuberculosistoβ-lactamantibiotics.EBioMedicine9:170-

179

39. Cavanaugh JS, Jou R, WU MH, Dalton T, Kurbatova E, Ershova J, Cegielski JP. 2017.

Susceptibilities of MDR Mycobacterium tuberculosis isolates to unconventional drugs

compared with their reported pharmacokinetic/pharmacodynamics parameters. J

AntimicrobChemother72:1678-1687

40. England,K.,H. I.Boshoff,K.Arora,D.Weiner,E.Dayao,D.Schimel,L.E.Via,andC.E.

Barry 3rd. 2012. Meropenem-clavulanic acid shows activity against Mycobacterium

tuberculosis in vivo. Antimicrob Agents Chemother. 56:3384-3387. doi:

10.1128/AAC.05690-11;10.1128/AAC.05690-11.

41. Forsman LD, Giske CG, Bruchfeld J, Schön T, Juréen P, Ängeby K. 2015.Meropenem-

Clavulanic acid has high in vitro activity against multidrug-resistant Mycobacterium

tuberculosis.AntimicrobAgentsChemother59:3630-3632.Doi:10.1128/AAC.00171-15

42. GonzaloX,Drobniewski F. 2013. Is there aplace for beta-lactams in the treatmentof

multidrug-resistant/extensively drug-resistant tuberculosis? Synergy between

meropenem and amoxicillin/clavulanate. J. Antimicrob Chemother. 68:366-369. doi:

10.1093/jac/dks395;10.1093/jac/dks395.

43. Horita Y, Maeda S, Kazumi Y, Doi N. 2014. In vitro susceptibility of Mycobacterium

tuberculosis isolates to anoral carbapenemaloneor in combinationwithβ-lactamase

inhibitors.AntimicrobAgentsChemother58(11);7010-14

44. SalaC,DharN,HartkoornRC,ZhangM,HaYA,SchneiderP,ColeST.2010.Simplemodel

39

2


10.1107/S0907444912048998;10.1107/S0907444912048998.




10.1021/cb4001603.

27. Schoonmaker MK, Bishai WR, Lamichhane G. 2014. Nonclassical transpeptidases of

mycobacterium tuberculosis alter cell size, morphology, the cytosolic matric, protein

localization,virulence,andresistance toβ-lactams.AmericanSociety formicrobiology.

Doi:10.1128/JB.01396-13

28. TribouletS,DubéeV, LecoqL,BougaultC,Mainardi JL,RiceLB,Ethève-quelquejeuM,

GutmannL,MarieA,DubostL,HugonnetJE,SImorreJP,ArthurM.2013.Kineticfeatures

of L,D-Transpeptidase inactivationcritical forβ-lactamantibacterialactivity.PLoSOne.

8(7):e67831



30. CraigWA.2003.Basicpharmacodynamicsofantibacterialswithclinicalapplicationstothe

useofbeta-lactams,glycopeptides,andlinezolid.Infect.Dis.Clin.NorthAm.17:479-501

31. MoherD,LiberatiA,TetzlaffJ,AltmanDG,PRISMAGroup.2009.Preferredreportingitems

forsystematicreviewsandmeta-analyses:thePRISMAstatement.JClinEpidemiol;62:

1006-1012.

32. Chambers,H.F.,D.Moreau,D.Yajko,C.Miick,C.Wagner,C.Hackbarth,S.Kocagoz,E.

Rosenberg,W. K.Hadley, andH.Nikaido. 1995. Canpenicillins andother beta-lactam

antibioticsbeusedtotreattuberculosis?Antimicrob.AgentsChemother.39:2620-2624.

33. KaushikA,MakkerN,PandeyP,ParrishN,SinghU,LamichaneG.2015.Carbapenemsand

Rifampicin exhibit synergy against Mycobacterium tuberculosis and Mycobacterium

abscessus.AntimicrobAgentsChemother59:6561-6567.Doi:10.1128/AAC.01158-15

34. SolapureS,DineshN,ShandilR,RamachandranV,SharmaS,BhattacharjeeD,GangulyS,

Reddy J, Ahuja V, Panduga V, Parab M, Vishwas KG, Kumar N, Balganesh M,

BalasubramanianV.2013.Invitroandinvivoefficacyofbeta-lactamsagainstreplicating

andslowlygrowing/nonreplicatingM.tuberculosis.AntimicrobAgentsChemother.doi:

10.1128/AAC.00023-13.



55:2597-2600.doi:10.1128/AAC.01824-10;10.1128/AAC.01824-10.

36. Watt,B.,J.R.Edwards,A.Rayner,A.J.Grindey,andG.Harris.1992.Invitroactivityof

meropenem and imipenem against mycobacteria: development of a daily antibiotic

dosingschedule.Tuber.LungDis.73:134-136.doi:10.1016/0962-8479(92)90145-A.

37. ZhangD,WangY,LuJ,PangY.2016.Invitroactivityofβ-lactamsincombinationwithβ-

lactamase inhibitors against multidrug-resistant Mycobacterium tuberculosis isolates.


38. CohenKA,El-HayT,WyresKL,WeissbrodO,MunsamyV,YanoverC,AharonovR,Shaham

O,ConwayTC,GoldschmidtY,BishaiWR,PymA.2016.Paradoxicalhypersusceptibilityof

drug-resistantMycobacteriumtuberculosistoβ-lactamantibiotics.EBioMedicine9:170-

179

39. Cavanaugh JS, Jou R, WU MH, Dalton T, Kurbatova E, Ershova J, Cegielski JP. 2017.

Susceptibilities of MDR Mycobacterium tuberculosis isolates to unconventional drugs

compared with their reported pharmacokinetic/pharmacodynamics parameters. J

AntimicrobChemother72:1678-1687

40. England,K.,H. I.Boshoff,K.Arora,D.Weiner,E.Dayao,D.Schimel,L.E.Via,andC.E.

Barry 3rd. 2012. Meropenem-clavulanic acid shows activity against Mycobacterium

tuberculosis in vivo. Antimicrob Agents Chemother. 56:3384-3387. doi:

10.1128/AAC.05690-11;10.1128/AAC.05690-11.

41. Forsman LD, Giske CG, Bruchfeld J, Schön T, Juréen P, Ängeby K. 2015.Meropenem-

Clavulanic acid has high in vitro activity against multidrug-resistant Mycobacterium

tuberculosis.AntimicrobAgentsChemother59:3630-3632.Doi:10.1128/AAC.00171-15




10.1093/jac/dks395;10.1093/jac/dks395.

43. Horita Y, Maeda S, Kazumi Y, Doi N. 2014. In vitro susceptibility of Mycobacterium

tuberculosis isolates to anoral carbapenemaloneor in combinationwithβ-lactamase

inhibitors.AntimicrobAgentsChemother58(11);7010-14

44. SalaC,DharN,HartkoornRC,ZhangM,HaYA,SchneiderP,ColeST.2010.Simplemodel

40

Chapter 2

fortestingdrugsagainstnonreplicatingMycobacteriumtuberculosis.AntimicrobAgents


45. Srivastava S, van Rijn SP,Wessels AMA, Alffenaar JWC,Gumbo T. 2016. Susceptibility

testing of antibiotics that degrade faster than the doubling time of slow-growing

mycobacteria: Ertapenem sterilizing effect ofMycobacterium tuberculosis. Antimicrob

AgentsChemother60:3193-3195.Doi:10.1128/AAC.02924-15.

46. VanRijnSP,SrivastavaS,WesselsMA,vanSoolingenD,AlffenaarJW,GumboT.2017.The

sterilizingeffectofertapenem-clavulanate inahollow fibermodelof tuberculosis and

implicationsonclinicaldosing.AntimicrobAgentsChemotherdoi:10.1128/AAC.02039-16

47. DeshpandeD,SrivastavaS,NuermbergerE,PasipanodyaJG,SwaminathanS,GumboT.

2016AFaropenem,Linezolid,andMoxifloxacinregimenforbothdrug-susceptibleand

multidrug-resistant tuberculosis in children: FLAME Path on the Milky Way. Clinical

InfectiousDiseases.63:95-102

48. GurumurthyM,VermaR,NaftalinCM,HeeKH,LuQ,TanKH,IssacS,LinW,TanA,Seng

K, Lee LS, Paton N. 2017. Activity of Faropenemwith and without rifampicin against

Mycobacterium tuberculosis: Evaluation in a whole-blood bactericidal activity trial. J

Antimicrobchemother.Doi:10.1093/jac/dkx081

49. DharN,DubeeV,BallellL,CuinetG,HugonnetJE,Signorino-geloF,BarrosD,ArthurM,

McKinney JD.2015.RapidCytolysisofMycobacterium tuberculosis by faropenem,and

orallybioavailablebeta-lactamantibiotic.AntimicrobAgentsChemother59:1308-1319.

50. Srivastava S,DeshpandeD, Pasipanodya J,Nuermberger E, Swaminathan S,GumboT.

2016.Optimal clinicaldosesof faropenem, linezolid,andmoxifloxacin inchildrenwith

disseminatedtuberculosis:goldilocks.ClinicalInfectiousDiseases63:102-9

51. KaushikA,AmmermanAC,TasneenR,Story-rollerE,DooleyKE,DormanSE,Nuermberger

EL,LamichhaneG.2017.Invitroandinvivoactivityofbiapenemagainstdrug-susceptible

andrifampicin-resistantMycobacteriumtuberculosis.JAntimicrobChemother72:2320-

2325

52. ChambersHF, Turner J, SchecterGF, KawamuraM,Hopewell PC. 2005. Imipenem for

treatmentoftuberculosisinmiceandhumans.AntimicrobAgentsChemother.49:2816-

2821.doi:10.1128/AAC.49.7.2816-2821.2005.

53. RullasJ,DharN,MckinneyJD,Garcia-PérezA,LelievreJ,DiaconAH,HugonnetJE,Arthur

M,Angulo-BarturenI,Barro-aguirreD,BallellL.2015.Combinationsofβ-lactamantibiotics

currentlyinclinicaltrialsareefficaciousinaDHP-I-deficientmousemodeloftuberculosis

infection.AntimicrobAgentsChemother.59:4997-4999.Doi:10.1128/AAC.01063-15

54. ArbexMA,Bonini EH, KawakamePirollaG,D’Ambrosio L, Centis R,MiglioriGB. 2016.

Effectiveness and safety of imipenem/clavulanate and linezolid to treat multidrug en

extensivelydrug-resistanttuberculosisatareferralhospitalinBrazil.RevPortPneumol

22(6):337-341






WhiteV,ZumlaA,MiglioriGB.2016.Effectivenessandsafetyofimipenem/clavulanate

addedtoanoptimizedbackgroundregimen(OBR)versusOBRcontrol regimens in the

treatment of multidrug-resistant and extensively drug-resistant tuberculosis. Clinical

InfectiousDiseases62(9):1188-90.Doi:10.1093/cid/ciw088

56. Payen,M.C.,S.DeWit,C.Martin,R.Sergysels,I.Muylle,Y.VanLaethem,andN.Clumeck.

2012. Clinical use of the meropenem-clavulanate combination for extensively drug-

resistant tuberculosis. Int. J. Tuberc. Lung Dis. 16:558-560. doi: 10.5588/ijtld.11.0414;

10.5588/ijtld.11.0414

57. PayenMC,MuylleI,VandenbergO,MathysV,DelforgeM,vandenWijngaertS,Clumeck

N,DewitS.2018.Meropenem-clavulanatefordrug-resistanttuberculosis:afollow-upof

relapse-freecases.IntJTubercLungDis22(1):34-39

58. DeLorenzoS,AlffenaarJW,SotgiuG,CentisR,D’AmbrosioL,TiberiS,BolhuisMS,van

Altena R, Viggiani P, Piana A, Spanevello A, Migliori GB. 2013. Efficacy and safety of

meropenem-clavulanateaddedtolinezolid-containingregimensinthetreatmentofMDR-

/XDR-TB.EurRespirJ41:1386-1392

59. PalmeroD,GonzálezMontanerP,CufréM,GarcíaA,VescovoM,PoggiS.2014.FirstSeries

ofPatientswithXDRandpre-XDRTBtreatedwithregimentsthatincludedmeropenem-

clavulanateinArgentina.ArchBronconeumol.51:e49-e52.

60. Tiberi S, Payen MC, Sotgiu G, D’Ambrosio L, Guizado VA, Alffenaar JW, Arbex MA,

CamineroJA,CentisR,DeLorenzoS,GagaM,GualanoG,AriasAJR,ScardigliA,Skrahina

A,SolovicA,SulisG,TadoliniM,AkkermanOW,ArrascueEA,AleksaA,AvchinkoV,Bonini

41

2


fortestingdrugsagainstnonreplicatingMycobacteriumtuberculosis.AntimicrobAgents









47. DeshpandeD,SrivastavaS,NuermbergerE,PasipanodyaJG,SwaminathanS,GumboT.

2016AFaropenem,Linezolid,andMoxifloxacinregimenforbothdrug-susceptibleand

multidrug-resistant tuberculosis in children: FLAME Path on the Milky Way. Clinical

InfectiousDiseases.63:95-102

48. GurumurthyM,VermaR,NaftalinCM,HeeKH,LuQ,TanKH,IssacS,LinW,TanA,Seng

K, Lee LS, Paton N. 2017. Activity of Faropenemwith and without rifampicin against

Mycobacterium tuberculosis: Evaluation in a whole-blood bactericidal activity trial. J

Antimicrobchemother.Doi:10.1093/jac/dkx081

49. DharN,DubeeV,BallellL,CuinetG,HugonnetJE,Signorino-geloF,BarrosD,ArthurM,

McKinney JD.2015.RapidCytolysisofMycobacterium tuberculosis by faropenem,and

orallybioavailablebeta-lactamantibiotic.AntimicrobAgentsChemother59:1308-1319.

50. Srivastava S,DeshpandeD, Pasipanodya J,Nuermberger E, Swaminathan S,GumboT.

2016.Optimal clinicaldosesof faropenem, linezolid,andmoxifloxacin inchildrenwith

disseminatedtuberculosis:goldilocks.ClinicalInfectiousDiseases63:102-9

51. KaushikA,AmmermanAC,TasneenR,Story-rollerE,DooleyKE,DormanSE,Nuermberger

EL,LamichhaneG.2017.Invitroandinvivoactivityofbiapenemagainstdrug-susceptible

andrifampicin-resistantMycobacteriumtuberculosis.JAntimicrobChemother72:2320-

2325

52. ChambersHF, Turner J, SchecterGF, KawamuraM,Hopewell PC. 2005. Imipenem for

treatmentoftuberculosisinmiceandhumans.AntimicrobAgentsChemother.49:2816-

2821.doi:10.1128/AAC.49.7.2816-2821.2005.

53. RullasJ,DharN,MckinneyJD,Garcia-PérezA,LelievreJ,DiaconAH,HugonnetJE,Arthur

M,Angulo-BarturenI,Barro-aguirreD,BallellL.2015.Combinationsofβ-lactamantibiotics

currentlyinclinicaltrialsareefficaciousinaDHP-I-deficientmousemodeloftuberculosis

infection.AntimicrobAgentsChemother.59:4997-4999.Doi:10.1128/AAC.01063-15

54. ArbexMA,Bonini EH, KawakamePirollaG,D’Ambrosio L, Centis R,MiglioriGB. 2016.

Effectiveness and safety of imipenem/clavulanate and linezolid to treat multidrug en

extensivelydrug-resistanttuberculosisatareferralhospitalinBrazil.RevPortPneumol

22(6):337-341






WhiteV,ZumlaA,MiglioriGB.2016.Effectivenessandsafetyofimipenem/clavulanate

addedtoanoptimizedbackgroundregimen(OBR)versusOBRcontrol regimens in the

treatment of multidrug-resistant and extensively drug-resistant tuberculosis. Clinical

InfectiousDiseases62(9):1188-90.Doi:10.1093/cid/ciw088




10.5588/ijtld.11.0414

57. PayenMC,MuylleI,VandenbergO,MathysV,DelforgeM,vandenWijngaertS,Clumeck

N,DewitS.2018.Meropenem-clavulanatefordrug-resistanttuberculosis:afollow-upof

relapse-freecases.IntJTubercLungDis22(1):34-39

58. DeLorenzoS,AlffenaarJW,SotgiuG,CentisR,D’AmbrosioL,TiberiS,BolhuisMS,van

Altena R, Viggiani P, Piana A, Spanevello A, Migliori GB. 2013. Efficacy and safety of

meropenem-clavulanateaddedtolinezolid-containingregimensinthetreatmentofMDR-

/XDR-TB.EurRespirJ41:1386-1392

59. PalmeroD,GonzálezMontanerP,CufréM,GarcíaA,VescovoM,PoggiS.2014.FirstSeries

ofPatientswithXDRandpre-XDRTBtreatedwithregimentsthatincludedmeropenem-

clavulanateinArgentina.ArchBronconeumol.51:e49-e52.

60. Tiberi S, Payen MC, Sotgiu G, D’Ambrosio L, Guizado VA, Alffenaar JW, Arbex MA,

CamineroJA,CentisR,DeLorenzoS,GagaM,GualanoG,AriasAJR,ScardigliA,Skrahina

A,SolovicA,SulisG,TadoliniM,AkkermanOW,ArrascueEA,AleksaA,AvchinkoV,Bonini

42

Chapter 2

EH,MarínFAC,CollahuazoLópezL,deVriesG,DoreS,KunstH,MatteelliA,MoschosC,

Palmiere F, Papavasileiou A, Spanevelo A, Vasquez DV, Viggiani P,White V, Zumla A,

Migliori GB. 2016. Effectiveness and safety of meropenem/clavulanate-containing

regimens in the treatment of MDR- and XDR-TB. Eur Respir J 47:1235-1243.

Doi:10.1183/13993003.02146-2015

61. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJWC,Migliori

GB.2015.Ertapenem in the treatmentofmultidrug-resistant tuberculosis: first clinical

experience.EurRespirJ47:333-336.Doi:10.1183/13993003.01278-2015

62. VanRijnSP,ZuurMA,vanAltenaR,AkkermanOW,ProostJH,deLangeWCM,Kerstjens

HAM,TouwDJ,vanderWerfTS,KosterinkJGW,AlffenaarJWC.2017.Pharmacokinetic

modelingandlimitedsamplingstrategiesbasedonhealthyvolunteersformonitoringof

ertapenem in patients with multidrug-resistant tuberculosis. Antimicrob Agents

Chemother61:e01783-16.Doi:10.1128/AAC.01783-16

63. ZuurM,GhimireS,BolhuisMS,WesselsAMA,vanAltenaR,deLangeWCM,Kosterink

JGW, Touw DJ, van der Werf TS, Akkerman OW, Alffenaar JWC. 2018. The

pharmacokinetics of 2000 mg Ertapenem in tuberculosis patients. Antimicrob Agents

Chemother.[Epubaheadofprint]

64. Cavaleri M, Manolis E. 2015. Hollow Fiber System for Tuberculosis: The European

medicinesAgencyexperience.ClinInfect.Dis.15;61Suppl1:S1-4

65. European medicines Agency (EMA). 2016. Qualification opinion; in-vitro hollow fiber

systemmodeloftuberculosis(HSF-TB).EMA/CHMP/SAWP/47290/2015.

66. DrusanoGL,SgambatiN,EichasA,BrownDL,KulawyR,LouieA.2010.Thecombination

ofrifampinplusmoxifloxacineissynergisticforsuppressionofresistancebutantagonistic

forcellkillofMycobacteriumtuberculosisasdeterminedinahollow-fiberinfectionmodel.

Mbio.10;1(3)

67. GumboT,LouieA,DezielMR,ParsonsLM,SalfingerM,DrusanoGL.2004.Selectionofa

moxifloxacindosethatsuppressesdrugresistanceinMycobacteriumtuberculosis,byuse

ofaninvitropharmacodynamicinfectionmodelandmathematicalmodeling.JInfectDis.

1;190(9)

68. AhmadKhanF,SalimMAH,DuCrosP,CasasEC,KharmraevA,SikhondzeW,BenedettiA,

BastosM, Lan Z, Jaramillo E, Falzon D, Menzies D. 2017. Effectiveness and safety of

standardized shorter regimens for multidrug-resistant tuberculosis: individual patient

dataandaggregatedatameta-analyses.EurRespirJ27;50(1)

69. CecileMagis-Escurra,RichardMAnthony,AdriGMvanderZanden,DickvanSoolingen,

Jan-Willem C Alffenaar. 2018. Pound foolish and penny wise—when will dosing of

rifampicinbeoptimised?TheLancetRespiratoryMedicine

43

2


EH,MarínFAC,CollahuazoLópezL,deVriesG,DoreS,KunstH,MatteelliA,MoschosC,

Palmiere F, Papavasileiou A, Spanevelo A, Vasquez DV, Viggiani P,White V, Zumla A,

Migliori GB. 2016. Effectiveness and safety of meropenem/clavulanate-containing

regimens in the treatment of MDR- and XDR-TB. Eur Respir J 47:1235-1243.

Doi:10.1183/13993003.02146-2015

61. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJWC,Migliori


experience.EurRespirJ47:333-336.Doi:10.1183/13993003.01278-2015









Chemother.[Epubaheadofprint]

64. Cavaleri M, Manolis E. 2015. Hollow Fiber System for Tuberculosis: The European

medicinesAgencyexperience.ClinInfect.Dis.15;61Suppl1:S1-4

65. European medicines Agency (EMA). 2016. Qualification opinion; in-vitro hollow fiber

systemmodeloftuberculosis(HSF-TB).EMA/CHMP/SAWP/47290/2015.

66. DrusanoGL,SgambatiN,EichasA,BrownDL,KulawyR,LouieA.2010.Thecombination

ofrifampinplusmoxifloxacineissynergisticforsuppressionofresistancebutantagonistic

forcellkillofMycobacteriumtuberculosisasdeterminedinahollow-fiberinfectionmodel.

Mbio.10;1(3)

67. GumboT,LouieA,DezielMR,ParsonsLM,SalfingerM,DrusanoGL.2004.Selectionofa

moxifloxacindosethatsuppressesdrugresistanceinMycobacteriumtuberculosis,byuse

ofaninvitropharmacodynamicinfectionmodelandmathematicalmodeling.JInfectDis.

1;190(9)

68. AhmadKhanF,SalimMAH,DuCrosP,CasasEC,KharmraevA,SikhondzeW,BenedettiA,

BastosM, Lan Z, Jaramillo E, Falzon D, Menzies D. 2017. Effectiveness and safety of

standardized shorter regimens for multidrug-resistant tuberculosis: individual patient

dataandaggregatedatameta-analyses.EurRespirJ27;50(1)

69. CecileMagis-Escurra,RichardMAnthony,AdriGMvanderZanden,DickvanSoolingen,

Jan-Willem C Alffenaar. 2018. Pound foolish and penny wise—when will dosing of

rifampicinbeoptimised?TheLancetRespiratoryMedicine

CHAPTER 3

Antimicrob Agents Chemother 2014 Jun; 58(6): 3481–3484.PMID: 24733468

S.P. van RijnA.M.A. Wessels

B. GreijdanusD.J. Touw

J.W.C. Alffenaar

Quantification and Validation of Ertapenem Using a Liquid

Chromatography-Tandem Mass Spectrometry Method

46

Chapter 3

AbstractBackground:Ertapenem,acarbapenem,reliesontime-dependentkilling.Therapeuticdrug

monitoring (TDM) should be considered, when used in specific populations, to achieve

optimal bactericidal activity and optimize drug-dosing regimens. No validated LC-MS/MS

methodhasbeenreportedusingdeuteratedertapenemasinternalstandard.Anewsimple

and robust LC-MS/MS method using a quadruple mass spectrometer was developed for

analysisofertapeneminhumanplasma,usingdeuteratedertapenemasinternalstandard.

The calibration curvewas linear over a range of 0.1 (LLOQ) to 125mg/L. The calculated

accuracy ranged from -2.4 % to 10.3%.Within-run CV ranged from 2.7 % to 11.8% and

between-runCVrangedfrom0%to8.4%.Freeze-thawstabilitybiasedbetween-3.3%and

0.1%.StorageofQCsamplesfor96hat4°Cdiffered-4.3to5.6%,storageatroomtemperature

for24h,biasedfrom-10.7%to-14.8%andstorageintheautosamplerbiasedbetween-2.9%

and -10.0%. A simple LC-MS/MS method to quantify ertapenem in human plasma using

deuteratedertapenemasinternalstandardhasbeenvalidated.Thismethodcanbeusedin

pharmacokineticstudiesandinclinicalstudiesbyperformingTDM.

IntroductionCarbapenems belong to the Beta-lactam antibiotics and are widely used against a broad

spectrum of aerobe and anaerobe gram-positive and gram-negative bacteria (1, 2).

Ertapenem,approvedbytheFDAin2001,isoneofthesecarbapenems.Sinceertapenemhas

anapproximatehalf-lifeof4h,itcanbeadministeredoncedaily.Therefore,ertapenemcan

be favored above other carbapenems (3). The pharmacodynamic (PD) parameter of

ertapenem correlates with time dependent killing, which means that the plasma

concentration of ertapenem has to exceed minimal inhibitory concentration (MIC) for a

percentageoftimeofitsdosinginterval(4).

Pharmacokineticdataobtainedinhealthyvolunteersaredifficulttoextrapolatetospecific

patientpopulations.Duetothishighvariabilityinpharmacokineticparameters,exposureof

ertapenemmightbesuboptimal inthesespecificpopulations (5-10).Sinceertapenemisa

time-dependent antibiotic, therapeutic drug monitoring (TDM) should therefore be

considered,whenused inspecificpopulations, toachieveoptimalbactericidalactivityand

optimize drug-dosing regimens. Yetmore PK studies have to be performed to determine

efficacyandsafetyofertapeneminspecificpopulation(11,12).

Ertapenem is being suggested for having potential use against M. tuberculosis (TB) (1).

However, according to Caminero et al. carbapenems are used as fifth line drugs in the

treatment of TB and can only be used in severe cases only, since carbapenems are

administeredintravenously,costsarehighandclinicalexperienceisrestricted(13).Clinical

studiesassessingefficacyorsafetyprofilesforcarbapenemsarescarce,butshowedfavorable

preliminaryresults (14-16).Beforeclinicalefficacy inTBcanbedeterminedadose-finding

studyshouldbeperformedtoevaluatepharmacokineticparametersinthisspecificpatient

population. Therefore, an analytical method to measure ertapenem concentrations is

mandatory.

There are a few methods published about LC-MS/MS quantification and validation of

ertapenem in human plasma (17, 18). Since LC-MS/MS is easy to use frequently in daily

routine,morepharmacokineticstudiesarebeingperformedtoquantifydrugs.Itistherefore

47

3

Quantification and Validation of Ertapenem Using a LC-MS/MS

AbstractBackground:Ertapenem,acarbapenem,reliesontime-dependentkilling.Therapeuticdrug

monitoring (TDM) should be considered, when used in specific populations, to achieve

optimal bactericidal activity and optimize drug-dosing regimens. No validated LC-MS/MS

methodhasbeenreportedusingdeuteratedertapenemasinternalstandard.Anewsimple

and robust LC-MS/MS method using a quadruple mass spectrometer was developed for

analysisofertapeneminhumanplasma,usingdeuteratedertapenemasinternalstandard.

The calibration curvewas linear over a range of 0.1 (LLOQ) to 125mg/L. The calculated

accuracy ranged from -2.4 % to 10.3%.Within-run CV ranged from 2.7 % to 11.8% and

between-runCVrangedfrom0%to8.4%.Freeze-thawstabilitybiasedbetween-3.3%and

0.1%.StorageofQCsamplesfor96hat4°Cdiffered-4.3to5.6%,storageatroomtemperature

for24h,biasedfrom-10.7%to-14.8%andstorageintheautosamplerbiasedbetween-2.9%

and -10.0%. A simple LC-MS/MS method to quantify ertapenem in human plasma using

deuteratedertapenemasinternalstandardhasbeenvalidated.Thismethodcanbeusedin

pharmacokineticstudiesandinclinicalstudiesbyperformingTDM.

IntroductionCarbapenems belong to the Beta-lactam antibiotics and are widely used against a broad

spectrum of aerobe and anaerobe gram-positive and gram-negative bacteria (1, 2).

Ertapenem,approvedbytheFDAin2001,isoneofthesecarbapenems.Sinceertapenemhas

anapproximatehalf-lifeof4h,itcanbeadministeredoncedaily.Therefore,ertapenemcan

be favored above other carbapenems (3). The pharmacodynamic (PD) parameter of

ertapenem correlates with time dependent killing, which means that the plasma

concentration of ertapenem has to exceed minimal inhibitory concentration (MIC) for a

percentageoftimeofitsdosinginterval(4).

Pharmacokineticdataobtainedinhealthyvolunteersaredifficulttoextrapolatetospecific

patientpopulations.Duetothishighvariabilityinpharmacokineticparameters,exposureof

ertapenemmightbesuboptimal inthesespecificpopulations (5-10).Sinceertapenemisa

time-dependent antibiotic, therapeutic drug monitoring (TDM) should therefore be

considered,whenused inspecificpopulations, toachieveoptimalbactericidalactivityand

optimize drug-dosing regimens. Yetmore PK studies have to be performed to determine

efficacyandsafetyofertapeneminspecificpopulation(11,12).

Ertapenem is being suggested for having potential use against M. tuberculosis (TB) (1).

However, according to Caminero et al. carbapenems are used as fifth line drugs in the

treatment of TB and can only be used in severe cases only, since carbapenems are

administeredintravenously,costsarehighandclinicalexperienceisrestricted(13).Clinical

studiesassessingefficacyorsafetyprofilesforcarbapenemsarescarce,butshowedfavorable

preliminaryresults (14-16).Beforeclinicalefficacy inTBcanbedeterminedadose-finding

studyshouldbeperformedtoevaluatepharmacokineticparametersinthisspecificpatient

population. Therefore, an analytical method to measure ertapenem concentrations is

mandatory.

There are a few methods published about LC-MS/MS quantification and validation of

ertapenem in human plasma (17, 18). Since LC-MS/MS is easy to use frequently in daily

routine,morepharmacokineticstudiesarebeingperformedtoquantifydrugs.Itistherefore

48

Chapter 3

importanttohaveestablishedstandardsinordertocompareresultsofPKstudiesbetween

laboratories.PresentvalidatedLC-MS/MSmethodsforertapenemareusingextensivesample

preparation,e.g.liquid-liquidextraction(LLE),solidphaseextraction(SPE)andnitrogengas

drying(17,18).Thesemethodsaretimeconsumingandlesscost-effective.Thechoiceofan

internal standard for LC-MS/MS is important as it corrects for extraction, injection and

ionizationvariability.Particularlythelatterone,ionsuppressionandionenhancement,isa

sourceofvariability.Onlyadeuteratedinternalstandardissuitabletocompensateforthis

andassurearobusthighthroughputbioanalyticalmethod(19).SincenoLC-MS/MSmethod

has been validated using a deuterated internal standard, the purpose of this study is to

developanewsimpleandrobustLC-MS/MSmethodusingaquadruplemassspectrometer

withoutextensivesampleprocessing,usingdeuteratedertapenemas internal standard to

quantifyconcentrationsofertapeneminhumanplasmaforpharmacokineticstudies.

MaterialandmethodsAnalysis

Ertapenem (Fig 1) and the internal standard used, ertapenem-D4, were purchased from

Alsachim(Illkirch,Graffenstaden,France).

Figure1.Chemicalstructureofertapenem

AcetonitrileforLC-MS/MSwaspurchasedfromBioSolve(Valkenswaard,TheNetherlands).

Thechemicalsused,includingmethanolLichrosolvandtrifluoroaceticanhydrideareofHPLC

oranalyticalgradeandwerepurchasedfromVWR(Amsterdam,TheNetherlands).Purified

waterwasobtainedfromaMilli-Qwaterpurifyingsystem(MilliporeCorporation,Billerica,

MA, USA). The precipitation reagent consisted of amixture ofmethanol and acetonitrile

(4:21,v/v).PooledhumanplasmasampleswithEDTAasanticoagulantandpooledhuman

serum samples were made available according to the standard operating procedures of

UniversityMedicalCenterGroningen.

Thecalibrationstandards,blankandQCsampleswerefullythawedatroomtemperature.To

100µLofeachsampleavolumeof500µLofprecipitationreagentsand10µLofertapenem-

D4(250mg/L)wereaddedinavial.Thesampleswerevortexedfor1minute.Topromote

proteinprecipitation,thevialswerestoredat-20°Cfor30minutes.Thevialswerecentrifuged

for5minutesat11000rpm.FiveµLoftheupperlayerwereinjectedintotheLC-MS/MS.QC

samplesandcalibrationstandardswerestoredat-20°C.

TheanalysiswasperformedonatriplequadrupoleLC-MS/MS(ThermoScientific,SanJose,

CAUSA)withaMSpumpPlus(Finnigan,surveyor)andautosampler(Finnigan,surveyor).The

massspectrometerwasaTSQQuantumAccessMaxmassspectrometer.Theautosampler

temperature was set at 10°C. Liquid chromatographic separation was performed by a

HyPURITYC18,analyticalcolumn,50*2.1mm,3-μm(ThermoScientific,Interscience,Breda,

TheNetherlands)andtemperaturewassetat20°C.Themobilephasehadaflowof300μL/

min and consisted of purified water, acetonitrile and an aqueous buffer (containing

ammoniumacetate10g/L,aceticacid35mg/Landtrifluoroaceticanhydride2mg/Lwater.

The method had a runtime of 4 minutes and the elution gradient is shown in Table 1.

Table1.Eluentgradient

Time(minutes) A(%) B(%) C(%)0.00 5 95 0

0.50 5 35 60

1.30 5 35 60

1.40 5 0 95

2.80 5 0 95

3.00 5 95 0

4.00 5 95 0

A:aqueousbuffer;B:purifiedwater,C:acetonitrile

49

3


importanttohaveestablishedstandardsinordertocompareresultsofPKstudiesbetween

laboratories.PresentvalidatedLC-MS/MSmethodsforertapenemareusingextensivesample

preparation,e.g.liquid-liquidextraction(LLE),solidphaseextraction(SPE)andnitrogengas

drying(17,18).Thesemethodsaretimeconsumingandlesscost-effective.Thechoiceofan

internal standard for LC-MS/MS is important as it corrects for extraction, injection and

ionizationvariability.Particularlythelatterone,ionsuppressionandionenhancement,isa

sourceofvariability.Onlyadeuteratedinternalstandardissuitabletocompensateforthis

andassurearobusthighthroughputbioanalyticalmethod(19).SincenoLC-MS/MSmethod

has been validated using a deuterated internal standard, the purpose of this study is to

developanewsimpleandrobustLC-MS/MSmethodusingaquadruplemassspectrometer

withoutextensivesampleprocessing,usingdeuteratedertapenemas internal standard to

quantifyconcentrationsofertapeneminhumanplasmaforpharmacokineticstudies.

MaterialandmethodsAnalysis

Ertapenem (Fig 1) and the internal standard used, ertapenem-D4, were purchased from

Alsachim(Illkirch,Graffenstaden,France).

Figure1.Chemicalstructureofertapenem

AcetonitrileforLC-MS/MSwaspurchasedfromBioSolve(Valkenswaard,TheNetherlands).

Thechemicalsused,includingmethanolLichrosolvandtrifluoroaceticanhydrideareofHPLC

oranalyticalgradeandwerepurchasedfromVWR(Amsterdam,TheNetherlands).Purified

waterwasobtainedfromaMilli-Qwaterpurifyingsystem(MilliporeCorporation,Billerica,

MA, USA). The precipitation reagent consisted of amixture ofmethanol and acetonitrile

(4:21,v/v).PooledhumanplasmasampleswithEDTAasanticoagulantandpooledhuman

serum samples were made available according to the standard operating procedures of

UniversityMedicalCenterGroningen.

Thecalibrationstandards,blankandQCsampleswerefullythawedatroomtemperature.To

100µLofeachsampleavolumeof500µLofprecipitationreagentsand10µLofertapenem-

D4(250mg/L)wereaddedinavial.Thesampleswerevortexedfor1minute.Topromote

proteinprecipitation,thevialswerestoredat-20°Cfor30minutes.Thevialswerecentrifuged

for5minutesat11000rpm.FiveµLoftheupperlayerwereinjectedintotheLC-MS/MS.QC

samplesandcalibrationstandardswerestoredat-20°C.

TheanalysiswasperformedonatriplequadrupoleLC-MS/MS(ThermoScientific,SanJose,

CAUSA)withaMSpumpPlus(Finnigan,surveyor)andautosampler(Finnigan,surveyor).The

massspectrometerwasaTSQQuantumAccessMaxmassspectrometer.Theautosampler

temperature was set at 10°C. Liquid chromatographic separation was performed by a

HyPURITYC18,analyticalcolumn,50*2.1mm,3-μm(ThermoScientific,Interscience,Breda,

TheNetherlands)andtemperaturewassetat20°C.Themobilephasehadaflowof300μL/

min and consisted of purified water, acetonitrile and an aqueous buffer (containing

ammoniumacetate10g/L,aceticacid35mg/Landtrifluoroaceticanhydride2mg/Lwater.

The method had a runtime of 4 minutes and the elution gradient is shown in Table 1.

Table1.Eluentgradient

Time(minutes) A(%) B(%) C(%)0.00 5 95 0

0.50 5 35 60

1.30 5 35 60

1.40 5 0 95

2.80 5 0 95

3.00 5 95 0

4.00 5 95 0

A:aqueousbuffer;B:purifiedwater,C:acetonitrile

50

Chapter 3

TheMSwasconfiguredontopositiveelectrospray ionizationmodeandSelectedReaction

Monitoring (SRM)witha sprayvoltageof3500V,a capillary temperatureof350°Canda

sheathgaspressureandauxiliarypressure35and5arbitraryunitsrespectively.

Masstransitionsforertapenemwere476.1m/z→432.1m/zandforertapenem-D4480.1

m/z→436.1m/z,usingascanwidthof0.5m/z.Collisionenergywasdeterminedon10eV

forbothtransitions.Peakheight integrationforallcomponentswascalculatedbyXcalibur

softwareversion2.0.7(ThermoFisher,SanJose,CA,USA).

Analyticalmethodvalidation

Validationofthemethodincludedselectivityandsensitivity,linearity,accuracyandprecision,

recovery and dilution integrity conform the guidance for Industry of the Food and Drug

Administration.SincetheFDAdidnotpostulateamaximumCVrequirementforstability,a

maximumCVof15%wasemployedaccordingtoEMAguidelines(20,21).

Insomecases,forexampleinpharmacokineticstudies,humanserumisbeingcollectedfor

the quantification of ertapenem. To check if there’s a difference between the analysis of

ertapeneminhumanplasmaandinhumanserum,amatrixcomparisonhasbeenperformed.

Thecalibrationcurveofertapenemconsistedof8sampleswithconcentrationsof0.1,0.5,

2.0,7.5,20,50,90and125mg/L.QualityControl(QC)sampleswith4differentconcentrations

ofertapenemwereused,whereasLLOQis0.1mg/L,LOWis2.5mg/L,MEDis40mg/Land

HIGH is 120 mg/L. For selectivity 6 pooled human plasma samples were examined for

interferenceandcomparedwithresponseofthelowerlimitofquantificationsamples(LLOQ).

Duringthreedays,eachdayasinglecalibrationcurveinplasmaandinserumwasanalyzed

andaccuracywasmeasuredbyevaluationof fivedeterminationsperQCsampleonthree

consecutive days. Precision is divided into within-run and between-run using the same

methodasaccuracy.ThecoefficientofvariationforLLOQmaynotexceed20%andforother

QClevelsnotexceed15%.

Therecoverywasdeterminedonthreelevels(LOW,MEDandHIGH)infivefold.Asprotein

precipitationisusedastheonlywayofsamplepreparationinthismethod,relativerecovery

wasmeasured by comparing the ratios of integrated peak heights of ertapenem and the

internalstandardoftheprocessedQCsampleswiththeaveragepeakheightsoftherecovery

samples.Recoverysamples(LOW,MEDandHIGH)werepost-extractionblanksamplesspiked

atthesameconcentrationsastheQCsamples.

Stabilityofertapenemwastestedindifferenttestconditions,includingstoragestabilityand

freeze-thawstability.StoragestabilityofertapenemwasexaminedbystoringQCsamplesat

roomtemperature(20°C-25°C)inarefrigeratorat4°Candaftersamplepreparationinthe

autosamplerat10°C. Stabilitywasalso testedusing three freeze-thawcyclesat -20°C.All

stability tests were done using three different QC levels (LOW, MED, HIGH) in five

determinations per concentration. Stability is defined as a change in concentration and

shouldbe≤15%.SinceFDAguidelineshavenorequirementsforthecoefficientofvariation

ofeachQCsample(LOW,MED,HIGH),EMAguidelinerequirementsaretakenintoaccount,

statingthatCVofeachQCsampleshouldnotexceed15%(20,21).

Todeterminethedilutionintegrity,onthreeconsecutivedays,asamplewithaconcentration

of500mgertapenem/Lplasmawasdiluted10timesandthenpreparedinfivefold.

Statistics

Results were analyzed using one-way ANOVA and validated Excel sheets (Microsoft,

Redmond,WA).

ResultsMeanretentiontimesofertapenemandertapenem-D4wereequalto1.5minutes.

Examiningtheselectivityofthisanalyticalmethod,therewerenointerferingpeaksobserved

attheretentiontimeofertapenemorertapenem-D4inanyofthesixlotsofpooledhuman

plasma(Fig.2).

Thecalibrationcurveinplasmawaslinearoverarangeof0.1(LLOQ)to125mg/Landthe

correlationcoefficient(R2)was0.9988.Thecalibrationcurveparametersareasfollows;slope,

0.487 ± 0.00519 (average ± standard deviation); intercept, 0.0149 ± 0.0257; average

regressioncoefficient,0.99762;correlationcoefficient,0.9981.

51

3


TheMSwasconfiguredontopositiveelectrospray ionizationmodeandSelectedReaction

Monitoring (SRM)witha sprayvoltageof3500V,a capillary temperatureof350°Canda

sheathgaspressureandauxiliarypressure35and5arbitraryunitsrespectively.

Masstransitionsforertapenemwere476.1m/z→432.1m/zandforertapenem-D4480.1

m/z→436.1m/z,usingascanwidthof0.5m/z.Collisionenergywasdeterminedon10eV

forbothtransitions.Peakheight integrationforallcomponentswascalculatedbyXcalibur

softwareversion2.0.7(ThermoFisher,SanJose,CA,USA).

Analyticalmethodvalidation

Validationofthemethodincludedselectivityandsensitivity,linearity,accuracyandprecision,

recovery and dilution integrity conform the guidance for Industry of the Food and Drug

Administration.SincetheFDAdidnotpostulateamaximumCVrequirementforstability,a

maximumCVof15%wasemployedaccordingtoEMAguidelines(20,21).

Insomecases,forexampleinpharmacokineticstudies,humanserumisbeingcollectedfor

the quantification of ertapenem. To check if there’s a difference between the analysis of

ertapeneminhumanplasmaandinhumanserum,amatrixcomparisonhasbeenperformed.

Thecalibrationcurveofertapenemconsistedof8sampleswithconcentrationsof0.1,0.5,

2.0,7.5,20,50,90and125mg/L.QualityControl(QC)sampleswith4differentconcentrations

ofertapenemwereused,whereasLLOQis0.1mg/L,LOWis2.5mg/L,MEDis40mg/Land

HIGH is 120 mg/L. For selectivity 6 pooled human plasma samples were examined for

interferenceandcomparedwithresponseofthelowerlimitofquantificationsamples(LLOQ).

Duringthreedays,eachdayasinglecalibrationcurveinplasmaandinserumwasanalyzed

andaccuracywasmeasuredbyevaluationof fivedeterminationsperQCsampleonthree

consecutive days. Precision is divided into within-run and between-run using the same

methodasaccuracy.ThecoefficientofvariationforLLOQmaynotexceed20%andforother

QClevelsnotexceed15%.

Therecoverywasdeterminedonthreelevels(LOW,MEDandHIGH)infivefold.Asprotein

precipitationisusedastheonlywayofsamplepreparationinthismethod,relativerecovery

wasmeasured by comparing the ratios of integrated peak heights of ertapenem and the

internalstandardoftheprocessedQCsampleswiththeaveragepeakheightsoftherecovery

samples.Recoverysamples(LOW,MEDandHIGH)werepost-extractionblanksamplesspiked

atthesameconcentrationsastheQCsamples.

Stabilityofertapenemwastestedindifferenttestconditions,includingstoragestabilityand

freeze-thawstability.StoragestabilityofertapenemwasexaminedbystoringQCsamplesat

roomtemperature(20°C-25°C)inarefrigeratorat4°Candaftersamplepreparationinthe

autosamplerat10°C. Stabilitywasalso testedusing three freeze-thawcyclesat -20°C.All

stability tests were done using three different QC levels (LOW, MED, HIGH) in five

determinations per concentration. Stability is defined as a change in concentration and

shouldbe≤15%.SinceFDAguidelineshavenorequirementsforthecoefficientofvariation

ofeachQCsample(LOW,MED,HIGH),EMAguidelinerequirementsaretakenintoaccount,

statingthatCVofeachQCsampleshouldnotexceed15%(20,21).

Todeterminethedilutionintegrity,onthreeconsecutivedays,asamplewithaconcentration

of500mgertapenem/Lplasmawasdiluted10timesandthenpreparedinfivefold.

Statistics

Results were analyzed using one-way ANOVA and validated Excel sheets (Microsoft,

Redmond,WA).

ResultsMeanretentiontimesofertapenemandertapenem-D4wereequalto1.5minutes.

Examiningtheselectivityofthisanalyticalmethod,therewerenointerferingpeaksobserved

attheretentiontimeofertapenemorertapenem-D4inanyofthesixlotsofpooledhuman

plasma(Fig.2).

Thecalibrationcurveinplasmawaslinearoverarangeof0.1(LLOQ)to125mg/Landthe

correlationcoefficient(R2)was0.9988.Thecalibrationcurveparametersareasfollows;slope,

0.487 ± 0.00519 (average ± standard deviation); intercept, 0.0149 ± 0.0257; average

regressioncoefficient,0.99762;correlationcoefficient,0.9981.

52

Chapter 3

Forcomparisonoftheanalysisinplasmaandinserum,thepeakheightratiosofertapenem

andtheinternalstandardinplasmawerecomparedtothoseinserum.Analyzingbothdata

setsusingPassing-Bablokregression,showednostatisticallysignificantdifferencebetween

thetwomatrices:y=1.01(0.95-1.02)x+0.00(-0.01-0.04)at95%confidencelevel.

Figure2.Chromatogram

(a)ErtapenemD4atLLQ,(b)ertapenematLLQand(c)blankplasma

Accuracyandprecision,dividedinwithinrunandbetweenrun,werecalculatedusingspiked

samples for 5 determinations per concentration on 3 consecutive days. The calculated

accuracyrangedfrom-2.4%to10.3%.Within-runprecisionrangedfrom2.7%to11.8%and

between-runprecisionrangedfrom0%to8.4%.Theresultsofaccuracyandprecisionforall

QClevelsareshowninTable2.

Table2.ConcentrationsofcalibrationstandardsandQCsamples.QCsamples LLOQ LOW MED HIGH

Concentration(mg/L) 0.1 2.5 40 120

Accuracy(%bias) -2.4 9.3 7.3 10.3

Within-runprecision(%CV) 11.8 5.6 3.1 2.7

Between-runprecision(%CV) 8.4 0 1.5 1.6

QCsamplesLOW(2.5mg/L),MED(40mg/L)andHIGH(120mg/L)wereusedtodetermine

recovery; relative recovery was 101.7%, 97.9% and 95.1% for these three samples,

respectively.Dilutionintegritywasdeterminedinfive-foldon3consecutivedays.Accuracy

was 1% and within-run and between-run precisions were 3.2% and 2.3%, respectively.

Stabilityofertapenemusingdifferenttestconditionsisshownintable3.

Table3.Stabilitytestingresultsforertapenem.Testcondition LOW MED HIGHBenchtop,RT*,24h(%bias) -11.9 -10.7 -14.8

Refrigerator,4°C,96h(%bias) 5.6 -0.1 -4.3

Freeze-thaw,-20°C,3cycles(%bias) -0.2 0.1 -3.3

Autosampler,10°C,24h(%bias) -10.0 -2.9 -4.8

*RTroomtemperature

Measured concentrations of QC samples (LOW,MED, HIGH) for the freeze-thaw stability

biased between -3.3% and 0.1% and therefore comply with the guidelines. Stability was

determinedbymeasuringQCsamplesstoredfor96hat4°Canddiffered-4.3to5.6%from

thenominalconcentrations.Afterstorageatroomtemperaturefor24h,theconcentrationof

ertapenem biased from -10.7% to -14.8%, compared to the initial concentrations. After

sample preparation the concentration of ertapenem stored in the autosampler biased

between-2.9%and-10.0%fromthenominalconcentrations.

53

3


Forcomparisonoftheanalysisinplasmaandinserum,thepeakheightratiosofertapenem

andtheinternalstandardinplasmawerecomparedtothoseinserum.Analyzingbothdata

setsusingPassing-Bablokregression,showednostatisticallysignificantdifferencebetween

thetwomatrices:y=1.01(0.95-1.02)x+0.00(-0.01-0.04)at95%confidencelevel.

Figure2.Chromatogram

(a)ErtapenemD4atLLQ,(b)ertapenematLLQand(c)blankplasma

Accuracyandprecision,dividedinwithinrunandbetweenrun,werecalculatedusingspiked

samples for 5 determinations per concentration on 3 consecutive days. The calculated

accuracyrangedfrom-2.4%to10.3%.Within-runprecisionrangedfrom2.7%to11.8%and

between-runprecisionrangedfrom0%to8.4%.Theresultsofaccuracyandprecisionforall

QClevelsareshowninTable2.

Table2.ConcentrationsofcalibrationstandardsandQCsamples.QCsamples LLOQ LOW MED HIGH

Concentration(mg/L) 0.1 2.5 40 120

Accuracy(%bias) -2.4 9.3 7.3 10.3

Within-runprecision(%CV) 11.8 5.6 3.1 2.7

Between-runprecision(%CV) 8.4 0 1.5 1.6

QCsamplesLOW(2.5mg/L),MED(40mg/L)andHIGH(120mg/L)wereusedtodetermine

recovery; relative recovery was 101.7%, 97.9% and 95.1% for these three samples,

respectively.Dilutionintegritywasdeterminedinfive-foldon3consecutivedays.Accuracy

was 1% and within-run and between-run precisions were 3.2% and 2.3%, respectively.

Stabilityofertapenemusingdifferenttestconditionsisshownintable3.

Table3.Stabilitytestingresultsforertapenem.Testcondition LOW MED HIGHBenchtop,RT*,24h(%bias) -11.9 -10.7 -14.8

Refrigerator,4°C,96h(%bias) 5.6 -0.1 -4.3

Freeze-thaw,-20°C,3cycles(%bias) -0.2 0.1 -3.3

Autosampler,10°C,24h(%bias) -10.0 -2.9 -4.8

*RTroomtemperature

Measured concentrations of QC samples (LOW,MED, HIGH) for the freeze-thaw stability

biased between -3.3% and 0.1% and therefore comply with the guidelines. Stability was

determinedbymeasuringQCsamplesstoredfor96hat4°Canddiffered-4.3to5.6%from

thenominalconcentrations.Afterstorageatroomtemperaturefor24h,theconcentrationof

ertapenem biased from -10.7% to -14.8%, compared to the initial concentrations. After

sample preparation the concentration of ertapenem stored in the autosampler biased

between-2.9%and-10.0%fromthenominalconcentrations.

54

Chapter 3

Discussion

Thisisthefirstdesignandvalidationofanew,simpleandrapidanalysismethodusingatriple

quadrupleLC-MS/MSforthequantificationofertapeneminhumanplasmaanddeuterated

ertapenemasinternalstandard.

ThisLC-MS/MSmethodwasvalidatedforaccuracyandprecisionaccordingtoFDAguidelines,

havingbiases<20%forLLOQand<15%forotherQClevels(20).Thecalibrationcurvewas

linearwithin a rangeof 0.1 (LLOQ) – 125µg/mL, compared toother studies,whichwere

validatedup to50µg/mLandhadaLLOQof respectively0.5and1.0µg/mL (17,18)This

methoduseddeuteratedertapenemasinternalstandard,resultinginbetterinter-day,intra-

dayvariationandaccuracy,comparedtomethodsofPickeringetal.andKoaletal,which

usedBeta-lactammonologuesasinternalstandard(17,18).

Matrix comparison showed no difference between the analysis of ertapenem in human

plasmaandinhumanserum.However,becauseofthepoorstabilityofertapenematroom

temperatureit’srecommendedtodrawwholeblood(withEDTAastheanticoagulant)asit

canbeplacedoniceforashorttimeduringtransportfromthenursingwardtotheanalyzing

laboratory.

AsmentionedintheintroductionamajoradvantageofthisLC-MS/MSmethodisthatasimple

proteinprecipitationisusedinsteadofLLE,SPEornitrogengasdrying,resultinginalesstime

consumingandalessexpensivemethod,comparedtootherLC-MS/MSmethods(17,18).The

runtimeisveryshortsincetheretentiontimeofertapenemis1.5minutes.Thisfacilitatesa

highsampletroughput.ThisisagreatadvantageforlaboratorieshavingonlyoneLCMSMS

tosupporttheirclinicalTDMservice.

Ertapeneminplasmastoredatroomtemperaturedecreaseswithinashortperiodtimewith

10to15%.Therefore,itiscrucialtostoresamplesinthefreezeruntilanalysisandtoprocess

samplescontainingertapenemwithinthevalidatedtimeframeofstabilitytoassureaccurate

andpreciseresults.Reinjectionofprocessedsamplesstoredat10°C intheautosampler is

toleratedwithin24hours.

Sinceertapenem isa timedependentantibiotic, it isnecessary thatplasmaconcentration

exceedsMICatleast40%ofitsdosinginterval.Toattainthistargetinpatientssuspectedto

have altered pharmacokinetics due to renal function, high variability in plasma proteins

ertapenemconcentrationmeasuringmaybeofhelp,especiallyifmoreresistantpathogens

are targetedwith higherMIC values. Thismethodmeets the criteria for TDMbut is also

suitableforclinicalpharmacokineticstudiesorclinicaltrialstofurtherinvestigatetheuseof

ertapeneminotherinfectiousdiseasesorotherspecificpatientpopulations.

ConclusionA simple LC-MS/MS method to quantify ertapenem in human plasma using deuterated

ertapenemasinternalstandardhasbeendevelopedandvalidated.Thismethodcanbeused

inpharmacokineticstudiesandinclinicalstudiesbyperformingTDM.

55

3


Discussion

Thisisthefirstdesignandvalidationofanew,simpleandrapidanalysismethodusingatriple

quadrupleLC-MS/MSforthequantificationofertapeneminhumanplasmaanddeuterated

ertapenemasinternalstandard.

ThisLC-MS/MSmethodwasvalidatedforaccuracyandprecisionaccordingtoFDAguidelines,

havingbiases<20%forLLOQand<15%forotherQClevels(20).Thecalibrationcurvewas

linearwithin a rangeof 0.1 (LLOQ) – 125µg/mL, compared toother studies,whichwere

validatedup to50µg/mLandhadaLLOQof respectively0.5and1.0µg/mL (17,18)This

methoduseddeuteratedertapenemasinternalstandard,resultinginbetterinter-day,intra-

dayvariationandaccuracy,comparedtomethodsofPickeringetal.andKoaletal,which

usedBeta-lactammonologuesasinternalstandard(17,18).

Matrix comparison showed no difference between the analysis of ertapenem in human

plasmaandinhumanserum.However,becauseofthepoorstabilityofertapenematroom

temperatureit’srecommendedtodrawwholeblood(withEDTAastheanticoagulant)asit

canbeplacedoniceforashorttimeduringtransportfromthenursingwardtotheanalyzing

laboratory.

AsmentionedintheintroductionamajoradvantageofthisLC-MS/MSmethodisthatasimple

proteinprecipitationisusedinsteadofLLE,SPEornitrogengasdrying,resultinginalesstime

consumingandalessexpensivemethod,comparedtootherLC-MS/MSmethods(17,18).The

runtimeisveryshortsincetheretentiontimeofertapenemis1.5minutes.Thisfacilitatesa

highsampletroughput.ThisisagreatadvantageforlaboratorieshavingonlyoneLCMSMS

tosupporttheirclinicalTDMservice.

Ertapeneminplasmastoredatroomtemperaturedecreaseswithinashortperiodtimewith

10to15%.Therefore,itiscrucialtostoresamplesinthefreezeruntilanalysisandtoprocess

samplescontainingertapenemwithinthevalidatedtimeframeofstabilitytoassureaccurate

andpreciseresults.Reinjectionofprocessedsamplesstoredat10°C intheautosampler is

toleratedwithin24hours.

Sinceertapenem isa timedependentantibiotic, it isnecessary thatplasmaconcentration

exceedsMICatleast40%ofitsdosinginterval.Toattainthistargetinpatientssuspectedto

have altered pharmacokinetics due to renal function, high variability in plasma proteins

ertapenemconcentrationmeasuringmaybeofhelp,especiallyifmoreresistantpathogens

are targetedwith higherMIC values. Thismethodmeets the criteria for TDMbut is also

suitableforclinicalpharmacokineticstudiesorclinicaltrialstofurtherinvestigatetheuseof

ertapeneminotherinfectiousdiseasesorotherspecificpatientpopulations.

ConclusionA simple LC-MS/MS method to quantify ertapenem in human plasma using deuterated

ertapenemasinternalstandardhasbeendevelopedandvalidated.Thismethodcanbeused

inpharmacokineticstudiesandinclinicalstudiesbyperformingTDM.

56

Chapter 3

References1. Hugonnet,J.E.,L.W.Tremblay,H.I.Boshoff,C.E.Barry3rd,andJ.S.Blanchard.2009.

Meropenem-clavulanate is effective against extensively drug-resistantMycobacterium


10.1126/science.1167498.

2. Gupta,R.,M.Lavollay,J.L.Mainardi,M.Arthur,W.R.Bishai,andG.Lamichhane.2010.

TheMycobacteriumtuberculosisproteinLdtMt2isanonclassicaltranspeptidaserequired

forvirulenceandresistancetoamoxicillin.Nat.Med.16:466-469.doi:10.1038/nm.2120;

10.1038/nm.2120.

3. Majumdar,A.K.,D.G.Musson,K.L.Birk,C.J.Kitchen,S.Holland,J.McCrea,G.Mistry,M.

Hesney, L. Xi, S. X. Li, R.Haesen, R.A. Blum,R. L. Lins,H.Greenberg, S.Waldman, P.

Deutsch, and J. D. Rogers. 2002. Pharmacokinetics of ertapenem in healthy young

volunteers.Antimicrob.AgentsChemother.46:3506-3511.



5. Burkhardt,O., V. Kumar, S. Schmidt, J. T. Kielstein, T.Welte, andH.Derendorf. 2010.

UnderdosingofertapenemincriticallyillpatientswithpneumoniaconfirmedbyMonte

Carlo simulations. Int. J. Antimicrob. Agents. 35:96-97. doi:

10.1016/j.ijantimicag.2009.09.007;10.1016/j.ijantimicag.2009.09.007.

6. Brink,A.J.,G.A.Richards,V.Schillack,S.Kiem,andJ.Schentag.2009.Pharmacokinetics

of once-daily dosing of ertapenem in critically ill patients with severe sepsis. Int. J.

Antimicrob. Agents. 33:432-436. doi: 10.1016/j.ijantimicag.2008.10.005;

10.1016/j.ijantimicag.2008.10.005.

7. Dailly,E.,J.F.Arnould,F.Fraissinet,E.Naux,M.A.LetarddelaBouraliere,R.Bouquie,G.

Deslandes, P. Jolliet, and R. Le Floch. 2013. Pharmacokinetics of ertapenem in burns

patients. Int. J. Antimicrob. Agents. 42:48-52. doi: 10.1016/j.ijantimicag.2013.02.021;

10.1016/j.ijantimicag.2013.02.021.

8. Chen,M.,A.N.Nafziger,G. L.Drusano,L.Ma,and J. S.Bertino Jr.2006.Comparative

pharmacokinetics and pharmacodynamic target attainment of ertapenem in normal-

weight, obese, and extremely obese adults. Antimicrob. Agents Chemother. 50:1222-

1227.doi:10.1128/AAC.50.4.1222-1227.2006.

9. Mistry,G.C.,A.K.Majumdar,S.Swan,D.Sica,A.Fisher,Y.Xu,M.Hesney, L.Xi, J.A.

Wagner,andP.J.Deutsch.2006.Pharmacokineticsofertapeneminpatientswithvarying

degreesofrenalinsufficiencyandinpatientsonhemodialysis.J.Clin.Pharmacol.46:1128-

1138.doi:10.1177/0091270006291839.

10. Abdel-Rahman,S.M.,G.L.Kearns,S.Topelberg,R.F.Jacobs,G.C.Mistry,A.Majumdar,

Y. Xu, J. A. Wagner, C. J. Kitchen, M. Groff, G. Herman, and J. L. Blumer. 2010.

Pharmacokinetics and tolerability of single-dose intravenous ertapenem in infants,

children, and adolescents. Pediatr. Infect. Dis. J. 29:1072-1076. doi:

10.1097/INF.0b013e3181e82608;10.1097/INF.0b013e3181e82608.

11. Wiskirchen, D. E., S. T. Housman, R. Quintiliani, D. P. Nicolau, and J. L. Kuti. 2013.

Comparative pharmacokinetics, pharmacodynamics, and tolerability of ertapenem 1

gram/day administered as a rapid 5-minute infusion versus the standard 30-minute

infusion in healthy adult volunteers. Pharmacotherapy. 33:266-274. doi:

10.1002/phar.1197;10.1002/phar.1197

12. Breilh,D.,C.Fleureau,J.B.Gordien,O.Joanes-Boyau,J.Texier-Maugein,S.Rapaport,E.

Boselli,G.Janvier,andM.C.Saux.2011.Pharmacokineticsoffreeertapenemincritically

illsepticpatients:intermittentversuscontinuousinfusion.MinervaAnestesiol.77:1058-

1062.

13. Caminero, J.A.,G. Sotgiu,A. Zumla, andG.B.Migliori. 2010.Bestdrug treatment for

multidrug-resistantandextensivelydrug-resistanttuberculosis.LancetInfect.Dis.10:621-

629.doi:10.1016/S1473-3099(10)70139-0;10.1016/S1473-3099(10)70139-0.

14. DeLorenzo,S.,J.W.Alffenaar,G.Sotgiu,R.Centis,L.D'Ambrosio,S.Tiberi,M.S.Bolhuis,

R.vanAltena,P.Viggiani,A.Piana,A.Spanevello,andG.B.Migliori.2012.Efficacyand

safetyofmeropenem/clavunateaddedtolinezolidcontainingregimensinthetreatment

ofM/XDR-TB.Eur.Respir.J.doi:10.1183/09031936.00124312.

15. Dauby, N., I. Muylle, F. Mouchet, R. Sergysels, and M. C. Payen. 2011.

Meropenem/clavulanate and linezolid treatment for extensively drug-resistant

tuberculosis. Pediatr. Infect. Dis. J. 30:812-813. doi: 10.1097/INF.0b013e3182154b05;

10.1097/INF.0b013e3182154b05.




57

3


References1. Hugonnet,J.E.,L.W.Tremblay,H.I.Boshoff,C.E.Barry3rd,andJ.S.Blanchard.2009.

Meropenem-clavulanate is effective against extensively drug-resistantMycobacterium


10.1126/science.1167498.

2. Gupta,R.,M.Lavollay,J.L.Mainardi,M.Arthur,W.R.Bishai,andG.Lamichhane.2010.

TheMycobacteriumtuberculosisproteinLdtMt2isanonclassicaltranspeptidaserequired

forvirulenceandresistancetoamoxicillin.Nat.Med.16:466-469.doi:10.1038/nm.2120;

10.1038/nm.2120.

3. Majumdar,A.K.,D.G.Musson,K.L.Birk,C.J.Kitchen,S.Holland,J.McCrea,G.Mistry,M.

Hesney, L. Xi, S. X. Li, R.Haesen, R.A. Blum,R. L. Lins,H.Greenberg, S.Waldman, P.

Deutsch, and J. D. Rogers. 2002. Pharmacokinetics of ertapenem in healthy young

volunteers.Antimicrob.AgentsChemother.46:3506-3511.



5. Burkhardt,O., V. Kumar, S. Schmidt, J. T. Kielstein, T.Welte, andH.Derendorf. 2010.

UnderdosingofertapenemincriticallyillpatientswithpneumoniaconfirmedbyMonte

Carlo simulations. Int. J. Antimicrob. Agents. 35:96-97. doi:

10.1016/j.ijantimicag.2009.09.007;10.1016/j.ijantimicag.2009.09.007.

6. Brink,A.J.,G.A.Richards,V.Schillack,S.Kiem,andJ.Schentag.2009.Pharmacokinetics

of once-daily dosing of ertapenem in critically ill patients with severe sepsis. Int. J.

Antimicrob. Agents. 33:432-436. doi: 10.1016/j.ijantimicag.2008.10.005;

10.1016/j.ijantimicag.2008.10.005.

7. Dailly,E.,J.F.Arnould,F.Fraissinet,E.Naux,M.A.LetarddelaBouraliere,R.Bouquie,G.

Deslandes, P. Jolliet, and R. Le Floch. 2013. Pharmacokinetics of ertapenem in burns

patients. Int. J. Antimicrob. Agents. 42:48-52. doi: 10.1016/j.ijantimicag.2013.02.021;

10.1016/j.ijantimicag.2013.02.021.

8. Chen,M.,A.N.Nafziger,G. L.Drusano,L.Ma,and J. S.Bertino Jr.2006.Comparative


weight, obese, and extremely obese adults. Antimicrob. Agents Chemother. 50:1222-

1227.doi:10.1128/AAC.50.4.1222-1227.2006.

9. Mistry,G.C.,A.K.Majumdar,S.Swan,D.Sica,A.Fisher,Y.Xu,M.Hesney, L.Xi, J.A.

Wagner,andP.J.Deutsch.2006.Pharmacokineticsofertapeneminpatientswithvarying

degreesofrenalinsufficiencyandinpatientsonhemodialysis.J.Clin.Pharmacol.46:1128-

1138.doi:10.1177/0091270006291839.

10. Abdel-Rahman,S.M.,G.L.Kearns,S.Topelberg,R.F.Jacobs,G.C.Mistry,A.Majumdar,

Y. Xu, J. A. Wagner, C. J. Kitchen, M. Groff, G. Herman, and J. L. Blumer. 2010.

Pharmacokinetics and tolerability of single-dose intravenous ertapenem in infants,

children, and adolescents. Pediatr. Infect. Dis. J. 29:1072-1076. doi:

10.1097/INF.0b013e3181e82608;10.1097/INF.0b013e3181e82608.

11. Wiskirchen, D. E., S. T. Housman, R. Quintiliani, D. P. Nicolau, and J. L. Kuti. 2013.

Comparative pharmacokinetics, pharmacodynamics, and tolerability of ertapenem 1

gram/day administered as a rapid 5-minute infusion versus the standard 30-minute

infusion in healthy adult volunteers. Pharmacotherapy. 33:266-274. doi:

10.1002/phar.1197;10.1002/phar.1197

12. Breilh,D.,C.Fleureau,J.B.Gordien,O.Joanes-Boyau,J.Texier-Maugein,S.Rapaport,E.

Boselli,G.Janvier,andM.C.Saux.2011.Pharmacokineticsoffreeertapenemincritically

illsepticpatients:intermittentversuscontinuousinfusion.MinervaAnestesiol.77:1058-

1062.

13. Caminero, J.A.,G. Sotgiu,A. Zumla, andG.B.Migliori. 2010.Bestdrug treatment for

multidrug-resistantandextensivelydrug-resistanttuberculosis.LancetInfect.Dis.10:621-

629.doi:10.1016/S1473-3099(10)70139-0;10.1016/S1473-3099(10)70139-0.

14. DeLorenzo,S.,J.W.Alffenaar,G.Sotgiu,R.Centis,L.D'Ambrosio,S.Tiberi,M.S.Bolhuis,

R.vanAltena,P.Viggiani,A.Piana,A.Spanevello,andG.B.Migliori.2012.Efficacyand

safetyofmeropenem/clavunateaddedtolinezolidcontainingregimensinthetreatment

ofM/XDR-TB.Eur.Respir.J.doi:10.1183/09031936.00124312.

15. Dauby, N., I. Muylle, F. Mouchet, R. Sergysels, and M. C. Payen. 2011.

Meropenem/clavulanate and linezolid treatment for extensively drug-resistant

tuberculosis. Pediatr. Infect. Dis. J. 30:812-813. doi: 10.1097/INF.0b013e3182154b05;

10.1097/INF.0b013e3182154b05.




10.5588/ijtld.11.0414.

17. Koal, T.,M. Deters, K. Resch, and V. Kaever. 2006. Quantification of the carbapenem

antibiotic ertapenem in human plasma by a validated liquid chromatography-mass

spectrometrymethod.Clin.Chim.Acta.364:239-245.doi:10.1016/j.cccn.2005.07.004.

18. Pickering,M.,andS.Brown.2013.QuantificationandvalidationofHPLC-UVandLC-MS

assays for therapeutic drug monitoring of ertapenem in human plasma. Biomed.

Chromatogr.27:568-574.doi:10.1002/bmc.2829;10.1002/bmc.2829.

19. Lowes,S.,J.Jersey,R.Shoup,F.Garofolo,N.Savoie,E.Mortz,S.Needham,M.C.Caturla,

R.Steffen,C.Sheldon,R.Hayes,T.Samuels,L.DiDonato,J.Kamerud,S.Michael,Z.J.Lin,

J.Hillier,M.Moussallie,L.deSouzaTeixeira,M.Rocci,M.Buonarati,J.Truog,S.Hussain,

R. Lundberg,A.Breau,T. Zhang, J. Jonker,N.Berger, S.Gagnon-Carignan,C.Nehls,R.

Nicholson,M.Hilhorst,S.Karnik,T.deBoer,R.Houghton,K.Smith,L.Cojocaru,M.Allen,

T. Harter, S. Fatmi, F. Sayyarpour, J. Vija, M. Malone, and D. Heller. 2011.

Recommendationson:internalstandardcriteria,stability,incurredsamplereanalysisand

recent 483s by the Global CRO Council for Bioanalysis. Bioanalysis. 3:1323-1332. doi:

10.4155/bio.11.135;10.4155/bio.11.135.

20. U.S.DepartmentofHealthandHumanServices,FoodandDrugAdministration.Guidance

for industry,bioanalyticalmethodvalidation.Rockville:CenterforDrugEvaluationand

Research,2001.

21. European Medicines Agency, Committee for Medicinal Products for Human Use.

Guideline on bioanalytical validation (EMEA/CHMP/EWP/192217/2009). London:

EuropeanMedicinesAgency,2011.

59

3


10.5588/ijtld.11.0414.

17. Koal, T.,M. Deters, K. Resch, and V. Kaever. 2006. Quantification of the carbapenem

antibiotic ertapenem in human plasma by a validated liquid chromatography-mass

spectrometrymethod.Clin.Chim.Acta.364:239-245.doi:10.1016/j.cccn.2005.07.004.

18. Pickering,M.,andS.Brown.2013.QuantificationandvalidationofHPLC-UVandLC-MS

assays for therapeutic drug monitoring of ertapenem in human plasma. Biomed.

Chromatogr.27:568-574.doi:10.1002/bmc.2829;10.1002/bmc.2829.

19. Lowes,S.,J.Jersey,R.Shoup,F.Garofolo,N.Savoie,E.Mortz,S.Needham,M.C.Caturla,

R.Steffen,C.Sheldon,R.Hayes,T.Samuels,L.DiDonato,J.Kamerud,S.Michael,Z.J.Lin,

J.Hillier,M.Moussallie,L.deSouzaTeixeira,M.Rocci,M.Buonarati,J.Truog,S.Hussain,

R. Lundberg,A.Breau,T. Zhang, J. Jonker,N.Berger, S.Gagnon-Carignan,C.Nehls,R.

Nicholson,M.Hilhorst,S.Karnik,T.deBoer,R.Houghton,K.Smith,L.Cojocaru,M.Allen,

T. Harter, S. Fatmi, F. Sayyarpour, J. Vija, M. Malone, and D. Heller. 2011.

Recommendationson:internalstandardcriteria,stability,incurredsamplereanalysisand

recent 483s by the Global CRO Council for Bioanalysis. Bioanalysis. 3:1323-1332. doi:

10.4155/bio.11.135;10.4155/bio.11.135.

20. U.S.DepartmentofHealthandHumanServices,FoodandDrugAdministration.Guidance

for industry,bioanalyticalmethodvalidation.Rockville:CenterforDrugEvaluationand

Research,2001.

21. European Medicines Agency, Committee for Medicinal Products for Human Use.

Guideline on bioanalytical validation (EMEA/CHMP/EWP/192217/2009). London:

EuropeanMedicinesAgency,2011.

CHAPTER 4

S.P. van Rijn*R. van Altena*O.W. Akkerman

D. van SoolingenT. van der LaanW.C.M de LangeJ.G.W. KosterinkT.S. van der WerfJ.W.C. Alffenaar

Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant

Tuberculosis

Eur Respir J. 2016 Apr; 47(4): 1229-34.PMID: 26743484


62

Chapter 4

AbstractTreatmentofmultidrugresistant(MDR)andextensivelydrugresistant(XDR)tuberculosis(TB)

isbecomingmorechallengingbecauseofincreasedlevelofdrugresistanceagainstsecond

line tuberculosis drugs. One promising group of antimicrobial drugs are carbapenems.

ErtapenemisanattractivecarbapenemforthetreatmentofMDRandXDR-TBbecause its

relativelonghalf-lifeenablesoncedailydosing.Aretrospectivestudywasperformedforall

MDR-TB suspected patients at the Tuberculosis Center Beatrixoord of UniversityMedical

Center Groningen (Haren, The Netherlands) who received ertapenem as part of their

treatmentregimenbetweenthefirstofDecember2010andthefirstofMarch2013.Safety

and pharmacokinetics were evaluated. Eighteen patients were treated with 1000 mg

ertapenemforameanof77days(range5-210).Sputumsmearandculturewereconverted

inallpatients.Drugexposurewasevaluated in12patients.ThemeanAUC0-24was544,9

(range 309 – 1130)mg*h/L. Themean Cmaxwas 127.5 (73.9 – 277.9)mg/L. In general,

ertapenemtreatmentwaswelltoleratedduringMDR-TBtreatmentandshowedafavourable

PK/PDprofileinMDR-TBpatients.Weconcludethatertapenemisahighlypromisingdrugfor

thetreatmentofMDR-TBthatwarrantsfurtherinvestigation.

IntroductionTreatmentofmultidrugresistant(MDR)andextensivelydrugresistant(XDR)tuberculosis(TB)


linetuberculosisdrugs.Newdrugsarebeingevaluatedinclinicaltrials,butonlybedaquiline

anddelamanidhaveenteredthemarkettodate.Therefore,antimicrobialdrugs,whichhave

beendeveloped and labeled for other bacterial infectionsmaybeof potential use in the

treatmentofMDR-TB.

One promising group of antimicrobial drugs are carbapenems [1, 2]. An early in vitro

experimentshowedthatimipenemandmeropenemwereactiveagainstM.tuberculosis[3].

Chambersandco-workersshowedthatimipenemhasanti-mycobacterialactivityinmiceand

humans [4]. Imipenem and meropenem are currently listed as group 5 drugs for the

treatment of MDR-TB [5]. More recently, clinical experience of carbapenems inMDR-TB

patientsshowedpromisingresults[6-7].

CarbapenemsarepoorsubstratesforbetalactamaseC(BLaC)duetorapidacylationandslow

deacylation. Therefore, unlike beta-lactams, they are not rapidly hydrolyzed by BLaC and

therefore maintain their potential activity against M. tuberculosis [8]. The binding of

carbapenems to the LD transpeptidases results in inhibition of the peptidoglycan

polymerization of the cell wall [9]. Combined with a beta lactamase inhibitor, such as

clavulanate,activityagainstM.tuberculosisishigher[10].

Efficacy of carbapenems is correlated with the percentage of time the free plasma drug

concentrationtranscendstheMIC(Tfree>MIC).Maximalbactericidalactivityisreachedifthe

time aboveMIC is at least 40% of dosing interval [11,12]. To reach this target for gram

positive,gramnegativeandanaerobicbacterialinfectionsertapenemisgivenintravenously

inadoseof1000mgoncedaily[13].Ertapenemhastheadvantageoverothercarbapenems

becauseofalonghalf-lifeof4henablingoncedailydosing[12],whichisattractiveforMDR-

TBtreatment.Anotheradvantageisthatertapenemisnotaffectedbydrug-druginteractions

asitisneithermetabolizedbycytochromeP450norasubstrateforP-glycoprotein[14].

Toincludeertapenemamongtheothercarbapenemsasagroup5drugforthetreatmentof

MDR-TBadditionalpharmacokineticandsafetydataareurgentlyneeded[15].Therefore,the

63

Pharmacokinetics of Ertapenem in Patients with Multidrug-Resistant Tuberculosis

4

AbstractTreatmentofmultidrugresistant(MDR)andextensivelydrugresistant(XDR)tuberculosis(TB)


line tuberculosis drugs. One promising group of antimicrobial drugs are carbapenems.

ErtapenemisanattractivecarbapenemforthetreatmentofMDRandXDR-TBbecause its

relativelonghalf-lifeenablesoncedailydosing.Aretrospectivestudywasperformedforall

MDR-TB suspected patients at the Tuberculosis Center Beatrixoord of UniversityMedical

Center Groningen (Haren, The Netherlands) who received ertapenem as part of their

treatmentregimenbetweenthefirstofDecember2010andthefirstofMarch2013.Safety

and pharmacokinetics were evaluated. Eighteen patients were treated with 1000 mg

ertapenemforameanof77days(range5-210).Sputumsmearandculturewereconverted

inallpatients.Drugexposurewasevaluated in12patients.ThemeanAUC0-24was544,9

(range 309 – 1130)mg*h/L. Themean Cmaxwas 127.5 (73.9 – 277.9)mg/L. In general,

ertapenemtreatmentwaswelltoleratedduringMDR-TBtreatmentandshowedafavourable

PK/PDprofileinMDR-TBpatients.Weconcludethatertapenemisahighlypromisingdrugfor

thetreatmentofMDR-TBthatwarrantsfurtherinvestigation.

IntroductionTreatmentofmultidrugresistant(MDR)andextensivelydrugresistant(XDR)tuberculosis(TB)


linetuberculosisdrugs.Newdrugsarebeingevaluatedinclinicaltrials,butonlybedaquiline

anddelamanidhaveenteredthemarkettodate.Therefore,antimicrobialdrugs,whichhave

beendeveloped and labeled for other bacterial infectionsmaybeof potential use in the

treatmentofMDR-TB.

One promising group of antimicrobial drugs are carbapenems [1, 2]. An early in vitro

experimentshowedthatimipenemandmeropenemwereactiveagainstM.tuberculosis[3].

Chambersandco-workersshowedthatimipenemhasanti-mycobacterialactivityinmiceand

humans [4]. Imipenem and meropenem are currently listed as group 5 drugs for the

treatment of MDR-TB [5]. More recently, clinical experience of carbapenems inMDR-TB

patientsshowedpromisingresults[6-7].

CarbapenemsarepoorsubstratesforbetalactamaseC(BLaC)duetorapidacylationandslow

deacylation. Therefore, unlike beta-lactams, they are not rapidly hydrolyzed by BLaC and

therefore maintain their potential activity against M. tuberculosis [8]. The binding of

carbapenems to the LD transpeptidases results in inhibition of the peptidoglycan

polymerization of the cell wall [9]. Combined with a beta lactamase inhibitor, such as

clavulanate,activityagainstM.tuberculosisishigher[10].

Efficacy of carbapenems is correlated with the percentage of time the free plasma drug

concentrationtranscendstheMIC(Tfree>MIC).Maximalbactericidalactivityisreachedifthe

time aboveMIC is at least 40% of dosing interval [11,12]. To reach this target for gram

positive,gramnegativeandanaerobicbacterialinfectionsertapenemisgivenintravenously

inadoseof1000mgoncedaily[13].Ertapenemhastheadvantageoverothercarbapenems

becauseofalonghalf-lifeof4henablingoncedailydosing[12],whichisattractiveforMDR-

TBtreatment.Anotheradvantageisthatertapenemisnotaffectedbydrug-druginteractions

asitisneithermetabolizedbycytochromeP450norasubstrateforP-glycoprotein[14].

Toincludeertapenemamongtheothercarbapenemsasagroup5drugforthetreatmentof

MDR-TBadditionalpharmacokineticandsafetydataareurgentlyneeded[15].Therefore,the

64

Chapter 4

objectiveofthisstudywastoevaluatepharmacokineticsandsafetyinpatientsthatreceived

ertapenemaspartoftheirtreatmentMDR-TBregimen.

Patientsandmethods

Patients

AllpatientssuspectedtoMDR-TBattheTuberculosisCenterBeatrixoordoftheUniversity

MedicalCenterGroningen(Haren,TheNetherlands)whoreceivedertapenemaspartoftheir

treatment regimen between first of December 2010 and the first of March 2013 were

includedinthisretrospectivestudy.ThestudywasevaluatedbytheMedicalEthicalReview

Boardof theUniversityMedicalCenterGroningen (metc2013-492). Theneed forwritten

informedconsentwaswaivedfortheretrospectivecollectionandanalysisofanonymousdata

becauseitwasnotrequiredunderDutchLaw(WMO).ForeachMDR-TBsuspect,age,gender,

weight, length, ethnicity, drug susceptibility pattern, localization of tuberculosis,

antiretroviraltherapy,sputumconversion,adverseeffectsinducedbyertapenem,dose,total

exposuretoertapenem,anddurationoftreatmentwerecollected.

DrugsusceptibilitytoErtapenem

Drugsusceptibilitytesting(DST)ofertapenemwasperformedwithandwithoutclavulanic

acidusingthemiddlebrook7H10agardilutionmethodattheDutchNationalTuberculosis

Reference Laboratory (National Institute for Public Health and the Environment RIVM),

Bilthoven,TheNetherlands)[16].

Pharmacokineticsandpharmacodynamics

All patients receivedertapenem in a dosageof 1000mgoncedaily, given as intravenous

infusionin30min.InallMDR-TBpatientsroutineplasmaconcentrationswerecollectedat

steadystatetoassessdrugexposuretoenableindividualizeddosing.Forplasmasamplinga

peripheral intravenous catheter was inserted. Patency of the peripheral catheter was

maintainedbyasalinedrip.Beforeabloodsamplewastaken,thedripwasstoppedandthe

first4mlsofbloodwerediscarded.Thesampleswerecollectedbeforeadministrationandat

t = 1, 2, 3, 4, 5, 6, 8, 12, hrs post-dosage and stored at –80 °C until analysis. Plasma

concentrationswereassessedandvalidatedusingavalidatedliquidchromatography-tandem

massspectrometry(LC-MS/MS)inthelaboratoryofClinicaltoxicologyandDrugsAnalysisof

the department of Clinical Pharmacy and Pharmacology at theUniversityMedical Center

Groningen[17].PopulationpharmacokineticparameterswerecalculatedusingtheKinPOP

module. Both KinFIT and KinPOP were part of the software package MWPharm 3.82

(Mediware,TheNetherlands).TheTfree>MICwascalculatedasthishasbeenproposedasthe

best pharmacokinetic/pharmacodynamic parameter to predict in vivo efficacy of

carbapenems[10].Freedrugconcentrationwasassumedtobe5%[12,18].Eucastminimal

inhibitoryconcentrationsforertapenem(non-speciesrelated)of0.5and1.0mg/Lwereused

tocalculateTfree>MIC.

Safetyandtolerability

Reported adverse effects (AE) in medical charts were used to evaluate the safety of

ertapenem.SpecificattentionwaspaidtoAE’smentionedinearlierstudies:i.e.diarrheaand

vomiting.TheNaranjoalgorithmwasusedtoevaluateforcausalitybetweenadverseeffects

thatoccurredandertapenem[19].

Statisticsandpharmacokineticevaluation

SPSS 20 was used as statistical software (SPSS, Virgina, IL). Correlation between

pharmacokineticparametersandpatientcharacteristicswereanalyzedusingtheSpearman

correlationcoefficient.MICdatawerestatisticalanalyzedusingamethodologyforcensored

MICdata[20].

Results

Patients

Eighteenpatientstreatedwithertapenem,meanage29(range13-66years),wereretrieved.

Ertapenem was part of the treatment regimen because of suspected extensive drug

resistance,intolerancetosecondlinedrugsorcombinationofboth.Basedontheresultsof

thedrugsusceptibilitytestertapenemwasdiscontinuedinthreepatientswhoappearedto

havedrugsusceptibleTB.Genderwasunequallydistributedbetweenpatientsas8weremale

(44.4%)and10patientswerefemale(55.5%).Themeanbodymassindexwas21.3(range

65


4

objectiveofthisstudywastoevaluatepharmacokineticsandsafetyinpatientsthatreceived

ertapenemaspartoftheirtreatmentMDR-TBregimen.

Patientsandmethods

Patients

AllpatientssuspectedtoMDR-TBattheTuberculosisCenterBeatrixoordoftheUniversity

MedicalCenterGroningen(Haren,TheNetherlands)whoreceivedertapenemaspartoftheir

treatment regimen between first of December 2010 and the first of March 2013 were

includedinthisretrospectivestudy.ThestudywasevaluatedbytheMedicalEthicalReview

Boardof theUniversityMedicalCenterGroningen (metc2013-492). Theneed forwritten

informedconsentwaswaivedfortheretrospectivecollectionandanalysisofanonymousdata

becauseitwasnotrequiredunderDutchLaw(WMO).ForeachMDR-TBsuspect,age,gender,

weight, length, ethnicity, drug susceptibility pattern, localization of tuberculosis,

antiretroviraltherapy,sputumconversion,adverseeffectsinducedbyertapenem,dose,total

exposuretoertapenem,anddurationoftreatmentwerecollected.

DrugsusceptibilitytoErtapenem

Drugsusceptibilitytesting(DST)ofertapenemwasperformedwithandwithoutclavulanic

acidusingthemiddlebrook7H10agardilutionmethodattheDutchNationalTuberculosis

Reference Laboratory (National Institute for Public Health and the Environment RIVM),

Bilthoven,TheNetherlands)[16].


All patients receivedertapenem in a dosageof 1000mgoncedaily, given as intravenous

infusionin30min.InallMDR-TBpatientsroutineplasmaconcentrationswerecollectedat

steadystatetoassessdrugexposuretoenableindividualizeddosing.Forplasmasamplinga

peripheral intravenous catheter was inserted. Patency of the peripheral catheter was

maintainedbyasalinedrip.Beforeabloodsamplewastaken,thedripwasstoppedandthe

first4mlsofbloodwerediscarded.Thesampleswerecollectedbeforeadministrationandat

t = 1, 2, 3, 4, 5, 6, 8, 12, hrs post-dosage and stored at –80 °C until analysis. Plasma

concentrationswereassessedandvalidatedusingavalidatedliquidchromatography-tandem

massspectrometry(LC-MS/MS)inthelaboratoryofClinicaltoxicologyandDrugsAnalysisof

the department of Clinical Pharmacy and Pharmacology at theUniversityMedical Center

Groningen[17].PopulationpharmacokineticparameterswerecalculatedusingtheKinPOP

module. Both KinFIT and KinPOP were part of the software package MWPharm 3.82

(Mediware,TheNetherlands).TheTfree>MICwascalculatedasthishasbeenproposedasthe

best pharmacokinetic/pharmacodynamic parameter to predict in vivo efficacy of

carbapenems[10].Freedrugconcentrationwasassumedtobe5%[12,18].Eucastminimal

inhibitoryconcentrationsforertapenem(non-speciesrelated)of0.5and1.0mg/Lwereused

tocalculateTfree>MIC.


Reported adverse effects (AE) in medical charts were used to evaluate the safety of

ertapenem.SpecificattentionwaspaidtoAE’smentionedinearlierstudies:i.e.diarrheaand

vomiting.TheNaranjoalgorithmwasusedtoevaluateforcausalitybetweenadverseeffects

thatoccurredandertapenem[19].

Statisticsandpharmacokineticevaluation

SPSS 20 was used as statistical software (SPSS, Virgina, IL). Correlation between

pharmacokineticparametersandpatientcharacteristicswereanalyzedusingtheSpearman

correlationcoefficient.MICdatawerestatisticalanalyzedusingamethodologyforcensored

MICdata[20].

Results

Patients

Eighteenpatientstreatedwithertapenem,meanage29(range13-66years),wereretrieved.

Ertapenem was part of the treatment regimen because of suspected extensive drug

resistance,intolerancetosecondlinedrugsorcombinationofboth.Basedontheresultsof

thedrugsusceptibilitytestertapenemwasdiscontinuedinthreepatientswhoappearedto

havedrugsusceptibleTB.Genderwasunequallydistributedbetweenpatientsas8weremale

(44.4%)and10patientswerefemale(55.5%).Themeanbodymassindexwas21.3(range

66

Chapter 4

13.7-32.6)kg/m2.PatientsoriginatedpredominantlyfromAfrica(11/18)andEurope(5/18).

PatientswereprimarilydiagnosedwithpulmonaryTB(13/18),extrapulmonarysiteswere

involvedin7patients.

Prescribeddosageofertapenemwas1000mgoncedailyinallpatients.Meantotaltreatment

duration of ertapenem was 77 days (range 5-210 days). Drug resistance pattern of the

patientstoanti-tuberculosisagentsareshownintable1.Mostprescribedanti-TBdrugswere:

moxifloxacin(17/18),injectable(16/18),linezolid(15/18),clofazimine(8/18),clarithromycin

(6/18)andpyrazinamide(5/18).

Intotal15patientscompletedtreatmentandwerecured.Threepatientswerelosttofollow

up.Allpatientswithpositivesputum-smearconvertedwithinameanperiodof17days(range

0-97days).CulturesremainednegativeaftercultureconversionandnorelapseofMDR-TB

wasobserved.

Table1.Resistancepattern

WHO ResistantN(%)

SensitiveN(%)

Group1 First-lineoraldrugs Isoniazid 17(94,4) 1(5,55) Rifampicin 15(83,3) 3(16,6) Pyrazinamide 8(44,4) 8(44,4) Ethambutol 11(61,1) 6(33,3) Rifabutin 13(72,2) 4(22,2)Group2 Injectableagents streptomycin 14(77,7) 4(22,2) Amikacin 3(16,6) 14(77,7) kanamycin 3(16,6) 14(77,7)

capreomycin 5(27,7) 12(66,6)

Group3 Fluorquinolones Moxifloxacin 2(11,1) 15(83,3)

Group4 OralbacteriostaticSecond-lineagents protionamide 7(38,8) 10(55,5)Group5 Agentswithunclear Linezolid 17(94,4) Roleintreatmentof Clarithromycin 3(16,6) 7(38,8) drugresistant-TB Clofamizine 8(44,4)Other Ertapenem 18(100)

DataarepresentedasN(%),N=18

DrugsusceptibilityofM.tuberculosistoertapenem

All the M. tuberculosis strains appeared susceptible to ertapenem. However, actual

determinationof theMICwascomplicatedby the fact thatertapenem itself appearedan

instablecompoundat37°C[17].Thiswasconfirmedbythefactthatafter7daysMICvalues

were lower than after 14 days. In addition, freshly prepared plates showed lowerMIC’s

comparedtoplatesstoredat4°C.Therefrigerator-storedplatesshowedlowerMIC’sthan

platesstoredatroomtemperature.IfertapenemwascombinedwithclavulanicacidallMIC’s

wereevenlower.

Figure1.Ertapenemplasmaconcentration-timecurvesin12patients


Theplasma concentration time curveswere obtained in 12patientswithMDR-TB. In the

remaining6patientsroutineplasmaconcentrationswerecollectedatatimepointatwhich

theydidnotyetreceiveertapenemorthisdrugwasnolongeradministered.Threepatients

hadmultipleplasmaconcentrationtimecurvesandthesewereconsistent.Themeancurve

isshowninfigure1.ThemeanAUC0-24was544,9(range309–1130)mg*h/L.Thesteady

statepharmacokineticparametersareshownintable2.

67


4

13.7-32.6)kg/m2.PatientsoriginatedpredominantlyfromAfrica(11/18)andEurope(5/18).

PatientswereprimarilydiagnosedwithpulmonaryTB(13/18),extrapulmonarysiteswere

involvedin7patients.

Prescribeddosageofertapenemwas1000mgoncedailyinallpatients.Meantotaltreatment

duration of ertapenem was 77 days (range 5-210 days). Drug resistance pattern of the

patientstoanti-tuberculosisagentsareshownintable1.Mostprescribedanti-TBdrugswere:

moxifloxacin(17/18),injectable(16/18),linezolid(15/18),clofazimine(8/18),clarithromycin

(6/18)andpyrazinamide(5/18).

Intotal15patientscompletedtreatmentandwerecured.Threepatientswerelosttofollow

up.Allpatientswithpositivesputum-smearconvertedwithinameanperiodof17days(range

0-97days).CulturesremainednegativeaftercultureconversionandnorelapseofMDR-TB

wasobserved.

Table1.Resistancepattern

WHO ResistantN(%)

SensitiveN(%)

Group1 First-lineoraldrugs Isoniazid 17(94,4) 1(5,55) Rifampicin 15(83,3) 3(16,6) Pyrazinamide 8(44,4) 8(44,4) Ethambutol 11(61,1) 6(33,3) Rifabutin 13(72,2) 4(22,2)Group2 Injectableagents streptomycin 14(77,7) 4(22,2) Amikacin 3(16,6) 14(77,7) kanamycin 3(16,6) 14(77,7)

capreomycin 5(27,7) 12(66,6)

Group3 Fluorquinolones Moxifloxacin 2(11,1) 15(83,3)

Group4 OralbacteriostaticSecond-lineagents protionamide 7(38,8) 10(55,5)Group5 Agentswithunclear Linezolid 17(94,4) Roleintreatmentof Clarithromycin 3(16,6) 7(38,8) drugresistant-TB Clofamizine 8(44,4)Other Ertapenem 18(100)

DataarepresentedasN(%),N=18

DrugsusceptibilityofM.tuberculosistoertapenem

All the M. tuberculosis strains appeared susceptible to ertapenem. However, actual

determinationof theMICwascomplicatedby the fact thatertapenem itself appearedan

instablecompoundat37°C[17].Thiswasconfirmedbythefactthatafter7daysMICvalues

were lower than after 14 days. In addition, freshly prepared plates showed lowerMIC’s

comparedtoplatesstoredat4°C.Therefrigerator-storedplatesshowedlowerMIC’sthan

platesstoredatroomtemperature.IfertapenemwascombinedwithclavulanicacidallMIC’s

wereevenlower.

Figure1.Ertapenemplasmaconcentration-timecurvesin12patients


Theplasma concentration time curveswere obtained in 12patientswithMDR-TB. In the

remaining6patientsroutineplasmaconcentrationswerecollectedatatimepointatwhich

theydidnotyetreceiveertapenemorthisdrugwasnolongeradministered.Threepatients

hadmultipleplasmaconcentrationtimecurvesandthesewereconsistent.Themeancurve

isshowninfigure1.ThemeanAUC0-24was544,9(range309–1130)mg*h/L.Thesteady

statepharmacokineticparametersareshownintable2.

68

Chapter 4

BasedonaMICof0.25mg/L,11outof12patientsexceededaminimumof40%-timeabove

MIC. In9patients theMDR-TBremainedsusceptiblewithaMICof0.5mg/L.Except for2

patients, none exceeded a minimum of 40%-time interval with a MIC of 1 mg/L. The

pharmacokineticpopulationmodel(KinPOP)ofertapenemshowedaclearanceof2.26(range

0.86-3,19)L/h/1.73m2andavolumeofdistributionof8.79(range4.76-13,57)L.

Table2.pharmacokineticparametersofErtapenem

StudyAUC0-24(h*mg/L) Cmax(mg/L) T1/2(h) Vd(L) CL(L/h)

1gIVinMDR-TBpatients

544.9(309–1130)

127.5(73.9–277.9)

2.4(2.047–3.528)

7.3(2.612–11.1)

2.1(0.0884–3.231)

1gIVinhealthyvolunteers[16]

572.1(572–672)

154.9(145–175)

4(3.8-4.4) 8.2 1.8

Dataarepresentedasmean(range).AUC0-24:Areaunderthecurve-timecurveupto24h.Cmax:highestobservedplasmaconcentration.Cl:clearance.T1/2:half-life.Vd:volumeofdistribution.


Ingeneral,ertapenemwastoleratedverywell.Inthreepatients,treatmentwithertapenem

wasstopped.OneofthesepatientssufferedfromCrohn’sdiseaseanddevelopedMDR-TB

aftermultipledosagesof infliximab,aTNFalpha-blocker [21-22].Thispatientexperienced

allergicfever,shortlyafteradministrationofertapenemandethambutol.Afterreintroduction

thishappenedagain(Naranjoscore=4).Bothadverseeventssubsidedafterwithdrawalof

theoffendingdrug.Inthesecondpatientertapenemwasstoppedafteranincreaseinliver

enzymes(ASAT:109/ALAT:255)after13daysoftreatmentwithertapenem.However,after

twomonths, while this patient was still on treatmentwithout ertapenem, liver enzymes

remainedelevated(Naranjoscore=1).Inonepatientkanamycin,linezolidandertapenem

werestoppedduetolinesepsis.Thiswasconsiderednot-to-berelatedtoertapenem.After

removal of the venous access port, the patient recovered. Ertapenem and a new venous

accessportwerenotreintroduced,sinceitwasnotindicatedanymore,duetolowbacillary

loadatthattimeandtheseIVdrugscouldbesubstitutedwithoralantimycobacterialdrugs.

Noneofthepatientsexperienceddiarrhea,vomitingordizziness.

DiscussionThisisthefirststudy,followingaclinicalreportofertapenem[6],presentingpharmacokinetic

andsafetydata inpatientswithMDR-TB. Incomparisonwithhealthyvolunteers,MDR-TB

patientsshowedlowerAUC0-24values.Meanvaluesofvolumeofdistributionandclearance

of MDR-TB patients were higher compared to healthy volunteers. Our observation is

consistentwithotherstudiesthatshowedalowerdrugexposureofertapeneminpatients

with infectiousdiseases [23-25].Moresurprisingwas the inter-variability inAUCbetween

patientswithMDR-TB.Otherantimycobacterialdrugsalsoshowhighlyvariabilityandlower

drug exposure in TB patients as well [2, 26-27]. It is not yet completely clear why drug

exposureislowerinTBpatients.Apparently,stageofdiseaseandalteredbodycomposition

maypotentiallyhelptoexplainthisobservation.

Since ertapenem belongs to the class of beta-lactams, ertapenem has a time-dependent

bactericidal activity. The Tfree>MIC is therefore important to evaluate the efficacy of

ertapenemagainstM.tuberculosis.Nicolauandcolleaguesindicatedthatincasemeropenem

showed40%T>MICbactericidalactivityisobservedwhereas20%T>MICappearedtohave

bacteriostaticactivity.OtherstudieshavementionedthisT>MICaswell[11-12,14].Protein

binding of ertapenem was assumed to be 5%, since ertapenem shows concentration-

dependent plasma protein binding. Healthy volunteers, whom average a peak plasma

concentrationof150mg/Laftertheendofinfusionof1gofertapenem,haveapercentage

of8%unboundprotein.Whentotaldrugplasmaconcentrationdeclinesbelow50mg/Lpeak

plasma concentration, the percentage of unbound ertapenem is circa 5% [18]. It is very

promisingtonoticethatthenon-speciesrelatedbreakpointofertapenemof0.5mg/Lwas

exceededformorethan40%ofthedayinthemajorityofpatientsassumingthatpatients

haveaproteinbindingof5%.AtahigherMICvalueof1mg/Lbacteriostaticactivitycouldbe

expected.Therefore,ertapenemseemsaveryattractivedrugforMDR-TBtreatment.Itseems

warrantedthatdosesofertapenemhigherthan1g/dayshouldbeusedinthetreatmentof

MDR-TB.However,invitroexperimentsevaluatingPK/PDtargetsforertapenemagainstM.

tuberculosishaveyettobeperformed.ThehollowfiberinfectionmodelissuitableforPK/PD

experimentsandhasalreadybeenusedsuccessfullybefore[28].

69


4

BasedonaMICof0.25mg/L,11outof12patientsexceededaminimumof40%-timeabove

MIC. In9patients theMDR-TBremainedsusceptiblewithaMICof0.5mg/L.Except for2

patients, none exceeded a minimum of 40%-time interval with a MIC of 1 mg/L. The

pharmacokineticpopulationmodel(KinPOP)ofertapenemshowedaclearanceof2.26(range

0.86-3,19)L/h/1.73m2andavolumeofdistributionof8.79(range4.76-13,57)L.

Table2.pharmacokineticparametersofErtapenem

StudyAUC0-24(h*mg/L) Cmax(mg/L) T1/2(h) Vd(L) CL(L/h)

1gIVinMDR-TBpatients

544.9(309–1130)

127.5(73.9–277.9)

2.4(2.047–3.528)

7.3(2.612–11.1)

2.1(0.0884–3.231)

1gIVinhealthyvolunteers[16]

572.1(572–672)

154.9(145–175)

4(3.8-4.4) 8.2 1.8

Dataarepresentedasmean(range).AUC0-24:Areaunderthecurve-timecurveupto24h.Cmax:highestobservedplasmaconcentration.Cl:clearance.T1/2:half-life.Vd:volumeofdistribution.


Ingeneral,ertapenemwastoleratedverywell.Inthreepatients,treatmentwithertapenem

wasstopped.OneofthesepatientssufferedfromCrohn’sdiseaseanddevelopedMDR-TB

aftermultipledosagesof infliximab,aTNFalpha-blocker [21-22].Thispatientexperienced

allergicfever,shortlyafteradministrationofertapenemandethambutol.Afterreintroduction

thishappenedagain(Naranjoscore=4).Bothadverseeventssubsidedafterwithdrawalof

theoffendingdrug.Inthesecondpatientertapenemwasstoppedafteranincreaseinliver

enzymes(ASAT:109/ALAT:255)after13daysoftreatmentwithertapenem.However,after

twomonths, while this patient was still on treatmentwithout ertapenem, liver enzymes

remainedelevated(Naranjoscore=1).Inonepatientkanamycin,linezolidandertapenem

werestoppedduetolinesepsis.Thiswasconsiderednot-to-berelatedtoertapenem.After

removal of the venous access port, the patient recovered. Ertapenem and a new venous

accessportwerenotreintroduced,sinceitwasnotindicatedanymore,duetolowbacillary

loadatthattimeandtheseIVdrugscouldbesubstitutedwithoralantimycobacterialdrugs.

Noneofthepatientsexperienceddiarrhea,vomitingordizziness.

DiscussionThisisthefirststudy,followingaclinicalreportofertapenem[6],presentingpharmacokinetic

andsafetydata inpatientswithMDR-TB. Incomparisonwithhealthyvolunteers,MDR-TB

patientsshowedlowerAUC0-24values.Meanvaluesofvolumeofdistributionandclearance

of MDR-TB patients were higher compared to healthy volunteers. Our observation is

consistentwithotherstudiesthatshowedalowerdrugexposureofertapeneminpatients

with infectiousdiseases [23-25].Moresurprisingwas the inter-variability inAUCbetween

patientswithMDR-TB.Otherantimycobacterialdrugsalsoshowhighlyvariabilityandlower

drug exposure in TB patients as well [2, 26-27]. It is not yet completely clear why drug

exposureislowerinTBpatients.Apparently,stageofdiseaseandalteredbodycomposition

maypotentiallyhelptoexplainthisobservation.

Since ertapenem belongs to the class of beta-lactams, ertapenem has a time-dependent

bactericidal activity. The Tfree>MIC is therefore important to evaluate the efficacy of

ertapenemagainstM.tuberculosis.Nicolauandcolleaguesindicatedthatincasemeropenem

showed40%T>MICbactericidalactivityisobservedwhereas20%T>MICappearedtohave

bacteriostaticactivity.OtherstudieshavementionedthisT>MICaswell[11-12,14].Protein

binding of ertapenem was assumed to be 5%, since ertapenem shows concentration-

dependent plasma protein binding. Healthy volunteers, whom average a peak plasma

concentrationof150mg/Laftertheendofinfusionof1gofertapenem,haveapercentage

of8%unboundprotein.Whentotaldrugplasmaconcentrationdeclinesbelow50mg/Lpeak

plasma concentration, the percentage of unbound ertapenem is circa 5% [18]. It is very

promisingtonoticethatthenon-speciesrelatedbreakpointofertapenemof0.5mg/Lwas

exceededformorethan40%ofthedayinthemajorityofpatientsassumingthatpatients

haveaproteinbindingof5%.AtahigherMICvalueof1mg/Lbacteriostaticactivitycouldbe

expected.Therefore,ertapenemseemsaveryattractivedrugforMDR-TBtreatment.Itseems

warrantedthatdosesofertapenemhigherthan1g/dayshouldbeusedinthetreatmentof

MDR-TB.However,invitroexperimentsevaluatingPK/PDtargetsforertapenemagainstM.

tuberculosishaveyettobeperformed.ThehollowfiberinfectionmodelissuitableforPK/PD

experimentsandhasalreadybeenusedsuccessfullybefore[28].

70

Chapter 4

BesidespharmacokineticsofertapeneminpatientswithMDR-TB,additionalsafetydataare

describedforthefirsttimeaswell.Onlyonepatient,withCrohn’sdisease,experiencedAE,

whichmightbepotentiallyrelatedtoertapenem.Onecanspeculatethismayberelatedto

aninfusionrelatedAE,duetoadevelopedimmunedisorderandeventuallyaconsequence

ofaninfliximabtreatment.DruginducedfeverisacommonAEofinfliximabinthetreatment

of Crohn’s Disease [29]. AE’s of other carbapenems, such as diarrhea, nausea, vomiting,

headache and rash arewell documented and found to bemild [1, 30]. According to the

product leaflet, ertapenem is given for a maximum of two consecutive weeks. Previous

studies explored the safety and tolerability of ertapenem for this period of time and

concludedthatadversesideeffectsweremildtomoderate[13].Inourstudyweshowedthat

AEdidnotincreaseduringprolongedtreatment.

ThemeasurementofactualMICvalueswascomplicatedby the fact thatertapenem isan

instablecompoundat37°C.AsdrugsusceptibilitytestingforM.tuberculosis takesat least

twoweeksat37°C,itishighlylikelythattheinitialdrugconcentrationdecreasesrapidlyin

time.Unfortunately,with the current drug susceptibility systems, e.g.MGITor plate, this

problemcannotbeovercome,asthedrugconcentrationinthemediumcannotbecorrected

foradecrease inconcentrationdue todegradationof thedrug.Recently thehollow fiber

infectionmodelsolvedthisproblemasdrugconcentrationscanbeincreasedtocorrectfor

degradation[31].Assystemsareexpensiveanddifficulttomanageitsnotlikelythatroutine

DSTwillbeperformedusinghollowfibersystems.AnotheralternativemaybetheuseofE-

tests[32].Thisismuchcheaperandeasiertoemploybutitisunclearifitcanhelptoovercome

theinstabilityofertapenem.

Themost important limitation of our study is the retrospective character and its limited

samplesize,andabsenceofcontrolgroup,therebypreventingameaningfulconclusionon

efficacyofertapenem.SecondlytheinabilitytodefineMICforertapeneminclinicalisolates

isanotherlimitation.Nevertheless,allpatientswerecuredandnorelapsewasnoticedafter

being treatedwith combination regimen including ertapenem. Likely the combination of

drugscontributedtosputumcultureconversionandfavorabletreatmentoutcome.Thisisin

line with recently published data on tolerability and outcomes in 5 patients receiving

ertapenem[6].

A recent editorial proposed a new classification of anti-tuberculosis drugs. Itmarked the

potentialofcarbapenemsasgroup5drugs,howevercarbapenemsarestillinneedofproper

evaluationandclinicalevidence[33].Beforeertapenemcanbelabeledasagroup5drugand

usedaspartofMDR-TB treatment,avalidprocedure to testdrugsusceptibilityhas tobe

madeavailable.Ideally,theuseofertapenemwouldbesupportedbytheresultsofaclinical

trial.

Inconclusionthisstudyprovidesnewknowledgeontheuseofertapeneminpatientswith

MDR-TB,presentingpharmacokineticandadditionalsafetydata.

71


4

BesidespharmacokineticsofertapeneminpatientswithMDR-TB,additionalsafetydataare

describedforthefirsttimeaswell.Onlyonepatient,withCrohn’sdisease,experiencedAE,

whichmightbepotentiallyrelatedtoertapenem.Onecanspeculatethismayberelatedto

aninfusionrelatedAE,duetoadevelopedimmunedisorderandeventuallyaconsequence

ofaninfliximabtreatment.DruginducedfeverisacommonAEofinfliximabinthetreatment

of Crohn’s Disease [29]. AE’s of other carbapenems, such as diarrhea, nausea, vomiting,

headache and rash arewell documented and found to bemild [1, 30]. According to the

product leaflet, ertapenem is given for a maximum of two consecutive weeks. Previous

studies explored the safety and tolerability of ertapenem for this period of time and

concludedthatadversesideeffectsweremildtomoderate[13].Inourstudyweshowedthat

AEdidnotincreaseduringprolongedtreatment.

ThemeasurementofactualMICvalueswascomplicatedby the fact thatertapenem isan

instablecompoundat37°C.AsdrugsusceptibilitytestingforM.tuberculosis takesat least

twoweeksat37°C,itishighlylikelythattheinitialdrugconcentrationdecreasesrapidlyin

time.Unfortunately,with the current drug susceptibility systems, e.g.MGITor plate, this

problemcannotbeovercome,asthedrugconcentrationinthemediumcannotbecorrected

foradecrease inconcentrationdue todegradationof thedrug.Recently thehollow fiber

infectionmodelsolvedthisproblemasdrugconcentrationscanbeincreasedtocorrectfor

degradation[31].Assystemsareexpensiveanddifficulttomanageitsnotlikelythatroutine

DSTwillbeperformedusinghollowfibersystems.AnotheralternativemaybetheuseofE-

tests[32].Thisismuchcheaperandeasiertoemploybutitisunclearifitcanhelptoovercome

theinstabilityofertapenem.

Themost important limitation of our study is the retrospective character and its limited

samplesize,andabsenceofcontrolgroup,therebypreventingameaningfulconclusionon

efficacyofertapenem.SecondlytheinabilitytodefineMICforertapeneminclinicalisolates

isanotherlimitation.Nevertheless,allpatientswerecuredandnorelapsewasnoticedafter

being treatedwith combination regimen including ertapenem. Likely the combination of

drugscontributedtosputumcultureconversionandfavorabletreatmentoutcome.Thisisin

line with recently published data on tolerability and outcomes in 5 patients receiving

ertapenem[6].

A recent editorial proposed a new classification of anti-tuberculosis drugs. Itmarked the

potentialofcarbapenemsasgroup5drugs,howevercarbapenemsarestillinneedofproper

evaluationandclinicalevidence[33].Beforeertapenemcanbelabeledasagroup5drugand

usedaspartofMDR-TB treatment,avalidprocedure to testdrugsusceptibilityhas tobe

madeavailable.Ideally,theuseofertapenemwouldbesupportedbytheresultsofaclinical

trial.

Inconclusionthisstudyprovidesnewknowledgeontheuseofertapeneminpatientswith

MDR-TB,presentingpharmacokineticandadditionalsafetydata.

72

Chapter 4

References

1. De Lorenzo S, Alffenaar JW, SotgiuG,et al. Efficacy and safety ofmeropenem/clavunate

addedtolinezolidcontainingregimensinthetreatmentofM/XDR-TB.EurRespirJ2012Sep

20.

2. PrangerAD,vanAltenaR,AarnoutseRE,etal.Evaluationofmoxifloxacinforthetreatment

oftuberculosis:3yearsofexperience.EurrespirJ2011;38(4):888-94.

3. WattB,EdwardsJR,RaynerA,etal. Invitroactivityofmeropenemandimipenemagainst

mycobacteria:Developmentofadailyantibioticdosingschedule.Tubercleandlungdisease.

1992;73(3):134-6.

4. ChambersHF,TurnerJ,SchecterGF,etal.Imipenemfortreatmentoftuberculosisinmiceand

humans.AntimicrobAgentsChemother2005;49(7):2816-21.

5. Caminero, J. A., G. Sotgiu, A. Zumla, and G. B. Migliori. 2010. Best drug treatment for

multidrug-resistant andextensively drug-resistant tuberculosis. Lancet Infect.Dis. 10:621-

629.doi:10.1016/S1473-3099(10)70139-0;10.1016/S1473-3099(10)70139-0.5.

6. Tiberi S,D’Ambrosio L,De Lorenzo S, et al. Ertapenem in the treatmentofMDR-TB: first

clinicalexperience.EurRespirJ2015inpress.

7. WintersN,Butler-LaporteG,MenziesD.EfficacyandsafetyofWorldHealthOrganization

group5drugsformultidrug-resistanttuberculosis treatment.EurRespir J.2015sept17. [

Epubadheadofprint]

8. Hugonnet J, Blanchard JS. Irreversible inhibition of the mycobacterium tuberculosis β-

lactamasebyclavulanate.Biochemistry2007;46(43):11998-2004.

9. CordillotM,DubeeV,TribouletS,etal.Invitrocross-linkingofMycobacteriumtuberculosis

peptidoglycanbyL,D-transpeptidasesand inactivationof theseenzymesbycarbapenems.


10. VezirisN,TruffotC,MainardiJL,etal.Activityofcarbapenemscombinedwithclavulanate

againstmurinetuberculosis.AntimicrobAgentsChemother2011Jun;55(6):2597-600.

11. NicolauDP.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.Clin Infect

Dis.2008Sep15;47Suppl1:S32-40.

12. ZhanelGG,WiebeR,Dilay L,etal. Comparative reviewof the carbapenems.Drugs 2007;

67(7):1027-52.

13. TepplerH,GesserRM,FriedlandIR,etal.SafetyandtolerabilityofErtapenem.JAntimicrob

Chemother2004Jun;53Suppl2:ii75-81.

14. NixDE,MajumdarAK,DiNubileMJ.PharmacokineticsandpharmacodynamicsofErtapenem:

Anoverviewforclinicians.JAntimicrobChemother.2004Jun;53Suppl2:ii23-8.

15. DooleyKE,ObukuEA,DurakovicN,etal.WorldHealthOrganizationgroup5drugsforthe

treatmentofdrug-resistanttuberculosis:unclearefficacyoruntappedpotential?JInfectDis.

2013May1:207(9):1352-8

16. Van Klingeren B, Dessens-Kroon M, van der Laan T, et al. Drug susceptibility testing of

mycobacterium tuberculosis complex by use of a high-throughput, reproducible, absolute

concentrationmethod.JClinMicrobiol.2007;45:2662-8

17. vanRijnSP,WesselsAM,GreijdanusB,etal.QuantificationandvalidationofErtapenemusing

aliquidchromatography-tandemmassspectrometrymethod.AntimicrobAgentsChemother.

2014Jun:58(6):3481-4.

18. MajumdarAK,MussonDG,BirkKL,etal.PharmacokineticsofErtapeneminhealthyyoung

volunteers.AntimicrobAgentsChemother2002Nov;46(11):3506-11.

19. NaranjoCA,BustoU,SellersEM,etal.Amethodforestimatingtheprobabilityofadverse

drugreactions.ClinPharmacolTher1981Aug;30(2):239-45.

20. VandeKassteeleJ,vanSanten-VerheuvelMG,KoedijkFD,etal.Newstatisticaltechniquefor

analyzingMIC-based susceptibility data. Antimicrob Agents Chemother. 2012Mar; 56(3):

1557-63

21. CantiniF,NiccoliL,GolettiD.Adalimub,etanercept,infliximab,andtheriskoftuberculosis:

data from clinical trials, national registries, and postmarketing surveillance. J Rheumatol

Suppl.2014May;91:47-55

73


4

References

1. De Lorenzo S, Alffenaar JW, SotgiuG,et al. Efficacy and safety ofmeropenem/clavunate

addedtolinezolidcontainingregimensinthetreatmentofM/XDR-TB.EurRespirJ2012Sep

20.

2. PrangerAD,vanAltenaR,AarnoutseRE,etal.Evaluationofmoxifloxacinforthetreatment

oftuberculosis:3yearsofexperience.EurrespirJ2011;38(4):888-94.

3. WattB,EdwardsJR,RaynerA,etal. Invitroactivityofmeropenemandimipenemagainst

mycobacteria:Developmentofadailyantibioticdosingschedule.Tubercleandlungdisease.

1992;73(3):134-6.

4. ChambersHF,TurnerJ,SchecterGF,etal.Imipenemfortreatmentoftuberculosisinmiceand

humans.AntimicrobAgentsChemother2005;49(7):2816-21.

5. Caminero, J. A., G. Sotgiu, A. Zumla, and G. B. Migliori. 2010. Best drug treatment for

multidrug-resistant andextensively drug-resistant tuberculosis. Lancet Infect.Dis. 10:621-

629.doi:10.1016/S1473-3099(10)70139-0;10.1016/S1473-3099(10)70139-0.5.

6. Tiberi S,D’Ambrosio L,De Lorenzo S, et al. Ertapenem in the treatment ofMDR-TB: first

clinicalexperience.EurRespirJ2015inpress.

7. WintersN,Butler-LaporteG,MenziesD.EfficacyandsafetyofWorldHealthOrganization

group5drugsformultidrug-resistanttuberculosistreatment.EurRespir J.2015sept17. [

Epubadheadofprint]

8. Hugonnet J, Blanchard JS. Irreversible inhibition of the mycobacterium tuberculosis β-

lactamasebyclavulanate.Biochemistry2007;46(43):11998-2004.

9. CordillotM,DubeeV,TribouletS,etal.Invitrocross-linkingofMycobacteriumtuberculosis

peptidoglycanbyL,D-transpeptidasesand inactivationof theseenzymesbycarbapenems.


10. VezirisN,TruffotC,MainardiJL,etal.Activityofcarbapenemscombinedwithclavulanate

againstmurinetuberculosis.AntimicrobAgentsChemother2011Jun;55(6):2597-600.

11. NicolauDP.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.Clin Infect

Dis.2008Sep15;47Suppl1:S32-40.

12. ZhanelGG,WiebeR,Dilay L,etal. Comparative reviewof the carbapenems.Drugs 2007;

67(7):1027-52.

13. TepplerH,GesserRM,FriedlandIR,etal.SafetyandtolerabilityofErtapenem.JAntimicrob

Chemother2004Jun;53Suppl2:ii75-81.

14. NixDE,MajumdarAK,DiNubileMJ.PharmacokineticsandpharmacodynamicsofErtapenem:

Anoverviewforclinicians.JAntimicrobChemother.2004Jun;53Suppl2:ii23-8.

15. DooleyKE,ObukuEA,DurakovicN,etal.WorldHealthOrganizationgroup5drugsforthe

treatmentofdrug-resistanttuberculosis:unclearefficacyoruntappedpotential?JInfectDis.

2013May1:207(9):1352-8

16. Van Klingeren B, Dessens-Kroon M, van der Laan T, et al. Drug susceptibility testing of

mycobacterium tuberculosis complex by use of a high-throughput, reproducible, absolute

concentrationmethod.JClinMicrobiol.2007;45:2662-8

17. vanRijnSP,WesselsAM,GreijdanusB,etal.QuantificationandvalidationofErtapenemusing

aliquidchromatography-tandemmassspectrometrymethod.AntimicrobAgentsChemother.

2014Jun:58(6):3481-4.

18. MajumdarAK,MussonDG,BirkKL,etal.PharmacokineticsofErtapeneminhealthyyoung

volunteers.AntimicrobAgentsChemother2002Nov;46(11):3506-11.

19. NaranjoCA,BustoU,SellersEM,etal.Amethodforestimatingtheprobabilityofadverse

drugreactions.ClinPharmacolTher1981Aug;30(2):239-45.

20. VandeKassteeleJ,vanSanten-VerheuvelMG,KoedijkFD,etal.Newstatisticaltechniquefor

analyzingMIC-based susceptibility data. Antimicrob Agents Chemother. 2012Mar; 56(3):

1557-63

21. CantiniF,NiccoliL,GolettiD.Adalimub,etanercept,infliximab,andtheriskoftuberculosis:

data from clinical trials, national registries, and postmarketing surveillance. J Rheumatol

Suppl.2014May;91:47-55

74

Chapter 4

22. BumbaceaD,ArendSM,EyubogluF,etal.Theriskoftuberculosisintransplantcandidates

andrecipients:aTBNETconsensusstatement.EurRespirJ.2012Oct:40(4):990-1013.

23. BrinkAJ,RichardsGA,SchillackV,etal.Pharmacokineticsofonce-dailydosingofErtapenem

incriticallyillpatientswithseveresepsis.IntJAntimicrobAgents2009May;33(5):432-6.

24. BurkhardtO,DerendorfH,WelteT.Ertapenem:Thenewcarbapenem5yearsafterfirstFDA

licensingforclinicalpractice.ExpertOpinPharmacother.2007Feb;8(2):237-56.

25. SauermannR,BurianB,BurianA,etal.TissuepharmacokineticsofErtapenematsteady-state

indiabeticpatientswithleginfections.JAntimicrobChemother.2012Dec4.

26. Alsaad N, van Altena R, Pranger AD, et al. Evaluation of co-trimoxazole in treatment of

multidrug-resistanttuberculosis.EurRespirJ.2012Oct25.

27. DongH,WangX,DongY,etal.Clinicalpharmacokinetic/pharmacodynamicprofileoflinezolid

inseverelyillintensivecareunitpatients.IntJAntimicrobAgents2011Oct;38(4):296-300.21.

Hess S, Hospach T, Nossal R, et al. Immune reconstitution after discontinuation of TNF

blockade.EurJPediatr.2011Oct;170(10):1337-42

28. GumboT,LouieA,DezielMR,etal.Selectionofamoxifloxacindosethatsuppressesdrug

resistanceinmycobacteriumtuberculosis,byuseofaninvitropharmacodynamicinfection

modelandmathematicalmodeling.JInfectDis.2004Nov1;190(9):1642-51.

29. HessS,HospachT,NossalR,etal.ImmunereconstitutionafterdiscontinuationofTNF

blockade.EurJPediatr.Oct;170(10):1337-42

30. ZhanelGG,SimorAE,VercaigneL,etal.CanadianCarbapenemDiscussionGroup.Imipenem

andmeropenem:Comparisonofinvitroactivity,pharmacokinetics,clinicaltrialsandadverse

effects.CanJInfectDis.1998Jul;9(4):215-28

31. Srivastava S., VanRijn SP, ShermanC, et al. SterilizingActivityof Ertapenem (ETP) agains

Mycobacterium tuberculosis (Mtb). Poster session presented at: ICAAC 2015; 66th annual

conferenceofInterscienceConferenceofAntimicrobialAgentsandChemotherapy;2015Sep

17-21;SanDiego,CA.

32. Esteban J, Ortiz A, Jiménez MS. Usefulness of E-test strips for testing susceptibility of

Mycobacterium tuberculosis complex strains. Eur J Clin Microbiol Infect Dis. 2005 Dec;

24(12):856-7

33. CamineroJA,ScardigliA.Classificationofantituberculosisdrugs:anewproposalbasedonthe

mostrecentevidence.EurRespirJ2015;46:887-893

75


4

22. BumbaceaD,ArendSM,EyubogluF,etal.Theriskoftuberculosisintransplantcandidates

andrecipients:aTBNETconsensusstatement.EurRespirJ.2012Oct:40(4):990-1013.

23. BrinkAJ,RichardsGA,SchillackV,etal.Pharmacokineticsofonce-dailydosingofErtapenem

incriticallyillpatientswithseveresepsis.IntJAntimicrobAgents2009May;33(5):432-6.

24. BurkhardtO,DerendorfH,WelteT.Ertapenem:Thenewcarbapenem5yearsafterfirstFDA

licensingforclinicalpractice.ExpertOpinPharmacother.2007Feb;8(2):237-56.

25. SauermannR,BurianB,BurianA,etal.TissuepharmacokineticsofErtapenematsteady-state

indiabeticpatientswithleginfections.JAntimicrobChemother.2012Dec4.

26. Alsaad N, van Altena R, Pranger AD, et al. Evaluation of co-trimoxazole in treatment of

multidrug-resistanttuberculosis.EurRespirJ.2012Oct25.

27. DongH,WangX,DongY,etal.Clinicalpharmacokinetic/pharmacodynamicprofileoflinezolid

inseverelyillintensivecareunitpatients.IntJAntimicrobAgents2011Oct;38(4):296-300.21.

Hess S, Hospach T, Nossal R, et al. Immune reconstitution after discontinuation of TNF

blockade.EurJPediatr.2011Oct;170(10):1337-42

28. GumboT,LouieA,DezielMR,etal.Selectionofamoxifloxacindosethatsuppressesdrug

resistanceinmycobacteriumtuberculosis,byuseofaninvitropharmacodynamicinfection

modelandmathematicalmodeling.JInfectDis.2004Nov1;190(9):1642-51.

29. HessS,HospachT,NossalR,etal.ImmunereconstitutionafterdiscontinuationofTNF

blockade.EurJPediatr.Oct;170(10):1337-42

30. ZhanelGG,SimorAE,VercaigneL,etal.CanadianCarbapenemDiscussionGroup.Imipenem

andmeropenem:Comparisonofinvitroactivity,pharmacokinetics,clinicaltrialsandadverse

effects.CanJInfectDis.1998Jul;9(4):215-28

31. Srivastava S., VanRijn SP, ShermanC, et al. SterilizingActivityof Ertapenem (ETP) agains

Mycobacterium tuberculosis (Mtb). Poster session presented at: ICAAC 2015; 66th annual

conferenceofInterscienceConferenceofAntimicrobialAgentsandChemotherapy;2015Sep

17-21;SanDiego,CA.

32. Esteban J, Ortiz A, Jiménez MS. Usefulness of E-test strips for testing susceptibility of

Mycobacterium tuberculosis complex strains. Eur J Clin Microbiol Infect Dis. 2005 Dec;

24(12):856-7

33. CamineroJA,ScardigliA.Classificationofantituberculosisdrugs:anewproposalbasedonthe

mostrecentevidence.EurRespirJ2015;46:887-893

CHAPTER 5

S. SrivastavaS.P. van Rijn

A.M.A. WesselsJ.W.C. Alffenaar

T. Gumbo

Susceptibility Testing of Antibiotics That Degrade Faster than the DoublingTime of Slow-Growing Mycobacteria: Ertapenem Sterilizing Effect Versus

Mycobacterium Tuberculosis

Antimicrob Agents Chemother. 2016 Apr 22; 60(5):3193-5.PMID: 26926650

78

Chapter 5

AbstractDrug susceptibility tests (DST) for Mycobacterium tuberculosis require at least 7 days of

incubation. Forunstable at 37°Cdrugs suchas ertapenem, it is likely that this drug couldbe

degraded before killing or inhibiting slow growing bacteria. This would alter the minimum

inhibitory concentrations (MICs) of the ertapenem, leading to falsely high MICs. Here, we

developedanewstrategytoperformDSTandMICsforsuchunstablecompounds.

Ertapenem,aβ-lactamagentofthecarbapenemclass,hasshownpromisingclinicalresultsand

favourable pharmacokinetics againstMycobacterium tuberculosis (Mtb) (1,2). The scourge of

multidrug-resistanttuberculosis(MDR-TB)andextensivelydrug-resistanttuberculosis(XDR-TB),

aglobalproblem,has increased theurgency for theuseof carbapenemssuchasertapenem,

meropenem,andfaropenem(2-4).RecentlythefirstphaseIIstudy(NCT02349841)evaluating

earlybactericidalactivityofmeropenemand faropenemhasbeencompletedand resultsare

expectedsoon.

Carbapenemsinhibitthepeptidasedomainofpenicillinbindingproteins,leadingtoautolysisand

peptidoglycanweakeningof thecellwall (5).Degradationofertapenemonstorage following

reconstitutionanddilutionistemperaturedependentandtheproposedin-useshelf-lifeis6hr

atroomtemperatureor24hoursat2to8°C(6).Mtbhasadoublingtimeofatleast24hours

under the best of circumstances (7,8). Mtb cell division is particularly slow; FtsZ, a protein

responsible for initiatingcelldivisionand recruitingproteins for formationofnewcellwall is

known tohavea rateofpolymerization that is at least 20 times slower inMtb compared to

Escherichiacoliforexample(9).InMtbatlowpH,thereplicationrateisupto10-20timesslower,

killofsuchsemidormantbacteriaisdefinedassterilizingeffect(7,8,10).Thus,microbialkilland

inhibitionofgrowthbythemosteffectiveofantibioticsisslowandtakesplaceoverseveraldays,

especiallybyβ-lactamsthatdependoncellwallturnover.

Drugsusceptibilitytests(DST)forMtbusingClinicalandLaboratoryStandardsInstitute(CLSI)and

theEuropeanCommitteeonAntimicrobial SusceptibilityTesting (EUCAST)approvedmethods

require at least 7 days of incubation (11). For drugs such as ertapenem, already appearing

unstableat37°C(2),itishighlylikelythatthisdrugcouldbedegradedbeforekillingorinhibiting

slow growing bacteria, especially semi dormant Mtb. This would be expected to alter the

minimuminhibitoryconcentrations(MICs)ofertapenem,leadingtofalselyhighMICsandfalse

resistance.Here,weshowtherapiddeclineofertapenemduringDSTandtherebywedeveloped

anewstrategytoperformDSTandMICsforsuchunstablecompounds.

79

5

Susceptibility testing of Antibiotics: Ertapenem Sterilizing Effect

AbstractDrug susceptibility tests (DST) for Mycobacterium tuberculosis require at least 7 days of

incubation. Forunstable at 37°Cdrugs suchas ertapenem, it is likely that thisdrug couldbe

degraded before killing or inhibiting slow growing bacteria. This would alter the minimum

inhibitory concentrations (MICs) of the ertapenem, leading to falsely high MICs. Here, we

developedanewstrategytoperformDSTandMICsforsuchunstablecompounds.

Ertapenem,aβ-lactamagentofthecarbapenemclass,hasshownpromisingclinicalresultsand

favourable pharmacokinetics againstMycobacterium tuberculosis (Mtb) (1,2). The scourge of

multidrug-resistanttuberculosis(MDR-TB)andextensivelydrug-resistanttuberculosis(XDR-TB),

aglobalproblem,has increased theurgency for theuseof carbapenemssuchasertapenem,

meropenem,andfaropenem(2-4).RecentlythefirstphaseIIstudy(NCT02349841)evaluating

earlybactericidal activityofmeropenemand faropenemhasbeencompletedand resultsare

expectedsoon.

Carbapenemsinhibitthepeptidasedomainofpenicillinbindingproteins,leadingtoautolysisand

peptidoglycanweakeningof thecellwall (5).Degradationofertapenemonstorage following

reconstitutionanddilutionistemperaturedependentandtheproposedin-useshelf-lifeis6hr

atroomtemperatureor24hoursat2to8°C(6).Mtbhasadoublingtimeofatleast24hours

under the best of circumstances (7,8). Mtb cell division is particularly slow; FtsZ, a protein

responsible for initiating celldivisionand recruitingproteins for formationofnewcellwall is

known tohavea rateofpolymerization that is at least 20 times slower inMtb compared to

Escherichiacoliforexample(9).InMtbatlowpH,thereplicationrateisupto10-20timesslower,

killofsuchsemidormantbacteriaisdefinedassterilizingeffect(7,8,10).Thus,microbialkilland

inhibitionofgrowthbythemosteffectiveofantibioticsisslowandtakesplaceoverseveraldays,

especiallybyβ-lactamsthatdependoncellwallturnover.

Drugsusceptibilitytests(DST)forMtbusingClinicalandLaboratoryStandardsInstitute(CLSI)and

theEuropeanCommitteeonAntimicrobial SusceptibilityTesting (EUCAST)approvedmethods

require at least 7 days of incubation (11). For drugs such as ertapenem, already appearing

unstableat37°C(2),itishighlylikelythatthisdrugcouldbedegradedbeforekillingorinhibiting

slow growing bacteria, especially semi dormant Mtb. This would be expected to alter the

minimuminhibitoryconcentrations(MICs)ofertapenem,leadingtofalselyhighMICsandfalse

resistance.Here,weshowtherapiddeclineofertapenemduringDSTandtherebywedeveloped

anewstrategytoperformDSTandMICsforsuchunstablecompounds.

80

Chapter 5

Ertapenem (purchased from SIGMA) was first dissolved in purified water and subsequently

diluted in Middlebrook 7H9 broth to the desired drug concentrations of 5.0 and 50 mg/L

respectively.Thetwosolutionswereincubatedat37ºC.After0,5,8,24,32and48hours,three

sampleswerecollectedfromeachsolutionandimmediatelystoredat-80ºCuntilfurtheranalysis.

Allsampleswerethenfullythawedatroomtemperatureandanalysedinduplicateusingafully

validatedassay(12).Thecalibrationcurveofertapenemwaslinearoverarangeof0.1to125

mg/L and the correlation coefficient was 0.999. The % coefficient of variation between the

replicatesforeachconcentrationateachtimepointwas2.7-11.2%.Figure1showsthedecrease

in ertapenem concentration in the solution at 37°C. After 5 hours of incubation, ertapenem

concentrationwasreducedby45.3%and40.7%incomparisonwiththeinitialconcentrationsof

5and50mg/L,respectively.After48hours,theconcentrationswere20.1%and26.8%ofthe

timezeroconcentrations.

Figure1.LC-MS/MSanalysisofertapenemtodeterminetherateofdegradationat370C.

Panel A and B, 5 and 50 mg/L initial concentration, respectively show the percentage of

ertapenemmeasuredafterincubationat370C.M.tuberculosisgrows“inslowmotion”compared

totheseotherbacteria(72timesslowerthanE.coliwheninlog-phasegrowthand720times

slower for semi dormantM. tuberculosis) (5). Whereas, as shown in the figure, longer the

incubationperiodmore isthedegradationofertapenem.Thiscouldeventually leadtofalsely

highertapenemMIC.

Mtb H37Ra (ATCC 25177) was used in the MIC and dose-response experiments. For each

experiment, one stock vialwas thawedandbacteria grown to logarithmic growthphase (log

phase growth) inMiddlebrook7H9broth enrichedwith 10%OADC for 4 days at 37°Cunder

shakingconditionsand5%CO2.Forsemidormantbacteriaunderacidicconditions, theday4

culturewas inoculated inMiddlebrook 7H9 acidified to pH of 5.8 bymeans of citric acid as

describedearlier (7).SterilizingeffectMICswere identifiedusingboth thebrothdilutionand

resazurincolorimetricassay(11,13).MtbinacidifiedMiddlebrook7H9brothwasexposedtothe

followingertapenemconcentrations,intriplicate:0,0.075,0.15,0.3,0.6,1,1.25,2,2.5,4,5,8,

16,32,and64mg/L.Thecultureswereincubatedat370Cwith5%CO2for7days.Inoneset,each

replicatereceivedadaily50%supplementationofertapenemconcentrationatvolumesof<1%.

Fortheresazurinassay,analiquotoftheday7cultureshadresazurin(0.001%v/v)addedtothe

samplesandplates,whichwerethenfurtherincubatedfor24hoursat37°Cunder5%CO2.The

ertapenem MIC without daily ertapenem supplementation was 64 mg/L, while that with

supplementationwas0.6mg/L.

Day7culturesdescribedabovethatwerenotusedforresazurinassayswerewashedtwicein

normalsalinetopreventdrugcarryover,andweresubsequentlyspreadonMiddlebrook7H10

agar,andincubatedfor3weeksat37°Cforenumerationofcolonyformingunits(CFU)counts.

InhibitorysigmoidEmaxcurvesforconcentrationversusCFU/mLundersterilizingeffectconditions

areshown inFigure2. Comparisonof the tworegressions,with thenullhypothesis that the

maximalkill(Emax)orefficacyandconcentrationmediating50%ofEmax(EC50)orpotencyrevealed

aratioofprobabilitiesof7.46andadifferenceincorrectedAkaikeInformationcriteriascoresof

4.02,whichmeansthattheefficacyandpotencydifferedwithsupplementation.TheEC50was

1.41 mg/L without ertapenem supplementation compared to 0.19 mg/L with daily

supplementation. Theefficacywas0.751 log10 CFU/mLwithoutdaily supplementation versus

2.38log10CFU/mLwithsupplementation.Thus,ertapenemdisplayspotentialforsterilizingeffect

thatwouldotherwisebemaskedbynotaccountingforthedegradation.

81

5


Ertapenem (purchased from SIGMA) was first dissolved in purified water and subsequently

diluted in Middlebrook 7H9 broth to the desired drug concentrations of 5.0 and 50 mg/L

respectively.Thetwosolutionswereincubatedat37ºC.After0,5,8,24,32and48hours,three

sampleswerecollectedfromeachsolutionandimmediatelystoredat-80ºCuntilfurtheranalysis.

Allsampleswerethenfullythawedatroomtemperatureandanalysedinduplicateusingafully

validatedassay(12).Thecalibrationcurveofertapenemwaslinearoverarangeof0.1to125

mg/L and the correlation coefficient was 0.999. The % coefficient of variation between the

replicatesforeachconcentrationateachtimepointwas2.7-11.2%.Figure1showsthedecrease

in ertapenem concentration in the solution at 37°C. After 5 hours of incubation, ertapenem

concentrationwasreducedby45.3%and40.7%incomparisonwiththeinitialconcentrationsof

5and50mg/L,respectively.After48hours,theconcentrationswere20.1%and26.8%ofthe

timezeroconcentrations.

Figure1.LC-MS/MSanalysisofertapenemtodeterminetherateofdegradationat370C.

Panel A and B, 5 and 50 mg/L initial concentration, respectively show the percentage of

ertapenemmeasuredafterincubationat370C.M.tuberculosisgrows“inslowmotion”compared

totheseotherbacteria(72timesslowerthanE.coliwheninlog-phasegrowthand720times

slower for semi dormantM. tuberculosis) (5). Whereas, as shown in the figure, longer the

incubationperiodmore isthedegradationofertapenem.Thiscouldeventually leadtofalsely

highertapenemMIC.

Mtb H37Ra (ATCC 25177) was used in the MIC and dose-response experiments. For each

experiment, one stock vialwas thawedandbacteria grown to logarithmic growthphase (log

phase growth) inMiddlebrook7H9broth enrichedwith 10%OADC for 4 days at 37°Cunder

shakingconditionsand5%CO2.Forsemidormantbacteriaunderacidicconditions, theday4

culturewas inoculated inMiddlebrook 7H9 acidified to pH of 5.8 bymeans of citric acid as

describedearlier (7).SterilizingeffectMICswere identifiedusingboth thebrothdilutionand

resazurincolorimetricassay(11,13).MtbinacidifiedMiddlebrook7H9brothwasexposedtothe

followingertapenemconcentrations,intriplicate:0,0.075,0.15,0.3,0.6,1,1.25,2,2.5,4,5,8,

16,32,and64mg/L.Thecultureswereincubatedat370Cwith5%CO2for7days.Inoneset,each

replicatereceivedadaily50%supplementationofertapenemconcentrationatvolumesof<1%.

Fortheresazurinassay,analiquotoftheday7cultureshadresazurin(0.001%v/v)addedtothe

samplesandplates,whichwerethenfurtherincubatedfor24hoursat37°Cunder5%CO2.The

ertapenem MIC without daily ertapenem supplementation was 64 mg/L, while that with

supplementationwas0.6mg/L.

Day7culturesdescribedabovethatwerenotusedforresazurinassayswerewashedtwicein

normalsalinetopreventdrugcarryover,andweresubsequentlyspreadonMiddlebrook7H10

agar,andincubatedfor3weeksat37°Cforenumerationofcolonyformingunits(CFU)counts.

InhibitorysigmoidEmaxcurvesforconcentrationversusCFU/mLundersterilizingeffectconditions

areshown inFigure2. Comparisonof the tworegressions,with thenullhypothesis that the

maximalkill(Emax)orefficacyandconcentrationmediating50%ofEmax(EC50)orpotencyrevealed

aratioofprobabilitiesof7.46andadifferenceincorrectedAkaikeInformationcriteriascoresof

4.02,whichmeansthattheefficacyandpotencydifferedwithsupplementation.TheEC50was

1.41 mg/L without ertapenem supplementation compared to 0.19 mg/L with daily

supplementation. Theefficacywas0.751 log10 CFU/mLwithoutdaily supplementation versus

2.38log10CFU/mLwithsupplementation.Thus,ertapenemdisplayspotentialforsterilizingeffect

thatwouldotherwisebemaskedbynotaccountingforthedegradation.

82

Chapter 5

Figure2.ErtapenemdoseresponseagainstslowlyreplicatingM.tuberculosis.

Asshowninthefigure,whenthedegradationratewastakenintoaccountbysupplementingthe

drugdaily,therewasbetterkillofsemi-dormantM.tuberculosisbyertapenem.

Here,firstweshowthatertapenemdegradesconsiderably,atrates>20-foldthedoublingtimes

ofMtbunderacidicconditions.Thiseffect is strikingwhenonecompared theMICswithand

withoutsupplementation.TheMICsintheabsenceofsupplementationwouldbeconsideredin

the resistance range by EUCAST breakpoints (14). Second, we show that ertapenem

supplementationbrings itwellwithin the susceptibility range. This suggests thatmostof the

published MICs for this drug are likely falsely high and rates of resistance are likely falsely

elevated.Inaddition,sincemanypeoplehaveusedMICstochoosewhichofthecarbapenems

wouldbebettersuitedfortreatmentofXDR-TB,gooddrugsmayhavebeendiscardedbecause

oftheartefactualmannerinwhichcurrentMICsareperformedforunstablemolecules.Third,

weshowthatertapenemislikelytohavegoodsterilizingeffectintuberculosis.Followuphollow

fiberstudiesforsterilizingeffecthavenowbeencompletedinordertoidentifytheertapenem

dose, which can be used against both drug resistant and sensitive Mtb (manuscript in

preparation).

ACKNOWLDGEMENTS

FUNDING

This work was supported by National Institutes of Health (NIH) via DP2 OD001886 and

R01AI079497toTawandaGumbo.

POTENTIALCONFLICTOFINTEREST

TawandaGumboisaconsultantforAstellasPharmaUSAandLuminaCaresolutions;andfounded

Jacaranda Biomed Inc. Jan-Willem Alffenaar received financial support from Merck, Pfizer,

AstellasandGileadforinvestigator-initiatedstudiesand/oracademicsymposia.

83

5


Figure2.ErtapenemdoseresponseagainstslowlyreplicatingM.tuberculosis.

Asshowninthefigure,whenthedegradationratewastakenintoaccountbysupplementingthe

drugdaily,therewasbetterkillofsemi-dormantM.tuberculosisbyertapenem.

Here,firstweshowthatertapenemdegradesconsiderably,atrates>20-foldthedoublingtimes

ofMtbunderacidicconditions.Thiseffect is strikingwhenonecompared theMICswithand

withoutsupplementation.TheMICsintheabsenceofsupplementationwouldbeconsideredin

the resistance range by EUCAST breakpoints (14). Second, we show that ertapenem

supplementationbrings itwellwithin the susceptibility range. This suggests thatmostof the

published MICs for this drug are likely falsely high and rates of resistance are likely falsely

elevated.Inaddition,sincemanypeoplehaveusedMICstochoosewhichofthecarbapenems

wouldbebettersuitedfortreatmentofXDR-TB,gooddrugsmayhavebeendiscardedbecause

oftheartefactualmannerinwhichcurrentMICsareperformedforunstablemolecules.Third,

weshowthatertapenemislikelytohavegoodsterilizingeffectintuberculosis.Followuphollow

fiberstudiesforsterilizingeffecthavenowbeencompletedinordertoidentifytheertapenem

dose, which can be used against both drug resistant and sensitive Mtb (manuscript in

preparation).

ACKNOWLDGEMENTS

FUNDING

This work was supported by National Institutes of Health (NIH) via DP2 OD001886 and

R01AI079497toTawandaGumbo.

POTENTIALCONFLICTOFINTEREST

TawandaGumboisaconsultantforAstellasPharmaUSAandLuminaCaresolutions;andfounded

Jacaranda Biomed Inc. Jan-Willem Alffenaar received financial support from Merck, Pfizer,

AstellasandGileadforinvestigator-initiatedstudiesand/oracademicsymposia.

84

Chapter 5

References1. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJW,MiglioriGB.

2015. Ertapenem in the treatment of multidrug-resistant tuberculosis: First clinical

experience.EurRespirJ.doi:10.1183/13993003.01278-2015

2. Van Rijn SP, Altena R, AkkermanOW, van Soolingen D, van der Laan T, de LangeWCM,

Kosterink JGW, vanderWerf TS,Alffenaar JWC. 2015. Pharmacokinetics of ertapenem in

patientswitgmultidrug-resistanttuberculosis.EurRespir J.doi:10.1183/13993003.01654-

2015

3. DeLorenzoS,AlffenaarJW,SotgiuG,CentisR,D'AmbrosioL,TiberiS,BolhuisMS,vanAltena

R,ViggianiP,PianaA,SpanevelloA,MiglioriGB.2013.Efficacyandsafetyofmeropenem-

clavulanate added to linezolid-containing regimens in the treatment of MDR-/XDR-TB.

Eur.Respir.J.41:1386-1392.

4. DhedaK,GumboT,GandhiNR,MurrayM,TheronG,UdwadiaZ,MiglioriGB,WarrenR.2014.

Globalcontroloftuberculosis: fromextensively-drugresistanttountreatabletuberculosis.

LancetRespirMed.2:321-338.

5. CordillotM,DubeeV,TribouletS,DubostL,MarieA,HugonnetJE,ArthurM,MainardiJL.

2013. In vitro cross-linking of Mycobacterium tuberculosis peptidoglycan by L,D-

transpeptidasesandinactivationoftheseenzymesbycarbapenems.AntimicrobAgentsand

Chemother.57:5940-45

6. Scientific discussion for the approval of Invanz.

http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-

_Scientific_Discussion/human/000389/WC500033918.pdf.

7. Gumbo T, Dona CS, Meek C, Leff R. 2009. Pharmacokinetics-pharmacodynamics of

pyrazinamideinanovel invitromodeloftuberculosisforsterilizingeffect:aparadigmfor

fasterassessmentofnewantituberculosisdrugs.AntimicrobialAgentsandChemotherapy.

53:3197-3204.

8. MusukaS,SrivastavaS,SiyambalapitiyageDonaCW,MeekC,LeffR,PasipanodyaJ,Gumbo

T. 2013. Thioridazine pharmacokinetic-pharmacodynamic parameters "wobble" during

treatmentoftuberculosis:atheoreticalbasisforshorter-durationcurativemonotherapywith

congeners.AntimicrobialAgentsandChemotherapy.57:5870-5877.

9. Hett EC, Rubin EJ. 2008. Bacterial growth and cell division: a mycobacterial perspective.

Microbiol.Mol.Biol.Rev.72:126-56,table.

10. MitchisonDA.1979.Basicmechanismsofchemotherapy.Chest.76:771-781.

11. Clinical and Laboratory Standards Institute. 2003. Susceptibility testing ofMycobacteria,

Nocardiae,andotheraerobicActinomycetes;ApprovedStandard.M24-A2

12. Van Rijn SP,Wessels AM,Greijdanus B, TouwDJ, Alffenaar JW. 2014.Quantification and

validationofertapenemusingaliquidchromatography-tandemmassspectrometrymethod.

AntimicrobialAgentsandChemotherapy.58:3481-3484.

13. Srivastava S, Pasipanodya J, Sherman CM, Meek C, Leff R, Gumbo T. 2015. Rapid drug

toleranceanddramaticsterilizingeffectofmoxifloxacinmonotherapyinanovelhollow-fiber

model of intracellular Mycobacterium kansasii disease.Antimicrobial Agents and

Chemotherapy.59:2273-2279.

14. VezirisN, Truffot C,Mainardi JL, Jarlier, V. 2011. Activity of carbapenems combinedwith

clavulanateagainstmurinetuberculosis.AntimicrobialAgentsandChemotherapy.55:2597-

2600.

85

5


References1. TiberiS,D’AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJW,MiglioriGB.


experience.EurRespirJ.doi:10.1183/13993003.01278-2015

2. Van Rijn SP, Altena R, AkkermanOW, van Soolingen D, van der Laan T, de LangeWCM,

Kosterink JGW, vanderWerf TS,Alffenaar JWC. 2015. Pharmacokinetics of ertapenem in

patientswitgmultidrug-resistanttuberculosis.EurRespir J.doi:10.1183/13993003.01654-

2015

3. DeLorenzoS,AlffenaarJW,SotgiuG,CentisR,D'AmbrosioL,TiberiS,BolhuisMS,vanAltena

R,ViggianiP,PianaA,SpanevelloA,MiglioriGB.2013.Efficacyandsafetyofmeropenem-

clavulanate added to linezolid-containing regimens in the treatment of MDR-/XDR-TB.

Eur.Respir.J.41:1386-1392.

4. DhedaK,GumboT,GandhiNR,MurrayM,TheronG,UdwadiaZ,MiglioriGB,WarrenR.2014.

Globalcontroloftuberculosis: fromextensively-drugresistanttountreatabletuberculosis.

LancetRespirMed.2:321-338.

5. CordillotM,DubeeV,TribouletS,DubostL,MarieA,HugonnetJE,ArthurM,MainardiJL.

2013. In vitro cross-linking of Mycobacterium tuberculosis peptidoglycan by L,D-

transpeptidasesandinactivationoftheseenzymesbycarbapenems.AntimicrobAgentsand

Chemother.57:5940-45

6. Scientific discussion for the approval of Invanz.

http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-

_Scientific_Discussion/human/000389/WC500033918.pdf.


pyrazinamideinanovel invitromodeloftuberculosisforsterilizingeffect:aparadigmfor

fasterassessmentofnewantituberculosisdrugs.AntimicrobialAgentsandChemotherapy.

53:3197-3204.

8. MusukaS,SrivastavaS,SiyambalapitiyageDonaCW,MeekC,LeffR,PasipanodyaJ,Gumbo

T. 2013. Thioridazine pharmacokinetic-pharmacodynamic parameters "wobble" during

treatmentoftuberculosis:atheoreticalbasisforshorter-durationcurativemonotherapywith

congeners.AntimicrobialAgentsandChemotherapy.57:5870-5877.

9. Hett EC, Rubin EJ. 2008. Bacterial growth and cell division: a mycobacterial perspective.

Microbiol.Mol.Biol.Rev.72:126-56,table.

10. MitchisonDA.1979.Basicmechanismsofchemotherapy.Chest.76:771-781.

11. Clinical and Laboratory Standards Institute. 2003. Susceptibility testing ofMycobacteria,

Nocardiae,andotheraerobicActinomycetes;ApprovedStandard.M24-A2

12. Van Rijn SP,Wessels AM,Greijdanus B, TouwDJ, Alffenaar JW. 2014.Quantification and

validationofertapenemusingaliquidchromatography-tandemmassspectrometrymethod.

AntimicrobialAgentsandChemotherapy.58:3481-3484.

13. Srivastava S, Pasipanodya J, Sherman CM, Meek C, Leff R, Gumbo T. 2015. Rapid drug

toleranceanddramaticsterilizingeffectofmoxifloxacinmonotherapyinanovelhollow-fiber

model of intracellular Mycobacterium kansasii disease.Antimicrobial Agents and

Chemotherapy.59:2273-2279.

14. VezirisN, Truffot C,Mainardi JL, Jarlier, V. 2011. Activity of carbapenems combinedwith

clavulanateagainstmurinetuberculosis.AntimicrobialAgentsandChemotherapy.55:2597-

2600.

CHAPTER 6

S.P. van Rijn*S. Srivastava*

A.M.A. WesselsD. Soolingen

J.W.C. AlffenaarT. Gumbo

Sterilizing Effect of Ertapenem-Clavulanate in a

Hollow-Fiber Model ofTuberculosis and Implications on

Clinical Dosing

Antimicrob Agents Chemother. 2017 Aug 24; 61(9).PMID: 28696238


88

Chapter 6

AbstractCarbapenems are now being explored for treatment of multi-drug resistant tuberculosis

(MDR-TB),especiallyinconjunctionwithclavulanate.Clinicaluseisconstrainedbytheneed

formultipleparenteraldosesperday,andlackofknowledgeoftheoptimaldoseforsterilizing

effect. Our objective was to identify the ertapenem exposure associated with optimal

sterilizingeffectandthendesignaonceadaydoseforclinicaluse.Weutilizedthehollow

fiber system model of tuberculosis in a 28-day exposure-response study of 8 different

ertapenem doses in combination with clavulanate. The systems were sampled at

predetermined time-points to verify the concentration-time profile and identify the total

bacterial burden. Inhibitory sigmoid Emax modelling was used to identify the relationship

between total bacterial burden and the drug exposure, and identify optimal exposures.

Contrarytotheliterature,ertapenem-clavulanatecombinationdemonstratedgoodmicrobial

kill and sterilizingeffect. In adose-fractionationhollow fiber study,efficacywas linked to

percentageofthe24-hourdosingintervalofertapenemconcentrationpersistingaboveMIC

(%TMIC). We performed 10,000MDR-TB patient computer-aided clinical trial simulations,

basedonMonteCarlomethods,to identifythedosesandschedulethatwouldachieveor

exceed%TMIC³40%.Weidentifiedanintravenousdoseof2gramsonceperdayasachieving

the target in 96% of patients. An ertapenem susceptibility breakpoint MIC 2 mg/L was

identifiedforthatdose.Anertapenemdoseof2goncedailyisthemostsuitabletobetested

inaphaseIIstudyofsterilizingeffectinMDR-TBpatients.

IntroductionTheemergenceofdrugresistanttuberculosis(TB),especiallymultidrug-resistantTB(MDR-

TB), extensively drug resistant (XDR-TB), and virtually incurable TB (termed totally drug-

resistant TB by some), is a global emergency that threatens to underminemany gains of

chemotherapy(1-4).Asaresult,thereiscurrentlyafourprongedeffort;(i)identificationof

new smallmolecules to kill drug resistantMycobacterium tuberculosis, (ii) repurposingof

antimicrobial drugs not currently used to treat TB into TB therapeutics, (iii) host-directed

therapy, and (iv) use of pharmacokinetic/pharmacodynamics science to optimize efficacy

while suppressing emergenceof acquireddrug resistance (5-8).Carbapenems, extensively

used to treatGram-negativebacteriaover the last30 years,havealsobeen shown tobe

effectiveagainstM.tuberculosisinvitroandinvivowheninthepresenceofaβ-lactamase

inhibitor(6,9).

Severalinitiativesareongoingtoexploretheaddedvalueofcarbapenemsgivenaspartofa

multidrugregimenforM/XDR-TB(10,11).InmurineTB,efficacyhasbeendemonstratedfor

meropenemandimipenemwithclavulanate;howeverertapenemwasnobetterthannon-

treated controls (9). In addition, ertapenemdemonstratedhighMICs, suggestingpossible

natural resistance. However, ertapenem degrades rapidly in in vitro growth media at

incubationtemperaturesusedtomeasureMICswithconventionalmethods(12).Wehave

sincedevelopedaMICassaythatcorrectsforthisdegradation,whichhasdemonstratedmuch

lowerMICs(12).Themainadvantageofertapenemtopatientscouldbeitshalf-lifeof4hours,

which could allow a once a day schedule, as opposed to 0.6-0.7 hrs formeropenemand

imipenemwhichnecessitatesmultipleandprolongedintravenousinfusionsperday(13).The

multiple infusions per day with meropenem and imipenem make it rather difficult to

administer long duration therapy in M/XDR-TB. Recently the first TB clinical data with

ertapenem showed that it was well tolerated as part of a salvage regimen for MDR-TB

patients(13,14).Unfortunately,theefficacyofthedrugcouldnotbeassessedasitwasused

inamultidrugregimen;moreover,itssterilizingeffectisunknown.

ThehollowfibersystemmodelofTB(HFS-TB)hasbeenusedtoexaminethesterilizingeffect

of anti-TBagents,definedas theability to kill either semi-dormantM. tuberculosis under

89

6

Sterilizing Effect of Ertapenem-Clavulanate in a Hollow-Fiber Model of Tuberculosis

AbstractCarbapenems are now being explored for treatment of multi-drug resistant tuberculosis

(MDR-TB),especiallyinconjunctionwithclavulanate.Clinicaluseisconstrainedbytheneed

formultipleparenteraldosesperday,andlackofknowledgeoftheoptimaldoseforsterilizing

effect. Our objective was to identify the ertapenem exposure associated with optimal





bacterial burden. Inhibitory sigmoid Emax modelling was used to identify the relationship

between total bacterial burden and the drug exposure, and identify optimal exposures.

Contrarytotheliterature,ertapenem-clavulanatecombinationdemonstratedgoodmicrobial

kill and sterilizingeffect. In adose-fractionationhollow fiber study,efficacywas linked to

percentageofthe24-hourdosingintervalofertapenemconcentrationpersistingaboveMIC

(%TMIC). We performed 10,000MDR-TB patient computer-aided clinical trial simulations,

basedonMonteCarlomethods,to identifythedosesandschedulethatwouldachieveor

exceed%TMIC³40%.Weidentifiedanintravenousdoseof2gramsonceperdayasachieving

the target in 96% of patients. An ertapenem susceptibility breakpoint MIC 2 mg/L was

identifiedforthatdose.Anertapenemdoseof2goncedailyisthemostsuitabletobetested

inaphaseIIstudyofsterilizingeffectinMDR-TBpatients.

IntroductionTheemergenceofdrugresistanttuberculosis(TB),especiallymultidrug-resistantTB(MDR-

TB), extensively drug resistant (XDR-TB), and virtually incurable TB (termed totally drug-

resistant TB by some), is a global emergency that threatens to underminemany gains of

chemotherapy(1-4).Asaresult,thereiscurrentlyafourprongedeffort;(i)identificationof

new smallmolecules to kill drug resistantMycobacterium tuberculosis, (ii) repurposingof

antimicrobial drugs not currently used to treat TB into TB therapeutics, (iii) host-directed

therapy, and (iv) use of pharmacokinetic/pharmacodynamics science to optimize efficacy

while suppressing emergenceof acquireddrug resistance (5-8).Carbapenems, extensively

used to treatGram-negativebacteriaover the last30 years,havealsobeen shown tobe

effectiveagainstM.tuberculosisinvitroandinvivowheninthepresenceofaβ-lactamase

inhibitor(6,9).

Severalinitiativesareongoingtoexploretheaddedvalueofcarbapenemsgivenaspartofa

multidrugregimenforM/XDR-TB(10,11).InmurineTB,efficacyhasbeendemonstratedfor

meropenemandimipenemwithclavulanate;howeverertapenemwasnobetterthannon-

treated controls (9). In addition, ertapenemdemonstratedhighMICs, suggestingpossible

natural resistance. However, ertapenem degrades rapidly in in vitro growth media at

incubationtemperaturesusedtomeasureMICswithconventionalmethods(12).Wehave

sincedevelopedaMICassaythatcorrectsforthisdegradation,whichhasdemonstratedmuch

lowerMICs(12).Themainadvantageofertapenemtopatientscouldbeitshalf-lifeof4hours,

which could allow a once a day schedule, as opposed to 0.6-0.7 hrs formeropenemand

imipenemwhichnecessitatesmultipleandprolongedintravenousinfusionsperday(13).The

multiple infusions per day with meropenem and imipenem make it rather difficult to

administer long duration therapy in M/XDR-TB. Recently the first TB clinical data with

ertapenem showed that it was well tolerated as part of a salvage regimen for MDR-TB

patients(13,14).Unfortunately,theefficacyofthedrugcouldnotbeassessedasitwasused

inamultidrugregimen;moreover,itssterilizingeffectisunknown.

ThehollowfibersystemmodelofTB(HFS-TB)hasbeenusedtoexaminethesterilizingeffect

of anti-TBagents,definedas theability to kill either semi-dormantM. tuberculosis under

90

Chapter 6

acidicconditionsorofnon-replicatingpersistersunderhypoxia(15-17). Itwasqualifiedby

the European Medicines Agency and editorially endorsed by the US Food and Drug

Administration (http://www.ema.europa.eu/docs/en_GB/document_library/Regulatory_an

d_procedural_guideline/2015/02/WC50018199.pdf).TheHFS-TBintandemwithcomputer-

aidedclinicaltrialsimulationswasfoundtohaveaforecastingaccuracyof>94%ofobserved

optimalexposuresanddosesinTBpatientsintheclinic(18-20).Thismakesthismodelideal

toidentifyoptimaldosesfortreatmentofM/XDR-TB,whichcandirectlybetranslatedinto

clinicaluse.Ourobjectivewastousethesemodelstoidentifytheoptimalsterilizingeffect

doseofertapenemfortreatmentofMDR-TB.

ResultsDose-effectHFS-TBstudyforsterilizingeffect

InthefirstHFS-TB,whichwasmainlyadoserangingstudy,weculturedM.tuberculosisH37Ra

underacidicconditionstoasemidormantstate,andtheinoculatedintoHFS-TBunitswith

circulating Middlebrook 7H9 acidified to a pH of 5.8, as described in the past (15, 21).

Differentertapenemexposures,basedonhumanequivalentdosesof0.25,0.5,1.0,2.0,3.0,

5.0,and10.0gramswereadministeredintothecentralcompartmentofduplicateHFS-TBvia

a computer-controlled syringepumpover30minutes, as inpatients;drug concentrations

achievedineachofthe16HFS-TBweremeasuredat8differenttimepointsoverthefirst24

hours.Clavulanatewasalsodispensedviasyringepumptoachieveapeakof3mg/Latthe

endof30minutesinfusion.Pharmacokineticmodellingofthemeasureddrugconcentrations,

revealedthatthelowestAkaikeInformationCriteriascores(22)wereforaonecompartment

model. The ertapenem total clearance (± standard deviation) was 4.11±1.83L, and the

volume of 22.55±4.0 L, which translates to a half-life of 3.80 hours. The regression for

observed concentrations versus pharmacokinetic model predicted concentrations had an

r2=0.997andtheslopewas0.996±0.006,whichisclosetounity.Thus,theonecompartment

modeldescribedthedatawell,withnobias.

Figure1showsthatertapenemachievedgoodsterilizingeffect.Thebacterialburdenatthe

start of therapywas 4.0 log10 CFU/mL. The data are presented as inhibitory sigmoid Emax

modelsbetween“nominal”humanequivalentdoseandmicrobialburden.

Figure1.Ertapenem-clavulanatedose-effectsterilizingeffectinthehollowfibermodel.

Drug treatmentsaredepictedas “nominal”humanequivalentdoses.Onday3, inhibitorysigmoidEmaxmodellingdemonstratednomodelconverge,andtherewasverylittlekill,thusregressionsforday3wereleftoutofthefigure.However,byday7therewasalreadygoodmicrobialkill,characterizedbymaximalkill(Emax)of1.13±0.34log10CFU/mL.Byday28,allertapenemtreatedHFS-TBcompletelysterilizedthebacteria.

InFigure1,therewasnomodelconvergenceonday3,whileonday28,attheendofthe

experiment, all ertapenem treated systems now had bacterial burden below limits of

detection.Allsystemsachieved%oftimeaboveMIC(%TMIC)for100%ofthedosinginterval;

thetroughat23.5hrswas>4mg/Linallsystems,andallachievedthesamemicrobialkillon

day28.

Ertapenemdose-fractionationstudyintheHFS-TB

Next, we performed a new HFS-TB, this time using M. tuberculosis H37Rv and a dose-

fractionationdesign, fora treatmentdurationof14days.Onmeasurementofertapenem

concentrations,similartothefirststudy,theconcentrationswerealsobestdescribedusinga

onecompartmentmodel;theobservedversuspredictedconcentrationsrevealedaslopeof

0.995±0.002(r2>0.999).Theconcentration-timeprofilesachievedwitheachdoseareshown

bydosingscheduleinFigures2A-C.,togetherwiththeertapenem(plusclavulanate2.5mg/L)

MICof4mg/L.InhibitorysigmoidEmaxmodelfittingbyexposureexpressedaseitherCmax/MIC

orAUC0-24/MICor%TMICrevealedAkaikeInformationCriteriascoresshowninTable1.

91

6


acidicconditionsorofnon-replicatingpersistersunderhypoxia(15-17). Itwasqualifiedby

the European Medicines Agency and editorially endorsed by the US Food and Drug

Administration (http://www.ema.europa.eu/docs/en_GB/document_library/Regulatory_an

d_procedural_guideline/2015/02/WC50018199.pdf).TheHFS-TBintandemwithcomputer-

aidedclinicaltrialsimulationswasfoundtohaveaforecastingaccuracyof>94%ofobserved

optimalexposuresanddosesinTBpatientsintheclinic(18-20).Thismakesthismodelideal

toidentifyoptimaldosesfortreatmentofM/XDR-TB,whichcandirectlybetranslatedinto

clinicaluse.Ourobjectivewastousethesemodelstoidentifytheoptimalsterilizingeffect

doseofertapenemfortreatmentofMDR-TB.

ResultsDose-effectHFS-TBstudyforsterilizingeffect

InthefirstHFS-TB,whichwasmainlyadoserangingstudy,weculturedM.tuberculosisH37Ra

underacidicconditionstoasemidormantstate,andtheinoculatedintoHFS-TBunitswith

circulating Middlebrook 7H9 acidified to a pH of 5.8, as described in the past (15, 21).


5.0,and10.0gramswereadministeredintothecentralcompartmentofduplicateHFS-TBvia

a computer-controlled syringepumpover30minutes, as inpatients;drug concentrations

achievedineachofthe16HFS-TBweremeasuredat8differenttimepointsoverthefirst24

hours.Clavulanatewasalsodispensedviasyringepumptoachieveapeakof3mg/Latthe

endof30minutesinfusion.Pharmacokineticmodellingofthemeasureddrugconcentrations,

revealedthatthelowestAkaikeInformationCriteriascores(22)wereforaonecompartment

model. The ertapenem total clearance (± standard deviation) was 4.11±1.83L, and the

volume of 22.55±4.0 L, which translates to a half-life of 3.80 hours. The regression for

observed concentrations versus pharmacokinetic model predicted concentrations had an

r2=0.997andtheslopewas0.996±0.006,whichisclosetounity.Thus,theonecompartment

modeldescribedthedatawell,withnobias.

Figure1showsthatertapenemachievedgoodsterilizingeffect.Thebacterialburdenatthe

start of therapywas 4.0 log10 CFU/mL. The data are presented as inhibitory sigmoid Emax

modelsbetween“nominal”humanequivalentdoseandmicrobialburden.

Figure1.Ertapenem-clavulanatedose-effectsterilizingeffectinthehollowfibermodel.

Drug treatmentsaredepictedas “nominal”humanequivalentdoses.Onday3, inhibitorysigmoidEmaxmodellingdemonstratednomodelconverge,andtherewasverylittlekill,thusregressionsforday3wereleftoutofthefigure.However,byday7therewasalreadygoodmicrobialkill,characterizedbymaximalkill(Emax)of1.13±0.34log10CFU/mL.Byday28,allertapenemtreatedHFS-TBcompletelysterilizedthebacteria.

InFigure1,therewasnomodelconvergenceonday3,whileonday28,attheendofthe

experiment, all ertapenem treated systems now had bacterial burden below limits of

detection.Allsystemsachieved%oftimeaboveMIC(%TMIC)for100%ofthedosinginterval;

thetroughat23.5hrswas>4mg/Linallsystems,andallachievedthesamemicrobialkillon

day28.

Ertapenemdose-fractionationstudyintheHFS-TB

Next, we performed a new HFS-TB, this time using M. tuberculosis H37Rv and a dose-

fractionationdesign, fora treatmentdurationof14days.Onmeasurementofertapenem

concentrations,similartothefirststudy,theconcentrationswerealsobestdescribedusinga

onecompartmentmodel;theobservedversuspredictedconcentrationsrevealedaslopeof

0.995±0.002(r2>0.999).Theconcentration-timeprofilesachievedwitheachdoseareshown

bydosingscheduleinFigures2A-C.,togetherwiththeertapenem(plusclavulanate2.5mg/L)

MICof4mg/L.InhibitorysigmoidEmaxmodelfittingbyexposureexpressedaseitherCmax/MIC

orAUC0-24/MICor%TMICrevealedAkaikeInformationCriteriascoresshowninTable1.

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Chapter 6

Figure2.DosefractionationstudytoPK/PDindexlinkedtoertapenemefficacy

TheconcentrationtimeprofilesareshownrelativetotheMIC.Symbolsindicatemeasuredconcentrations,andthelinesmodeledprofile.A.Concentration-timeprofilesofertapenemidentified in theHFS-TBwithevery8hrdosingschedule.B.Concentration-timeprofilesofertapenemidentifiedintheHFS-TBwithevery12hrdosingschedule.C.Concentration-timeprofilesofertapenemidentifiedintheHFS-TBwithonceadaydosingschedule.Giventheconcentrationrange,thescaleobscuresthetimethatconcentrationspersistedaboveMICforsomeofdoses.Fortheblueopencircles,thelowestconcentration,thetimeaboveMICwas0hrs.Forthedoseshownbycayennetrinagles,thetimewas3hr,fortheblackopendiamondsitwas8.32hr,whilefortheopenmagentasquaresitwas11.7hr.Therestcanbereadoffthegraph. D. Inhibitory sigmoidEmaxmodel for%TMIC versusbacterialburden.Onday7, themaximalkill(Emax)was1.14log10CFU/mL,consistentwithfindingsinthefirstHFS-TBdose-effectstudy.Thestudywasforonly14days.Examinationofthecurvesoneachdayshowsthat80%ofmaximalkilloccursarounda%TMICof40%onallsamplingdays,exceptday3whenitoccurswithlowerexposures.

Table1.AkaikeInformationCriteriaScoresforPK/PDindexversusertapenemsterilizingeffect.

Day3 Day7 Day10 Day14AUC0-24/MIC -30.26 -18.99 -31.41 -5.112Cmax/MIC -30.63 -17.19 -31.55 -2.144%TMIC -60.39 -62.96 -49.39 -45.06

Thelowestscoreswerefor%TMIC,whichmeansthisisthePK/PDindexlinkedtomicrobialkill.

Figure2DshowstheinhibitorysigmoidEmaxMICcurvesforeachsamplingdaybasedon%TMIC.

Based on day 10, which had the highest r2 of 0.94, the relationship between %TMIC and

bacterialburdenwas:

log10CFU/mL=5.68-%TMIC2.56/[23.522.56+%TMIC

2.56]

Fromthisrelationship,wecalculatedtheEC80asa%TMICof40.41%ofthedosinginterval.

Indeed,thiscanbereadoffFigure2Daswell,whichshowsthatonegetsthesameexposure

foroptimalkillwhicheversamplingdayisexamined.

MonteCarlosimulationstoidentifyoptimalertapenemdose

InTBpatients,pharmacokineticvariabilityisoneofthemostimportantdriversofsterilizing

effect(23-29).Therefore,inordertoidentifytheoptimalertapenemdoseforpulmonaryTB,

we performedMonte Carlo simulations of 10,000 patientswith pulmonary TB, using the

pharmacokineticparameterestimatesandbetween-patientvariabilityindicesshowninTable

2basedonBurkhardtetal(30-32).Wealsoaccountedfortheertapenempenetrationinto

epithelialliningfluid(ELF)of7.48±8.17%(whichmirrorsthenon-proteinboundconcentration

of5-15%),andthatinlungtissueof23.6±12.3%(33).Weperformedsimulationstodetermine

howmuch1.0gonceaday,or1.0gtwiceaday,or2.0gonceaday,or2.0gtwiceaday,or

3.0g once a daywould achieve or exceed the target exposure, which is %TMIC of 40.41%

associated with optimal sterilizing effect in ELF of patients. For internal validation, we

compared the pharmacokinetic parameters in the 10,000 simulated patients to those of

Burkhardt et al in Table 2, which shows that the simulations faithfully recapitulated the

pharmacokinetic parameters and variability. As an extra external validation step, we

comparedthepharmacokineticparametersinthesimulationstothoseweactuallyobserved

inourMDR-TBpatientsintheNetherlands,asshowninTable2(13).Table2showsthatthe

pharmacokineticparametersandvarianceinoursimulationswerevirtuallyidenticaltothose

weobserved inpatients.Therefore, the simulationswereaccurate in reproducingwhat is

identifiedintheclinic.

93

6


Figure2.DosefractionationstudytoPK/PDindexlinkedtoertapenemefficacy

TheconcentrationtimeprofilesareshownrelativetotheMIC.Symbolsindicatemeasuredconcentrations,andthelinesmodeledprofile.A.Concentration-timeprofilesofertapenemidentified in theHFS-TBwithevery8hrdosingschedule.B.Concentration-timeprofilesofertapenemidentifiedintheHFS-TBwithevery12hrdosingschedule.C.Concentration-timeprofilesofertapenemidentifiedintheHFS-TBwithonceadaydosingschedule.Giventheconcentrationrange,thescaleobscuresthetimethatconcentrationspersistedaboveMICforsomeofdoses.Fortheblueopencircles,thelowestconcentration,thetimeaboveMICwas0hrs.Forthedoseshownbycayennetrinagles,thetimewas3hr,fortheblackopendiamondsitwas8.32hr,whilefortheopenmagentasquaresitwas11.7hr.Therestcanbereadoffthegraph. D. Inhibitory sigmoidEmaxmodel for%TMIC versusbacterialburden.Onday7, themaximalkill(Emax)was1.14log10CFU/mL,consistentwithfindingsinthefirstHFS-TBdose-effectstudy.Thestudywasforonly14days.Examinationofthecurvesoneachdayshowsthat80%ofmaximalkilloccursarounda%TMICof40%onallsamplingdays,exceptday3whenitoccurswithlowerexposures.

Table1.AkaikeInformationCriteriaScoresforPK/PDindexversusertapenemsterilizingeffect.

Day3 Day7 Day10 Day14AUC0-24/MIC -30.26 -18.99 -31.41 -5.112Cmax/MIC -30.63 -17.19 -31.55 -2.144%TMIC -60.39 -62.96 -49.39 -45.06

Thelowestscoreswerefor%TMIC,whichmeansthisisthePK/PDindexlinkedtomicrobialkill.

Figure2DshowstheinhibitorysigmoidEmaxMICcurvesforeachsamplingdaybasedon%TMIC.

Based on day 10, which had the highest r2 of 0.94, the relationship between %TMIC and

bacterialburdenwas:

log10CFU/mL=5.68-%TMIC2.56/[23.522.56+%TMIC

2.56]

Fromthisrelationship,wecalculatedtheEC80asa%TMICof40.41%ofthedosinginterval.

Indeed,thiscanbereadoffFigure2Daswell,whichshowsthatonegetsthesameexposure

foroptimalkillwhicheversamplingdayisexamined.

MonteCarlosimulationstoidentifyoptimalertapenemdose

InTBpatients,pharmacokineticvariabilityisoneofthemostimportantdriversofsterilizing

effect(23-29).Therefore,inordertoidentifytheoptimalertapenemdoseforpulmonaryTB,

we performedMonte Carlo simulations of 10,000 patientswith pulmonary TB, using the

pharmacokineticparameterestimatesandbetween-patientvariabilityindicesshowninTable

2basedonBurkhardtetal(30-32).Wealsoaccountedfortheertapenempenetrationinto

epithelialliningfluid(ELF)of7.48±8.17%(whichmirrorsthenon-proteinboundconcentration

of5-15%),andthatinlungtissueof23.6±12.3%(33).Weperformedsimulationstodetermine

howmuch1.0gonceaday,or1.0gtwiceaday,or2.0gonceaday,or2.0gtwiceaday,or

3.0g once a daywould achieve or exceed the target exposure, which is %TMIC of 40.41%

associated with optimal sterilizing effect in ELF of patients. For internal validation, we

compared the pharmacokinetic parameters in the 10,000 simulated patients to those of

Burkhardt et al in Table 2, which shows that the simulations faithfully recapitulated the

pharmacokinetic parameters and variability. As an extra external validation step, we

comparedthepharmacokineticparametersinthesimulationstothoseweactuallyobserved

inourMDR-TBpatientsintheNetherlands,asshowninTable2(13).Table2showsthatthe

pharmacokineticparametersandvarianceinoursimulationswerevirtuallyidenticaltothose

weobserved inpatients.Therefore, the simulationswereaccurate in reproducingwhat is

identifiedintheclinic.

94

Chapter 6

Table2.Comparisonofpharmacokineticparameterandconcentrationestimatesandrangesin10,000simulatedpatientstothoseactuallyobservedinpatientstreatedwith1G.

SubroutinePRIORbasedonliterature(±SD)

10,000simulatedTBpatients

ObservedinMDR-TBpatients

TotalclearanceinL/hr 2.63±0.83 2.6(0.02-6.00) 2.1(0.09–3.23)VolumeinL 10.6±2.51 11(1.2-19) 7.3(2.61–11.10)Half-lifeinhours - 2.8(2.20-3.70) 2.4(2.05–3.53)AUC0-24inmg*h/L - 448(166-4255) 545(309–1130)

Figure3Ashowsthetargetattainmentprobability(TAP)foreachdoseanddosingschedule

astheMICchanges.Ononeextreme,the1gmonceadaydosehadaTAPlessthan90%once

theMICwas1mg/L,whilethedoseof3gmtwiceadayachievedahighTAPuntil8mg/L,then

fellprecipitouslyat16mg/L.For2gmaday,theTAPfellatanMICof2mg/L.Thismeansthat

thesusceptibilitybreakpointforertapenemplusclavulanatewillfallbetweenMICof1and

16mg/L,andwilldependonthefinaldosechosen.

SinceMICvariabilityisalsoanimportantdeterminantoftherapyresponseinTBpatients(25,

34-36),wealsotookintoaccounttheMICdistribution.Figure3BshowstheertapenemMIC

distribution from 33 MDR-TB patients isolates in the Netherlands, in the presence of

clavulanate.Figure3BshowsthatallisolateswouldhaveMICsbetweengreaterthan1mg/L

andbelow128mg/L.However,inthepastwehaveshownthatertapenemdegradesduring

theMICtesting,andifonceaccountsforthedegradation,thereisa4-tubedilutiondecrease

inMICs; ifclavulanate isaddedandertapenemissupplementedthere isa7-tubedilution

difference(12).Thus,wetransformedtheMICsforthe33clinical isolatesdownby4-tube

dilutionsaswell,asshowninFigure3B.Inthatscenario,only6.5%of isolateshadanMIC

greaterthan1mg/L.

SummationofallTAPstoaccountfordistributionofMICsgivestheproportionof10,000TB

patientswhowouldachieve theargett exposureof%TMICof 40%, termed the cumulative

fractionofresponse(CFR).Figure3CshowstheCFRsfortheonceadaydosingschedulefor

bothobservedMICsandtransformedMICs.ForthetransformedMICsthedoseof2gmaday

hadaCFRof96%.

Figure 3. Target attainment probability and cumulative fraction of response for variousertapenemdoses.

A.Targetattainmentprobabilityfor%TMICof40%asM.tuberculosisMICchanges.NodoseordosingscheduleiseffectiveonceMICsare16mg/L.B.ErtapenemMICdistributioninisolatesfrom the Netherlands, with and without transformation to account for ertapenemdegradation.C.Proportionof10,000patientswhoachievedorexceeded%TMICof40%withonceadaydosing.TheproportionishighlysensitivetotheMIC,andfellonsensitivityanalysis,aworst-casescenario.D.Proportionof10,000patientswhoachievedorexceeded%TMICof40%withtwiceadaydosing.Thetwiceadaydosingscheduleachievedthetargetinhigherproportionsofpatients,evenonsensitivityanalysis.However,giventhehardshipoftwiceaday administration of therapy in TB, we chose the 2gm once a day dose as being mostpractical. WealsoperformedertapenemplusclavalunateMICsin4clinical isolatesincubatedat4°C

versus37°Ctotryandslowdowndrugdegradation:MICswerelowerat4°Cby4,2,3,and

2-tube dilutions. Therefore, we performed sensitivity testing by examining CFR is MIC

transformationwasonly2-tubedilution(worstcasescenario).Figure3Cshowsthatthedose

of2gmonceadaywouldnotachievethetarget in90%ofpatients;nevertheless itwould

achievethisin63%ofpatients,whichisstillreasonable.Figure3Dshowstheresultsoftwice

aweekdosingschedule;aswouldbeexpectedfroma%TMICdrivendrug,thisdosingschedule

95

6


Table2.Comparisonofpharmacokineticparameterandconcentrationestimatesandrangesin10,000simulatedpatientstothoseactuallyobservedinpatientstreatedwith1G.

SubroutinePRIORbasedonliterature(±SD)

10,000simulatedTBpatients

ObservedinMDR-TBpatients

TotalclearanceinL/hr 2.63±0.83 2.6(0.02-6.00) 2.1(0.09–3.23)VolumeinL 10.6±2.51 11(1.2-19) 7.3(2.61–11.10)Half-lifeinhours - 2.8(2.20-3.70) 2.4(2.05–3.53)AUC0-24inmg*h/L - 448(166-4255) 545(309–1130)

Figure3Ashowsthetargetattainmentprobability(TAP)foreachdoseanddosingschedule

astheMICchanges.Ononeextreme,the1gmonceadaydosehadaTAPlessthan90%once

theMICwas1mg/L,whilethedoseof3gmtwiceadayachievedahighTAPuntil8mg/L,then

fellprecipitouslyat16mg/L.For2gmaday,theTAPfellatanMICof2mg/L.Thismeansthat

thesusceptibilitybreakpointforertapenemplusclavulanatewillfallbetweenMICof1and

16mg/L,andwilldependonthefinaldosechosen.

SinceMICvariabilityisalsoanimportantdeterminantoftherapyresponseinTBpatients(25,

34-36),wealsotookintoaccounttheMICdistribution.Figure3BshowstheertapenemMIC

distribution from 33 MDR-TB patients isolates in the Netherlands, in the presence of

clavulanate.Figure3BshowsthatallisolateswouldhaveMICsbetweengreaterthan1mg/L

andbelow128mg/L.However,inthepastwehaveshownthatertapenemdegradesduring

theMICtesting,andifonceaccountsforthedegradation,thereisa4-tubedilutiondecrease

inMICs; ifclavulanate isaddedandertapenemissupplementedthere isa7-tubedilution

difference(12).Thus,wetransformedtheMICsforthe33clinical isolatesdownby4-tube

dilutionsaswell,asshowninFigure3B.Inthatscenario,only6.5%of isolateshadanMIC

greaterthan1mg/L.

SummationofallTAPstoaccountfordistributionofMICsgivestheproportionof10,000TB

patientswhowouldachieve theargett exposureof%TMICof 40%, termed the cumulative

fractionofresponse(CFR).Figure3CshowstheCFRsfortheonceadaydosingschedulefor

bothobservedMICsandtransformedMICs.ForthetransformedMICsthedoseof2gmaday

hadaCFRof96%.

Figure 3. Target attainment probability and cumulative fraction of response for variousertapenemdoses.

A.Targetattainmentprobabilityfor%TMICof40%asM.tuberculosisMICchanges.NodoseordosingscheduleiseffectiveonceMICsare16mg/L.B.ErtapenemMICdistributioninisolatesfrom the Netherlands, with and without transformation to account for ertapenemdegradation.C.Proportionof10,000patientswhoachievedorexceeded%TMICof40%withonceadaydosing.TheproportionishighlysensitivetotheMIC,andfellonsensitivityanalysis,aworst-casescenario.D.Proportionof10,000patientswhoachievedorexceeded%TMICof40%withtwiceadaydosing.Thetwiceadaydosingscheduleachievedthetargetinhigherproportionsofpatients,evenonsensitivityanalysis.However,giventhehardshipoftwiceaday administration of therapy in TB, we chose the 2gm once a day dose as being mostpractical. WealsoperformedertapenemplusclavalunateMICsin4clinical isolatesincubatedat4°C

versus37°Ctotryandslowdowndrugdegradation:MICswerelowerat4°Cby4,2,3,and

2-tube dilutions. Therefore, we performed sensitivity testing by examining CFR is MIC

transformationwasonly2-tubedilution(worstcasescenario).Figure3Cshowsthatthedose

of2gmonceadaywouldnotachievethetarget in90%ofpatients;nevertheless itwould

achievethisin63%ofpatients,whichisstillreasonable.Figure3Dshowstheresultsoftwice

aweekdosingschedule;aswouldbeexpectedfroma%TMICdrivendrug,thisdosingschedule

96

Chapter 6

performedbetter.Thedoseof1.0gmtwiceadaywouldachievetargetexposurein99%of

patients,andonsensitivitytestingwouldstillachievethis in70%ofpatients. Thedoseof

2gmtwiceadaywouldachieve>90%evenonsensitivitytesting.

DiscussionThisisthefirststudythatshowedtheertapenem-clavulanateefficacyandsterilizingeffect,

unlikefindingsinthemurinemodel,likelybecausetheHFS-TBmimickedthehalf-lifeof4hrs

encountered in patients unlike that of 1.0 hours in mice. We were able to recapitulate

ertapenem’spharmacokinetics,anditshalf-lifeof4hrs,asencounteredinTBpatients,which

likelyexplainsbetterefficacyinthismodelcomparedtothatencounteredinmiceinwhich

ertapenemhalf-lifeis1hour.Moreover,dosagessimulatedinthemodelwereinarangethat

would likelybetolerable inpatients.Thisstudyshowedtheadvantageofthehollowfiber

system, namely a better recapitulation of human like pharmacokinetics, and ofmicrobial

sterilizingeffectconditions.TheMonteCarlosimulationsthenintroducedthevariabilitythat

wouldbeencounteredforpharmacokineticparametersbetweenpatientsandMICsbetween

M.tuberculosisstrains.Ourtwo-stepexternalvalidationapproachinthesimulationsensured

thatoursimulationsreflectedclinicalreality;sensitivitytestingaccountedforanyuncertainty

inMICdistribution.Thisallowedustoperformdose-effectstudiesthattakeintoaccountthe

exposure-effectrelationshipasdescribedinthehollowfibermodel,theessentialaspectsof

drugbehaviourinpatientssuchaspharmacokineticvariabilityandthedrugpenetrationratios

to lungs that are important in determining efficacy, and susceptibility ofM. tuberculosis

isolatesencounteredinhospitals.Thisapproach,inmanyexperimentsbasedonthesameM.

tuberculosis isolateweused in thecurrent study,hasbeen found tobe>94%accurate in

identifying clinicaldoses thatareoptimal inTBpatientsbasedon recentpresentation for

regulatoryapproval(19).

Ertapenem–clavulanatemayplayanimportantroleintheintensivephaseofTBtreatment

duetoitssterilizingeffect.Inaddition,theintravenousadministrationmaybemoresuitable

fortheintensivephaseinwhichM/XDR-TBpatientarelikelytobeadministeredinaTBclinic.

AscarbapenemsarealreadypartoftheWHOlistofTBdrugsforM/XDR-TB,thenextstepis

toexploretheuseofertapenem–clavulanateinpatients,usingthe2gonceadaydosewe

identified.Recently,ithasbeenshownthatmeropenem–clavulanatehaspromisingactivity

againstMDR-TBinvitro(37,38).Indeed,imipenem-clavulanateandmeropenem-clavulanate

wereassociatedwithatreatmentsuccessof>57%andcultureconversion>60%,inarecent

systemic analysis of five studies (39). However, since clavulanate is administered as oral

amoxicillin-clavulanate, gastrointestinal side effects may become a problem if this

formulationisadministeredforaprolongeddurationmultipletimesadaywithmeropenem

orimipenem,whichwouldcompromiseabsorptionofotheroraldrugs.Unfortunately,the

currentsuppliersofcarbapenems,arenotinterestedindevelopinganinfusedcombination

of carbapenem and clavulanate. The main advantage of ertapenem is its long half-life

enablingoncedailydosing,whichwouldalsoallowaonceadayclavulanatedose,potentially

reducingside-effects.Thismayevenfacilitatedosinginanoutpatientsetting.Patientsmay

present at the clinic once a day for their drug administration as part of direct observed

treatment,orcouldreceivetreatmentasaonceadayinfusionathomewhensputumculture

negativeinthosecountrieswherethedrugisalreadypartofhomecarefortreatmentofother

chronic infections. Ertapenemhas a labelled infusion timeof only 30minutes,whichwill

facilitatearelativelyshortstayattheoutpatientclinic.Evenmorerapidinfusionhavebeen

exploredandshowedsimilardrugexposureandtolerability(40).Weshowthatadoseof2g

givenoncedailycouldcontributetoaneffectiveregimen.Ertapenemuptoadoseof3ghas

been administered to healthy volunteers (41). Moreover, doses up to 2 g have been

administratedin30minwithoutanyadditionalcomplications(42).However,thereisaneed

for a prospective phase II study exploring safety and efficacy of 2g ertapenem with

clavulanateonceadayinMDR-TBpatients.

Ontheotherhand,theamountoftimeclavulanatehastobearoundtokeeppotentiatingthe

ertapenem is still unclear. Thus, the target concentration to aim with dosing is unclear.

Clavulanate has a shorter half-life compared to ertapenem. However, penetration into

bronchialmucosais118%,anditsproteinbindingisminimalat20%,andlikelyaneffective

concentrationremainsatsiteofeffectevenwhendosedonce.Sinceclavulanate isrenally

eliminated,between-patientvariabilityinsystemicclearance,whichisabout58%,isdriven

mainly by renal function: the lower the creatinine clearance, the less it is cleared (43).

Separate dose-effect studies on clavulanate role will need to be studied, after which

simulationssimilartothecurrentonescanbeperformed.

97

6


performedbetter.Thedoseof1.0gmtwiceadaywouldachievetargetexposurein99%of

patients,andonsensitivitytestingwouldstillachievethis in70%ofpatients. Thedoseof

2gmtwiceadaywouldachieve>90%evenonsensitivitytesting.

DiscussionThisisthefirststudythatshowedtheertapenem-clavulanateefficacyandsterilizingeffect,

unlikefindingsinthemurinemodel,likelybecausetheHFS-TBmimickedthehalf-lifeof4hrs

encountered in patients unlike that of 1.0 hours in mice. We were able to recapitulate

ertapenem’spharmacokinetics,anditshalf-lifeof4hrs,asencounteredinTBpatients,which

likelyexplainsbetterefficacyinthismodelcomparedtothatencounteredinmiceinwhich

ertapenemhalf-lifeis1hour.Moreover,dosagessimulatedinthemodelwereinarangethat

would likelybetolerable inpatients.Thisstudyshowedtheadvantageofthehollowfiber

system, namely a better recapitulation of human like pharmacokinetics, and ofmicrobial

sterilizingeffectconditions.TheMonteCarlosimulationsthenintroducedthevariabilitythat

wouldbeencounteredforpharmacokineticparametersbetweenpatientsandMICsbetween

M.tuberculosisstrains.Ourtwo-stepexternalvalidationapproachinthesimulationsensured

thatoursimulationsreflectedclinicalreality;sensitivitytestingaccountedforanyuncertainty

inMICdistribution.Thisallowedustoperformdose-effectstudiesthattakeintoaccountthe

exposure-effectrelationshipasdescribedinthehollowfibermodel,theessentialaspectsof

drugbehaviourinpatientssuchaspharmacokineticvariabilityandthedrugpenetrationratios

to lungs that are important in determining efficacy, and susceptibility ofM. tuberculosis

isolatesencounteredinhospitals.Thisapproach,inmanyexperimentsbasedonthesameM.

tuberculosis isolateweused in thecurrent study,hasbeen found tobe>94%accurate in

identifying clinicaldoses thatareoptimal inTBpatientsbasedon recentpresentation for

regulatoryapproval(19).

Ertapenem–clavulanatemayplayanimportantroleintheintensivephaseofTBtreatment

duetoitssterilizingeffect.Inaddition,theintravenousadministrationmaybemoresuitable

fortheintensivephaseinwhichM/XDR-TBpatientarelikelytobeadministeredinaTBclinic.

AscarbapenemsarealreadypartoftheWHOlistofTBdrugsforM/XDR-TB,thenextstepis

toexploretheuseofertapenem–clavulanateinpatients,usingthe2gonceadaydosewe

identified.Recently,ithasbeenshownthatmeropenem–clavulanatehaspromisingactivity

againstMDR-TBinvitro(37,38).Indeed,imipenem-clavulanateandmeropenem-clavulanate

wereassociatedwithatreatmentsuccessof>57%andcultureconversion>60%,inarecent

systemic analysis of five studies (39). However, since clavulanate is administered as oral

amoxicillin-clavulanate, gastrointestinal side effects may become a problem if this

formulationisadministeredforaprolongeddurationmultipletimesadaywithmeropenem

orimipenem,whichwouldcompromiseabsorptionofotheroraldrugs.Unfortunately,the

currentsuppliersofcarbapenems,arenotinterestedindevelopinganinfusedcombination

of carbapenem and clavulanate. The main advantage of ertapenem is its long half-life

enablingoncedailydosing,whichwouldalsoallowaonceadayclavulanatedose,potentially

reducingside-effects.Thismayevenfacilitatedosinginanoutpatientsetting.Patientsmay

present at the clinic once a day for their drug administration as part of direct observed

treatment,orcouldreceivetreatmentasaonceadayinfusionathomewhensputumculture

negativeinthosecountrieswherethedrugisalreadypartofhomecarefortreatmentofother

chronic infections. Ertapenemhas a labelled infusion timeof only 30minutes,whichwill

facilitatearelativelyshortstayattheoutpatientclinic.Evenmorerapidinfusionhavebeen

exploredandshowedsimilardrugexposureandtolerability(40).Weshowthatadoseof2g

givenoncedailycouldcontributetoaneffectiveregimen.Ertapenemuptoadoseof3ghas

been administered to healthy volunteers (41). Moreover, doses up to 2 g have been

administratedin30minwithoutanyadditionalcomplications(42).However,thereisaneed

for a prospective phase II study exploring safety and efficacy of 2g ertapenem with

clavulanateonceadayinMDR-TBpatients.

Ontheotherhand,theamountoftimeclavulanatehastobearoundtokeeppotentiatingthe

ertapenem is still unclear. Thus, the target concentration to aim with dosing is unclear.

Clavulanate has a shorter half-life compared to ertapenem. However, penetration into

bronchialmucosais118%,anditsproteinbindingisminimalat20%,andlikelyaneffective

concentrationremainsatsiteofeffectevenwhendosedonce.Sinceclavulanate isrenally

eliminated,between-patientvariabilityinsystemicclearance,whichisabout58%,isdriven

mainly by renal function: the lower the creatinine clearance, the less it is cleared (43).

Separate dose-effect studies on clavulanate role will need to be studied, after which

simulationssimilartothecurrentonescanbeperformed.

98

Chapter 6

Finally,pharmacokinetic/pharmacodynamics-basedsusceptibilitybreakpoints inTB,mostly

derived from hollow fiber model monotherapy studies, have been shown to be highly

accurateindelineatingTBpatientswhofailorrespondtocombinationtherapy(25,34,35).

The 2mg/L ertapenem susceptibility breakpointwe identified for the dose of 2gm a day

shouldbethususedbycliniciansasdecision-makingtooltodetermineifapatientwillrespond

toertapenem therapy. Thisbreakpointwill differ from theepidemiological cut-off value,

whichmaybemoreusefulforepidemiologicaltrackingofacquiredertapenemresistance,as

opposedtoclinicaldecision-making.

Therearesomelimitationstoourstudy.First,weusedtwoisolatesM.tuberculosisforthe

sterilizingeffectexperiments.Inclusionofalargernumberofisolatescouldchangethatfinal

targetexposureassociatedwithoptimalefficacy.However,hollowfiberstudiesinthepast

whenweusedthesestrainswerefoundtobepredictiveoftheoptimalexposuretargetsin

patients for sterilizing effect (15, 24, 25, 44-46). A second limitation is that we used

pharmacokineticdatafromcriticallyillpatientsaspriordataforourMonteCarlosimulations.

Type of disease that a patient has can alter the pharmacokinetic parameters, so that TB

patients could have different pharmacokinetics. However, as shown in Table 2, the

pharmacokineticparameterestimatesinsimulatedpatients,andtheAUC0-24achievedwith

1g doses, were virtually identical to those we have identified in TB patients in the

Netherlands,aspartoftherapeuticdrugmonitoring.Thisvalidatesthatsimulatedpatients

had pharmacokinetic parameters and concentrations similar to those encountered in TB

patients.

Inconclusion,wehaveshownbysimulationofhumandrugexposureofdifferentdosagein

aninvitroinfectionmodelofM.tuberculosisthatertapenem–clavulanatemaybeavaluable

assettoTBtreatment.Basedonavailablepharmacokineticdata,wehaveidentifiedthatthe

doseofertapenemmostsuitabletobetestedinaphaseIIstudyis2goncedaily.AnMIC2

mg/Lshouldbeusedtodefineresistancetothisdrug.

2

Materialsandmethods

We used M. tuberculosis H37Ra (ATCC #25177) and H37Rv (ATCC #27294) for our

experiments,withgrowthandstorageconditionsdescribedbefore(15).Theseisolateshave

beenusedintheHFS-TBbefore,withgoodforecastingaccuracyoftheclinic.Ertapenemwas

purchased fromMerck Sharp & Dohme. Clavulanate was purchased from Sigma-Aldrich.

Drugs were dissolved in sterile water, and syringe filtered for further use. Hollow fiber

cartridgeswerepurchasedfromFiberCell(Frederick,MD,USA).

ThehollowfibersystemmodelofTB

ConstructionoftheHFS-TBtomeasuresterilizingeffecthasbeendescribedindetailinthe

past (15). The system recapitulates concentration-time profiles of drugs encountered in

patients,takingintoaccountthepenetrationintolungs.Inthesterilizingeffectstudies,semi-

dormantM.tuberculosisgrowingintheMiddlebrook7H9brothacidifiedusingaceticacidto

a pH of 5.8 was used, which grow at a rate 8-10-fold slower than log-phase growthM.

tuberculosis(47).TheHFS-TBinthismodeluseacidifiedMiddlebrook7H9brothwithoutoleic

acid,albuminorcatalasebutwith20%dextrose.Theperipheralcompartmentofeachof16

HFS-TB units with circulating acidified Middlebrook 7H9 broth was inoculated with M.

tuberculosis.AllHFS-TBwereincubatedat37°Cunder5%CO2fortheentiretyofthestudy.


5.0, and 10.0 grams were administered into the central compartment via a computer-

controlledsyringepumpover30minutes,asinpatients.Theconcentrationsachievedwith

thedoseswereanAUC0-24of0,25,50,100,200,250,500,1000mg*h/L.Thereweretwo

replicateshollowfibersystemsforeachdoseorAUC0-24.Clavulanatewasalsodispensedvia

syringepumptoachieveapeakof3mg/Lattheendof30minutesinfusion.Mediainflow

andoutflowweresettomimictheertapenemhalf-lifeof4hoursencounteredinpatients;

wetookintoaccountthedegradationrateofthedrugthatwehaveidentifiedinthepast.We

recapitulated pharmacokinetics as described in the INVANZ® ertapenem for injection) for

intravenous(IV)orintramuscular(IM),packageinsert.

ThecentralcompartmentsofeachHFS-TBweresampledsixtimesduringthefirst24hrs,and

ertapenem concentrations measured using a liquid chromatography-tandem mass

spectrometry(LC-MS/MS)methodasdescribedinthepast(48)inordertoverifythathuman-

99

6


Finally,pharmacokinetic/pharmacodynamics-basedsusceptibilitybreakpoints inTB,mostly

derived from hollow fiber model monotherapy studies, have been shown to be highly

accurateindelineatingTBpatientswhofailorrespondtocombinationtherapy(25,34,35).

The 2mg/L ertapenem susceptibility breakpointwe identified for the dose of 2gm a day

shouldbethususedbycliniciansasdecision-makingtooltodetermineifapatientwillrespond

toertapenem therapy. Thisbreakpointwill differ from theepidemiological cut-off value,

whichmaybemoreusefulforepidemiologicaltrackingofacquiredertapenemresistance,as

opposedtoclinicaldecision-making.

Therearesomelimitationstoourstudy.First,weusedtwoisolatesM.tuberculosisforthe

sterilizingeffectexperiments.Inclusionofalargernumberofisolatescouldchangethatfinal

targetexposureassociatedwithoptimalefficacy.However,hollowfiberstudiesinthepast

whenweusedthesestrainswerefoundtobepredictiveoftheoptimalexposuretargetsin

patients for sterilizing effect (15, 24, 25, 44-46). A second limitation is that we used

pharmacokineticdatafromcriticallyillpatientsaspriordataforourMonteCarlosimulations.

Type of disease that a patient has can alter the pharmacokinetic parameters, so that TB

patients could have different pharmacokinetics. However, as shown in Table 2, the

pharmacokineticparameterestimatesinsimulatedpatients,andtheAUC0-24achievedwith

1g doses, were virtually identical to those we have identified in TB patients in the

Netherlands,aspartoftherapeuticdrugmonitoring.Thisvalidatesthatsimulatedpatients

had pharmacokinetic parameters and concentrations similar to those encountered in TB

patients.

Inconclusion,wehaveshownbysimulationofhumandrugexposureofdifferentdosagein

aninvitroinfectionmodelofM.tuberculosisthatertapenem–clavulanatemaybeavaluable

assettoTBtreatment.Basedonavailablepharmacokineticdata,wehaveidentifiedthatthe

doseofertapenemmostsuitabletobetestedinaphaseIIstudyis2goncedaily.AnMIC2

mg/Lshouldbeusedtodefineresistancetothisdrug.

Materialsandmethods

We used M. tuberculosis H37Ra (ATCC #25177) and H37Rv (ATCC #27294) for our

experiments,withgrowthandstorageconditionsdescribedbefore(15).Theseisolateshave

beenusedintheHFS-TBbefore,withgoodforecastingaccuracyoftheclinic.Ertapenemwas

purchased fromMerck Sharp & Dohme. Clavulanate was purchased from Sigma-Aldrich.

Drugs were dissolved in sterile water, and syringe filtered for further use. Hollow fiber

cartridgeswerepurchasedfromFiberCell(Frederick,MD,USA).

ThehollowfibersystemmodelofTB

ConstructionoftheHFS-TBtomeasuresterilizingeffecthasbeendescribedindetailinthe

past (15). The system recapitulates concentration-time profiles of drugs encountered in

patients,takingintoaccountthepenetrationintolungs.Inthesterilizingeffectstudies,semi-

dormantM.tuberculosisgrowingintheMiddlebrook7H9brothacidifiedusingaceticacidto

a pH of 5.8 was used, which grow at a rate 8-10-fold slower than log-phase growthM.

tuberculosis(47).TheHFS-TBinthismodeluseacidifiedMiddlebrook7H9brothwithoutoleic

acid,albuminorcatalasebutwith20%dextrose.Theperipheralcompartmentofeachof16

HFS-TB units with circulating acidified Middlebrook 7H9 broth was inoculated with M.

tuberculosis.AllHFS-TBwereincubatedat37°Cunder5%CO2fortheentiretyofthestudy.


5.0, and 10.0 grams were administered into the central compartment via a computer-

controlledsyringepumpover30minutes,asinpatients.Theconcentrationsachievedwith

thedoseswereanAUC0-24of0,25,50,100,200,250,500,1000mg*h/L.Thereweretwo

replicateshollowfibersystemsforeachdoseorAUC0-24.Clavulanatewasalsodispensedvia

syringepumptoachieveapeakof3mg/Lattheendof30minutesinfusion.Mediainflow

andoutflowweresettomimictheertapenemhalf-lifeof4hoursencounteredinpatients;

wetookintoaccountthedegradationrateofthedrugthatwehaveidentifiedinthepast.We

recapitulated pharmacokinetics as described in the INVANZ® ertapenem for injection) for

intravenous(IV)orintramuscular(IM),packageinsert.

ThecentralcompartmentsofeachHFS-TBweresampledsixtimesduringthefirst24hrs,and

ertapenem concentrations measured using a liquid chromatography-tandem mass

spectrometry(LC-MS/MS)methodasdescribedinthepast(48)inordertoverifythathuman-

100

Chapter 6

likepharmacokineticshadbeenachieved.Ertapenemconcentrationsweremodelledusinga

onecompartmentpharmacokineticmodelwithfirstorderinputandelimination,usingADAPT

5software,asdescribedinthepast(15,24,25,44-46).Theseactualexposuresachievedin

theHFS-TBweresubsequentlyused in thePK/PDanalyses. Inorder toenumerate theM.

tuberculosisburdenasCFU/mL,theperipheralcompartmentofeachHFS-TBwassampledon

days0,3,7,14,21,and28.Sampleswerewashedandprocessedasdescribedinthepast(15)

andspreadonMiddlebrook7H10agarsupplementedwith10%Oleicacid-dextrose-catalase.

The cultures were incubated for 21 days at 370C with 5% CO2 before the colonies were

counted.

Identificationofoptimalertapenemdoseusingcomputer-aidedclinicaltrialsimulations.

Forthedomainofinput,weutilizedthepharmacokineticparameterestimatesandbetween-

patient variability indices identifiedbyBurkhardtetal (31).Weperformed simulations to

determinehowmuch1.0gonceaday,or1.0gtwiceaday,or2.0gonceaday,or2.0gtwicea

day,or3.0gonceadayor3.0gmtwiceadaywouldachieveorexceedthetargetexposure,

whichis%TMICassociatedwithoptimalsterilizingeffectinlungtissueofpatients.

Acknowledgements

None.

Funding

ThisworkwassupportedbytheNationalInstituteofGeneralMedicalSciencesoftheNational

InstitutesofHealthNewInnovatorAwardDP2OD001886-01toandtheNationalInstituteof

AllergyandInfectiousDiseasesawardR01AI079497toTawandaGumbo.

Transparencydeclaration

TGisaconsultantforLuminaCaresolutions,andfoundedJacarandaBiomed,Inc.JWAreports

personalfeesfromPfizer,Astellas,MSD,Gilead,grantsfromPfizer,Astellas,MSD,alloutside

thesubmittedwork.Allotherauthorshavenoconflictofinterest.

References 1. DhedaK,GumboT,MaartensG,DooleyKE,McNerneyR,MurrayM,FurinJ,NardellEA,

LondonL,LessemE,TheronG,vanHeldenP,NiemannS,MerkerM,DowdyD,VanRieA,

SiuGK,PasipanodyaJG,RodriguesC,ClarkTG,SirgelFA,EsmailA,LinHH,AtreSR,Schaaf

HS,ChangKC,LangeC,NahidP,UdwadiaZF,HorsburghCR,Jr.,ChurchyardGJ,Menzies

D,HesselingAC,NuermbergerE,McIlleronH,FennellyKP,GoemaereE,JaramilloE,Low

M, Jara CM, Padayatchi N, Warren RM. 2017. The epidemiology, pathogenesis,

transmission, diagnosis, and management of multidrug-resistant, extensively drug-

resistant, and incurable tuberculosis. Lancet Respir Med doi:10.1016/S2213-

2600(17)30079-6.

2. DhedaK,GumboT,GandhiNR,MurrayM,TheronG,UdwadiaZ,MiglioriGB,WarrenR.

2014. Global control of tuberculosis: from extensively drug-resistant to untreatable

tuberculosis.LancetRespirMed2:321-338.

3. LangeC,Abubakar I,AlffenaarJW,BothamleyG,CamineroJA,CarvalhoAC,ChangKC,

CodecasaL,CorreiaA,CruduV,DaviesP,DedicoatM,DrobniewskiF,DuarteR,EhlersC,

ErkensC,GolettiD,GuntherG,IbraimE,KampmannB,KuksaL,deLW,vanLF,vanLJ,

MatteelliA,MenziesD,MonederoI,RichterE,Rusch-GerdesS,SandgrenA,ScardigliA,

SkrahinaA,TortoliE,VolchenkovG,WagnerD,vanderWerfMJ,WilliamsB,YewWW,

Zellweger JP, Cirillo DM. 2014. Management of patients with multidrug-

resistant/extensively drug-resistant tuberculosis in Europe: a TBNET consensus

statement.EurRespirJ44:23-63.

4. UdwadiaZF,AmaleRA,AjbaniKK,RodriguesC.2012.Totallydrug-resistanttuberculosis

inIndia.ClinInfectDis54:579-581.

5. AlsaadN,WilffertB,vanAltenaR,deLangeWC,vanderWerfTS,KosterinkJG,Alffenaar

JW. 2014. Potential antimicrobial agents for the treatment of multidrug-resistant

tuberculosis.EurRespirJ43:884-897.

6. Hugonnet JE, Tremblay LW,BoshoffHI,BarryCE, III, Blanchard JS. 2009.Meropenem-


Science323:1215-1218.

101

6


likepharmacokineticshadbeenachieved.Ertapenemconcentrationsweremodelledusinga

onecompartmentpharmacokineticmodelwithfirstorderinputandelimination,usingADAPT

5software,asdescribedinthepast(15,24,25,44-46).Theseactualexposuresachievedin

theHFS-TBweresubsequentlyused in thePK/PDanalyses. Inorder toenumerate theM.

tuberculosisburdenasCFU/mL,theperipheralcompartmentofeachHFS-TBwassampledon

days0,3,7,14,21,and28.Sampleswerewashedandprocessedasdescribedinthepast(15)

andspreadonMiddlebrook7H10agarsupplementedwith10%Oleicacid-dextrose-catalase.

The cultures were incubated for 21 days at 370C with 5% CO2 before the colonies were

counted.

Identificationofoptimalertapenemdoseusingcomputer-aidedclinicaltrialsimulations.

Forthedomainofinput,weutilizedthepharmacokineticparameterestimatesandbetween-

patient variability indices identifiedbyBurkhardtetal (31).Weperformed simulations to

determinehowmuch1.0gonceaday,or1.0gtwiceaday,or2.0gonceaday,or2.0gtwicea

day,or3.0gonceadayor3.0gmtwiceadaywouldachieveorexceedthetargetexposure,

whichis%TMICassociatedwithoptimalsterilizingeffectinlungtissueofpatients.

Acknowledgements

None.

Funding

ThisworkwassupportedbytheNationalInstituteofGeneralMedicalSciencesoftheNational

InstitutesofHealthNewInnovatorAwardDP2OD001886-01toandtheNationalInstituteof

AllergyandInfectiousDiseasesawardR01AI079497toTawandaGumbo.

Transparencydeclaration

TGisaconsultantforLuminaCaresolutions,andfoundedJacarandaBiomed,Inc.JWAreports

personalfeesfromPfizer,Astellas,MSD,Gilead,grantsfromPfizer,Astellas,MSD,alloutside

thesubmittedwork.Allotherauthorshavenoconflictofinterest.

References 1. DhedaK,GumboT,MaartensG,DooleyKE,McNerneyR,MurrayM,FurinJ,NardellEA,

LondonL,LessemE,TheronG,vanHeldenP,NiemannS,MerkerM,DowdyD,VanRieA,

SiuGK,PasipanodyaJG,RodriguesC,ClarkTG,SirgelFA,EsmailA,LinHH,AtreSR,Schaaf

HS,ChangKC,LangeC,NahidP,UdwadiaZF,HorsburghCR,Jr.,ChurchyardGJ,Menzies

D,HesselingAC,NuermbergerE,McIlleronH,FennellyKP,GoemaereE,JaramilloE,Low

M, Jara CM, Padayatchi N, Warren RM. 2017. The epidemiology, pathogenesis,

transmission, diagnosis, and management of multidrug-resistant, extensively drug-

resistant, and incurable tuberculosis. Lancet Respir Med doi:10.1016/S2213-

2600(17)30079-6.

2. DhedaK,GumboT,GandhiNR,MurrayM,TheronG,UdwadiaZ,MiglioriGB,WarrenR.

2014. Global control of tuberculosis: from extensively drug-resistant to untreatable

tuberculosis.LancetRespirMed2:321-338.

3. LangeC,Abubakar I,AlffenaarJW,BothamleyG,CamineroJA,CarvalhoAC,ChangKC,

CodecasaL,CorreiaA,CruduV,DaviesP,DedicoatM,DrobniewskiF,DuarteR,EhlersC,

ErkensC,GolettiD,GuntherG,IbraimE,KampmannB,KuksaL,deLW,vanLF,vanLJ,

MatteelliA,MenziesD,MonederoI,RichterE,Rusch-GerdesS,SandgrenA,ScardigliA,

SkrahinaA,TortoliE,VolchenkovG,WagnerD,vanderWerfMJ,WilliamsB,YewWW,

Zellweger JP, Cirillo DM. 2014. Management of patients with multidrug-

resistant/extensively drug-resistant tuberculosis in Europe: a TBNET consensus

statement.EurRespirJ44:23-63.

4. UdwadiaZF,AmaleRA,AjbaniKK,RodriguesC.2012.Totallydrug-resistanttuberculosis

inIndia.ClinInfectDis54:579-581.

5. AlsaadN,WilffertB,vanAltenaR,deLangeWC,vanderWerfTS,KosterinkJG,Alffenaar

JW. 2014. Potential antimicrobial agents for the treatment of multidrug-resistant

tuberculosis.EurRespirJ43:884-897.

6. Hugonnet JE, Tremblay LW,BoshoffHI,BarryCE, III, Blanchard JS. 2009.Meropenem-


Science323:1215-1218.

102

Chapter 6

7. BolhuisMS,vanderLaanT,KosterinkJG,vanderWerfTS,vanSD,AlffenaarJW.2014.In

vitrosynergybetweenlinezolidandclarithromycinagainstMycobacteriumtuberculosis.

EurRespirJ44:808-811.

8. Pasipanodya J, Gumbo T. 2011. An oracle: antituberculosis pharmacokinetics-

pharmacodynamics,clinicalcorrelation,andclinicaltrialsimulationstopredictthefuture.


9. VezirisN,TruffotC,MainardiJL,JarlierV.2011.Activityofcarbapenemscombinedwith

clavulanate against murine tuberculosis. Antimicrobial Agents and Chemotherapy

55:2597-2600.

10. TiberiS,SotgiuG,D'AmbrosioL,CentisR,AbdoArbexM,AlarconArrascueE,Alffenaar

JW,CamineroJA,GagaM,GualanoG,SkrahinaA,SolovicI,SulisG,TadoliniM,Alarcon

GuizadoV,DeLorenzoS,RobyAriasAJ,ScardigliA,AkkermanOW,AleksaA,Artsukevich

J,AuchynkaV,BoniniEH,ChongMarinFA,CollahuazoLopezL,deVriesG,DoreS,Kunst

H,MatteelliA,MoschosC,PalmieriF,PapavasileiouA,PayenMC,PianaA,SpanevelloA,

Vargas Vasquez D, Viggiani P, White V, Zumla A, Migliori GB. 2016. Comparison of

effectiveness and safety of imipenem/clavulanate- versus meropenem/clavulanate-

containingregimensinthetreatmentofMDR-andXDR-TB.EurRespirJ47:1758-1766.

11. TiberiS,PayenMC,SotgiuG,D'AmbrosioL,AlarconGuizadoV,AlffenaarJW,AbdoArbex

M,CamineroJA,CentisR,DeLorenzoS,GagaM,GualanoG,RobyAriasAJ,ScardigliA,

SkrahinaA,Solovic I,SulisG,TadoliniM,AkkermanOW,AlarconArrascueE,AleskaA,

AvchinkoV,BoniniEH,ChongMarinFA,CollahuazoLopezL,deVriesG,DoreS,KunstH,

Matteelli A,Moschos C, Palmieri F, Papavasileiou A, Spanevello A, Vargas Vasquez D,

Viggiani P, White V, Zumla A, Migliori GB. 2016. Effectiveness and safety of

meropenem/clavulanate-containingregimensinthetreatmentofMDR-andXDR-TB.Eur

RespirJ47:1235-1243.

12. SrivastavaS,vanRijnSP,WesselsAM,AlffenaarJW,GumboT.2016.SusceptibilityTesting

ofAntibioticsThatDegradeFasterthantheDoublingTimeofSlow-GrowingMycobacteria:

Ertapenem Sterilizing Effect versus Mycobacterium tuberculosis. Antimicrob Agents

Chemother60:3193-3195.

13. vanRijnSP,vanAltenaR,AkkermanOW,vanSoolingenD,vanderLaanT,deLangeWC,

Kosterink JG, vanderWerf TS,Alffenaar JW. 2016. Pharmacokinetics of ertapenem in

patientswithmultidrug-resistanttuberculosis.EurRespirJ47:1229-1234.

14. TiberiS,D'AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJW,Migliori


experience.EurRespirJ47:333-336.


pyrazinamideinanovelinvitromodeloftuberculosisforsterilizingeffect:aparadigmfor

fasterassessmentofnewantituberculosisdrugs.AntimicrobAgentsChemother53:3197-

3204.

16. Heifets L, Lindholm-Levy P. 1992. Pyrazinamide sterilizing activity in vitro against

semidormant Mycobacterium tuberculosis bacterial populations. AmRevRespirDis

145:1223-1225.

17. MitchisonDA.1985.Theactionofantituberculosisdrugsinshort-coursechemotherapy.

Tubercle66:219-225.

18. Gumbo T, Pasipanodya JG, Nuermberger E, Romero K, Hanna D. 2015. Correlations

BetweentheHollowFiberModelofTuberculosisandTherapeuticEventsinTuberculosis

Patients:LearnandConfirm.ClinInfectDis61Suppl1:S18-24.

19. Gumbo T, Pasipanodya JG, Romero K, Hanna D, Nuermberger E. 2015. Forecasting

accuracyofthehollowfibermodeloftuberculosisforclinicaltherapeuticoutcomes.Clin

InfectDis61Suppl1:S25-31.

20. PasipanodyaJG,NuermbergerE,RomeroK,HannaD,GumboT.2015.SystematicAnalysis

ofHollowFiberModelofTuberculosisExperiments.ClinInfectDis61Suppl1:S10-17.

21. GumboT,LenaertsAJ,HannaD,RomeroK,NuermbergerE.2015.Nonclinicalmodelsfor

antituberculosisdrugdevelopment:alandscapeanalysis.JInfectDis211Suppl3:S83-95.

22. AkaikeH.1974.Anewlookatthestatisticalmodelidentification,vol19,p716-723.

23. Swaminathan S, Pasipanodya JG, Ramachandran G, Hemanth Kumar AK, Srivastava S,

DeshpandeD,NuermbergerE,GumboT.2016.DrugConcentrationThresholdsPredictive

ofTherapyFailureandDeathinChildrenWithTuberculosis:BreadCrumbTrailsinRandom

Forests.ClinInfectDis63:S63-S74.

103

6


7. BolhuisMS,vanderLaanT,KosterinkJG,vanderWerfTS,vanSD,AlffenaarJW.2014.In

vitrosynergybetweenlinezolidandclarithromycinagainstMycobacteriumtuberculosis.

EurRespirJ44:808-811.

8. Pasipanodya J, Gumbo T. 2011. An oracle: antituberculosis pharmacokinetics-

pharmacodynamics,clinicalcorrelation,andclinicaltrialsimulationstopredictthefuture.



clavulanate against murine tuberculosis. Antimicrobial Agents and Chemotherapy

55:2597-2600.

10. TiberiS,SotgiuG,D'AmbrosioL,CentisR,AbdoArbexM,AlarconArrascueE,Alffenaar

JW,CamineroJA,GagaM,GualanoG,SkrahinaA,SolovicI,SulisG,TadoliniM,Alarcon

GuizadoV,DeLorenzoS,RobyAriasAJ,ScardigliA,AkkermanOW,AleksaA,Artsukevich

J,AuchynkaV,BoniniEH,ChongMarinFA,CollahuazoLopezL,deVriesG,DoreS,Kunst

H,MatteelliA,MoschosC,PalmieriF,PapavasileiouA,PayenMC,PianaA,SpanevelloA,

Vargas Vasquez D, Viggiani P, White V, Zumla A, Migliori GB. 2016. Comparison of

effectiveness and safety of imipenem/clavulanate- versus meropenem/clavulanate-

containingregimensinthetreatmentofMDR-andXDR-TB.EurRespirJ47:1758-1766.

11. TiberiS,PayenMC,SotgiuG,D'AmbrosioL,AlarconGuizadoV,AlffenaarJW,AbdoArbex

M,CamineroJA,CentisR,DeLorenzoS,GagaM,GualanoG,RobyAriasAJ,ScardigliA,

SkrahinaA,Solovic I,SulisG,TadoliniM,AkkermanOW,AlarconArrascueE,AleskaA,

AvchinkoV,BoniniEH,ChongMarinFA,CollahuazoLopezL,deVriesG,DoreS,KunstH,

Matteelli A,Moschos C, Palmieri F, Papavasileiou A, Spanevello A, Vargas Vasquez D,

Viggiani P, White V, Zumla A, Migliori GB. 2016. Effectiveness and safety of

meropenem/clavulanate-containingregimensinthetreatmentofMDR-andXDR-TB.Eur

RespirJ47:1235-1243.

12. SrivastavaS,vanRijnSP,WesselsAM,AlffenaarJW,GumboT.2016.SusceptibilityTesting

ofAntibioticsThatDegradeFasterthantheDoublingTimeofSlow-GrowingMycobacteria:

Ertapenem Sterilizing Effect versus Mycobacterium tuberculosis. Antimicrob Agents



Kosterink JG, vanderWerf TS,Alffenaar JW. 2016. Pharmacokinetics of ertapenem in

patientswithmultidrug-resistanttuberculosis.EurRespirJ47:1229-1234.

14. TiberiS,D'AmbrosioL,DeLorenzoS,ViggianiP,CentisR,SotgiuG,AlffenaarJW,Migliori




pyrazinamideinanovelinvitromodeloftuberculosisforsterilizingeffect:aparadigmfor

fasterassessmentofnewantituberculosisdrugs.AntimicrobAgentsChemother53:3197-

3204.

16. Heifets L, Lindholm-Levy P. 1992. Pyrazinamide sterilizing activity in vitro against

semidormant Mycobacterium tuberculosis bacterial populations. AmRevRespirDis

145:1223-1225.

17. MitchisonDA.1985.Theactionofantituberculosisdrugsinshort-coursechemotherapy.

Tubercle66:219-225.

18. Gumbo T, Pasipanodya JG, Nuermberger E, Romero K, Hanna D. 2015. Correlations

BetweentheHollowFiberModelofTuberculosisandTherapeuticEventsinTuberculosis

Patients:LearnandConfirm.ClinInfectDis61Suppl1:S18-24.

19. Gumbo T, Pasipanodya JG, Romero K, Hanna D, Nuermberger E. 2015. Forecasting

accuracyofthehollowfibermodeloftuberculosisforclinicaltherapeuticoutcomes.Clin

InfectDis61Suppl1:S25-31.

20. PasipanodyaJG,NuermbergerE,RomeroK,HannaD,GumboT.2015.SystematicAnalysis

ofHollowFiberModelofTuberculosisExperiments.ClinInfectDis61Suppl1:S10-17.

21. GumboT,LenaertsAJ,HannaD,RomeroK,NuermbergerE.2015.Nonclinicalmodelsfor

antituberculosisdrugdevelopment:alandscapeanalysis.JInfectDis211Suppl3:S83-95.

22. AkaikeH.1974.Anewlookatthestatisticalmodelidentification,vol19,p716-723.

23. Swaminathan S, Pasipanodya JG, Ramachandran G, Hemanth Kumar AK, Srivastava S,

DeshpandeD,NuermbergerE,GumboT.2016.DrugConcentrationThresholdsPredictive

ofTherapyFailureandDeathinChildrenWithTuberculosis:BreadCrumbTrailsinRandom

Forests.ClinInfectDis63:S63-S74.

104

Chapter 6

24. PasipanodyaJG,McIlleronH,BurgerA,WashPA,SmithP,GumboT.2013.Serumdrug

concentrations predictive of pulmonary tuberculosis outcomes. J Infect Dis 208:1464-

1473.

25. Chigutsa E, Pasipanodya JG, VisserME, vanHelden PD, Smith PJ, Sirgel FA,Gumbo T,

McIlleronH. 2015. Impact of nonlinear interactions of pharmacokinetics andMICs on

sputum bacillary kill rates as amarker of sterilizing effect in tuberculosis. Antimicrob

AgentsChemother59:38-45.

26. ModongoC,PasipanodyaJG,MagaziBT,SrivastavaS,ZetolaNM,WilliamsSM,SirugoG,

GumboT.2016.Artificial intelligenceandamikacinexposurespredictiveofoutcomein

multidrug-resistant tuberculosis patients. Antimicrob Agents Chemother

doi:10.1128/AAC.00962-16.

27. PasipanodyaJG,SrivastavaS,GumboT.2012.Meta-analysisofclinicalstudiessupports

the pharmacokinetic variability hypothesis for acquired drug resistance and failure of

antituberculosistherapy.ClinInfectDis55:169-177.

28. SrivastavaS,PasipanodyaJG,RamachandranG,DeshpandeD,ShufordS,CrosswellHE,

CirrincioneKN,ShermanCM,SwaminathanS,GumboT.2016.ALong-termCo-perfused

Disseminated Tuberculosis-3D Liver Hollow Fiber Model for Both Drug Efficacy and

HepatotoxicityinBabies.EBioMedicine6:126-138.

29. Srivastava S, Pasipanodya JG, Meek C, Leff R, Gumbo T. 2011. Multidrug-resistant

tuberculosis not due to noncompliance but to between-patient pharmacokinetic

variability.JInfectDis204:1951-1959.

30. BurkhardtO,KumarV,KatterweD,Majcher-PeszynskaJ,DrewelowB,DerendorfH,Welte

T. 2007. Ertapenem in critically ill patients with early-onset ventilator-associated

pneumonia: pharmacokineticswith special consideration of free-drug concentration. J

AntimicrobChemother59:277-284.

31. GumboT,Angulo-BarturenI,Ferrer-BazagaS.2015.Pharmacokinetic-pharmacodynamic

and dose-response relationships of antituberculosis drugs: recommendations and

standardsforindustryandacademia.JInfectDis211Suppl3:S96-S106.

32. RomeroK,SinhaV,AllerheiligenS,DanhofM,PinheiroJ,KruhlakN,WangY,WangSJ,

SauerJM,MarierJF,CorriganB,RogersJ,LambersHeerspinkHJ,GumboT,VisP,Watkins

P,MorrisonT,GillespieW,GordonMF,StephensonD,HannaD,PfisterM,LalondeR,

Colatsky T. 2014. Modeling and simulation for medical product development and

evaluation: highlights from the FDA-C-Path-ISOP 2013 workshop. J Pharmacokinet

Pharmacodyn41:545-552.

33. BurkhardtO,Majcher-PeszynskaJ,BornerK,MundkowskiR,DrewelowB,DerendorfH,

Welte T. 2005. Penetration of ertapenem into different pulmonary compartments of

patientsundergoinglungsurgery.JClinPharmacol45:659-665.

34. GumboT,ChigutsaE,PasipanodyaJ,VisserM,vanHeldenPD,SirgelFA,McIlleronH.2014.

The pyrazinamide susceptibility breakpoint above which combination therapy fails. J


35. GumboT,PasipanodyaJG,WashP,BurgerA,McIlleronH.2014.Redefiningmultidrug-

resistant tuberculosis based on clinical response to combination therapy. Antimicrob


36. Zheng X, Zheng R, Hu Y, Werngren J, Davies FL, Mansjo M, Xu B, Hoffner S. 2016.

DeterminationofMinimumInhibitoryConcentration (MIC)Breakpoints forsecond-line

drugsassociatedwithclinicaloutcomesinmultidrug-resistanttuberculosistreatmentin

China.AntimicrobialAgentsandChemotherapy.

37. DaviesForsmanL,GiskeCG,BruchfeldJ,SchonT,JureenP,AngebyK.2015.Meropenem-

clavulanate has high in vitro activity against multidrug-resistant Mycobacterium

tuberculosis.IntJMycobacteriol4Suppl1:80-81.



meropenemandamoxicillin/clavulanate.JAntimicrobChemother68:366-369.

39. SotgiuG,D'AmbrosioL,CentisR,TiberiS,EspositoS,DoreS,SpanevelloA,MiglioriGB.

2016.Carbapenems to TreatMultidrug andExtensivelyDrug-Resistant Tuberculosis:A

SystematicReview.IntJMolSci17:373.

40. Wiskirchen DE, Housman ST, Quintiliani R, Nicolau DP, Kuti JL. 2013. Comparative

pharmacokinetics, pharmacodynamics, and tolerability of ertapenem 1 gram/day

administered as a rapid 5-minute infusion versus the standard 30-minute infusion in

healthyadultvolunteers.Pharmacotherapy33:266-274.

41. MajumdarAK,MussonDG,BirkKL,KitchenCJ,HollandS,McCreaJ,MistryG,HesneyM,

XiL,LiSX,HaesenR,BlumRA,LinsRL,GreenbergH,WaldmanS,DeutschP,RogersJD.

2002.Pharmacokineticsofertapenem inhealthyyoungvolunteers.AntimicrobAgents


105

6


24. PasipanodyaJG,McIlleronH,BurgerA,WashPA,SmithP,GumboT.2013.Serumdrug

concentrations predictive of pulmonary tuberculosis outcomes. J Infect Dis 208:1464-

1473.

25. Chigutsa E, Pasipanodya JG, VisserME, vanHelden PD, Smith PJ, Sirgel FA,Gumbo T,

McIlleronH. 2015. Impact of nonlinear interactions of pharmacokinetics andMICs on

sputum bacillary kill rates as amarker of sterilizing effect in tuberculosis. Antimicrob


26. ModongoC,PasipanodyaJG,MagaziBT,SrivastavaS,ZetolaNM,WilliamsSM,SirugoG,

GumboT.2016.Artificial intelligenceandamikacinexposurespredictiveofoutcomein

multidrug-resistant tuberculosis patients. Antimicrob Agents Chemother

doi:10.1128/AAC.00962-16.

27. PasipanodyaJG,SrivastavaS,GumboT.2012.Meta-analysisofclinicalstudiessupports

the pharmacokinetic variability hypothesis for acquired drug resistance and failure of

antituberculosistherapy.ClinInfectDis55:169-177.

28. SrivastavaS,PasipanodyaJG,RamachandranG,DeshpandeD,ShufordS,CrosswellHE,

CirrincioneKN,ShermanCM,SwaminathanS,GumboT.2016.ALong-termCo-perfused

Disseminated Tuberculosis-3D Liver Hollow Fiber Model for Both Drug Efficacy and

HepatotoxicityinBabies.EBioMedicine6:126-138.

29. Srivastava S, Pasipanodya JG, Meek C, Leff R, Gumbo T. 2011. Multidrug-resistant

tuberculosis not due to noncompliance but to between-patient pharmacokinetic

variability.JInfectDis204:1951-1959.



pneumonia: pharmacokineticswith special consideration of free-drug concentration. J


31. GumboT,Angulo-BarturenI,Ferrer-BazagaS.2015.Pharmacokinetic-pharmacodynamic

and dose-response relationships of antituberculosis drugs: recommendations and

standardsforindustryandacademia.JInfectDis211Suppl3:S96-S106.

32. RomeroK,SinhaV,AllerheiligenS,DanhofM,PinheiroJ,KruhlakN,WangY,WangSJ,

SauerJM,MarierJF,CorriganB,RogersJ,LambersHeerspinkHJ,GumboT,VisP,Watkins

P,MorrisonT,GillespieW,GordonMF,StephensonD,HannaD,PfisterM,LalondeR,

Colatsky T. 2014. Modeling and simulation for medical product development and

evaluation: highlights from the FDA-C-Path-ISOP 2013 workshop. J Pharmacokinet

Pharmacodyn41:545-552.

33. BurkhardtO,Majcher-PeszynskaJ,BornerK,MundkowskiR,DrewelowB,DerendorfH,

Welte T. 2005. Penetration of ertapenem into different pulmonary compartments of

patientsundergoinglungsurgery.JClinPharmacol45:659-665.

34. GumboT,ChigutsaE,PasipanodyaJ,VisserM,vanHeldenPD,SirgelFA,McIlleronH.2014.

The pyrazinamide susceptibility breakpoint above which combination therapy fails. J


35. GumboT,PasipanodyaJG,WashP,BurgerA,McIlleronH.2014.Redefiningmultidrug-

resistant tuberculosis based on clinical response to combination therapy. Antimicrob


36. Zheng X, Zheng R, Hu Y, Werngren J, Davies FL, Mansjo M, Xu B, Hoffner S. 2016.

DeterminationofMinimumInhibitoryConcentration (MIC)Breakpoints forsecond-line

drugsassociatedwithclinicaloutcomesinmultidrug-resistanttuberculosistreatmentin

China.AntimicrobialAgentsandChemotherapy.

37. DaviesForsmanL,GiskeCG,BruchfeldJ,SchonT,JureenP,AngebyK.2015.Meropenem-

clavulanate has high in vitro activity against multidrug-resistant Mycobacterium

tuberculosis.IntJMycobacteriol4Suppl1:80-81.



meropenemandamoxicillin/clavulanate.JAntimicrobChemother68:366-369.

39. SotgiuG,D'AmbrosioL,CentisR,TiberiS,EspositoS,DoreS,SpanevelloA,MiglioriGB.

2016.Carbapenems to TreatMultidrug andExtensivelyDrug-Resistant Tuberculosis:A

SystematicReview.IntJMolSci17:373.

40. Wiskirchen DE, Housman ST, Quintiliani R, Nicolau DP, Kuti JL. 2013. Comparative

pharmacokinetics, pharmacodynamics, and tolerability of ertapenem 1 gram/day

administered as a rapid 5-minute infusion versus the standard 30-minute infusion in

healthyadultvolunteers.Pharmacotherapy33:266-274.



2002.Pharmacokineticsofertapenem inhealthyyoungvolunteers.AntimicrobAgents


106

Chapter 6

42. TepplerH,GesserRM,FriedlandIR,WoodsGL,MeibohmA,HermanG,MistryG,Isaacs

R.2004.Safetyandtolerabilityofertapenem.JAntimicrobChemother53Suppl2:ii75-81.

43. CarlierM, NoeM, DeWaele JJ, Stove V, Verstraete AG, Lipman J, Roberts JA. 2013.

Population pharmacokinetics and dosing simulations of amoxicillin/clavulanic acid in

criticallyillpatients.JAntimicrobChemother68:2600-2608.

44. Gumbo T, Louie A, Deziel MR, Liu W, Parsons LM, Salfinger M, Drusano GL. 2007.

Concentration-dependent Mycobacterium tuberculosis killing and prevention of

resistancebyrifampin.AntimicrobAgentsChemother51:3781-3788.

45. Gumbo T, Louie A, Liu W, Brown D, Ambrose PG, Bhavnani SM, Drusano GL. 2007.

Isoniazidbactericidalactivityandresistanceemergence:integratingpharmacodynamics

and pharmacogenomics to predict efficacy in different ethnic populations. Antimicrob


46. SrivastavaS,MusukaS,ShermanC,MeekC,LeffR,GumboT.2010.Efflux-pump-derived

multipledrugresistancetoethambutolmonotherapyinMycobacteriumtuberculosisand

thepharmacokineticsandpharmacodynamicsofethambutol.JInfectDis201:1225-1231.

47. Musuka S, Srivastava S, Siyambalapitiyage Dona CW, Meek C, Leff R, Pasipanodya J,

GumboT.2013.Thioridazinepharmacokinetic-pharmacodynamicparameters"Wobble"

during treatment of tuberculosis: a theoretical basis for shorter-duration curative

monotherapywithcongeners.AntimicrobAgentsChemother57:5870-5877.

48. vanRijnSP,WesselsAM,GreijdanusB,TouwDJ,AlffenaarJW.2014.Quantificationand

validation of ertapenem using a liquid chromatography-tandem mass spectrometry

method.AntimicrobialAgentsandChemotherapy58:3481-3484.

42. TepplerH,GesserRM,FriedlandIR,WoodsGL,MeibohmA,HermanG,MistryG,Isaacs

R.2004.Safetyandtolerabilityofertapenem.JAntimicrobChemother53Suppl2:ii75-81.

43. CarlierM, NoeM, DeWaele JJ, Stove V, Verstraete AG, Lipman J, Roberts JA. 2013.

Population pharmacokinetics and dosing simulations of amoxicillin/clavulanic acid in

criticallyillpatients.JAntimicrobChemother68:2600-2608.

44. Gumbo T, Louie A, Deziel MR, Liu W, Parsons LM, Salfinger M, Drusano GL. 2007.

Concentration-dependent Mycobacterium tuberculosis killing and prevention of

resistancebyrifampin.AntimicrobAgentsChemother51:3781-3788.

45. Gumbo T, Louie A, Liu W, Brown D, Ambrose PG, Bhavnani SM, Drusano GL. 2007.

Isoniazidbactericidalactivityandresistanceemergence:integratingpharmacodynamics

and pharmacogenomics to predict efficacy in different ethnic populations. Antimicrob


46. SrivastavaS,MusukaS,ShermanC,MeekC,LeffR,GumboT.2010.Efflux-pump-derived

multipledrugresistancetoethambutolmonotherapyinMycobacteriumtuberculosisand

thepharmacokineticsandpharmacodynamicsofethambutol.JInfectDis201:1225-1231.

47. Musuka S, Srivastava S, Siyambalapitiyage Dona CW, Meek C, Leff R, Pasipanodya J,

GumboT.2013.Thioridazinepharmacokinetic-pharmacodynamicparameters"Wobble"

during treatment of tuberculosis: a theoretical basis for shorter-duration curative

monotherapywithcongeners.AntimicrobAgentsChemother57:5870-5877.



method.AntimicrobialAgentsandChemotherapy58:3481-3484.

CHAPTER 7

Antimicrob Agents Chemother. 2017 Mar 24; 61(4).PMID: 28137814


S.P. van Rijn*M.A. Zuur*

R. van AltenaO.W. Akkerman

J.H. ProostW.C.M de Lange

H.A.M. KerstjensD.J. Touw

T.S. van der WerfJ.G.W. KosterinkJ.W.C. Alffenaar

Pharmacokinetic Modelling and Limited Sampling Strategies Based onHealthy Volunteers for Monitoring of

Ertapenem in Patients with Multidrug-resistant Tuberculosis

110

Chapter 7

AbstractErtapenem is a broad spectrum carbapenem antibiotic and is being explored against

Mycobacterium tuberculosis. Carbapenems have anti-bacterial activity when the plasma

concentration exceeds the minimal inhibitory concentration at least 40% of the time

(40%T>MIC).Toassess40%T>MIC inmultidrug-resistant tuberculosis (MDR-TB)patients,a

limitedsamplingstrategywasdevelopedusingapopulationpharmacokineticmodelbased

on healthy volunteers. A two-compartment population pharmacokinetic model was

developedfromdatainforty-twohealthyvolunteers,usinganiterativetwo-stageBayesian

method.ExternalvalidationwasperformedbyBayesian fittingof themodeldeveloped in

volunteers to the individual data of MDR-TB patients (AUC0-24h,fit) using the developed

populationmodelforvolunteersasaprior.AMonteCarlosimulation(n=1000)wasusedto

evaluatelimitedsamplingstrategies.Additionally,thefreeertapenemfraction(f)40%T>MIC

for MDR-TB patients was estimated with the population pharmacokinetic model. The

developedpopulationpharmacokineticmodelwasshowntoestimatetheAUC0-24hinMDR-

TBpatientswithanoverestimationof6.8(range:-17.2–30.7)%.Thebestperforminglimited

samplingstrategy,withatime-restrictionof0-6h,wasfoundtobesamplingat1and5h(R2=

0.78,meanpredictionerror=-0.33%andarootmeansquareerror=5.5%).Drugexposure

wasoverestimatedbyameanpercentageof4.2(-15.2–23.6)%.Consideringafreefraction

of5%andtheMICsetat0.5mg/L,9outof12patientswouldhaveexceededaminimumoff

40%T>MIC.Apopulationpharmacokineticmodelandlimitedsamplingstrategy,developed

usingdatafromhealthyvolunteers,showedtobeadequatetopredictertapenemexposure

inMDR-TBpatients.

IntroductionErtapenem isabroad spectrumcarbapenemantibiotic,usedagainsta rangeof infectious

diseases(1).Likeforallotherbeta-lactamantimicrobialproducts,theefficacyofertapenem

ischaracterizedbytime-dependentkilling.Carbapenemshaveanti-bacterialactivitywhenthe

plasmaconcentrationexceedstheminimalinhibitoryconcentrationatleast40%ofthetime

(40%T>MIC)(1,2).Althoughnotyetstudiedintuberculosis(TB)patients,free40%T>MICis

expectedtobeanimportantpharmacodynamicparameter(3).Carbapenemsincombination

withclavulanicacidhascreatedinterestsinceactivitywasshowninamurinemodelofTB(3).

Additionally,arecentstudyshowedthatcarbapenemsefficientlyinactivatedpeptidoglycan

cross-linkinginM.tuberculosis(3,4)andarecentmeropenemamoxicillin/clavulanicacidEBA

studyshowedactivityofcarbapenemsinTB(5).Recentlyanewsusceptibilitytestingmethod

toestimatetheMICofertapenemhasbeenintroduced(6)showingthatertapenemmightbe

morepotent in vitro thanpreviously thoughtbecause its chemicaldegradationhadnever

beenconsidered(7).Todateonlyalimitednumberofmultidrug-resistanttuberculosis(MDR-

TB)patientshavebeentreatedwithertapenemaspartofamultidrugregimen.Basedonthis

data, the drug appeared well tolerated during prolonged treatment (8,9). However,

ertapenemisnotyetaddedtotheWorldHealthOrganization(WHO)listofanti-TBdrugs,in

contrasttoimipenemandmeropenem.

Pharmacokineticsofertapenemhavetypicallybeenstudiedinhealthyvolunteers(10),people

withobesity(11,12),patientswithrenalfailure(13-15)andcriticallyillpatientswithvarious

pathologies (16-18). Lowerdrug exposurewasobserved in obese individuals (12), and an

increaseindoseintervalwasneededinpatientswithrenalinsufficiencywithanestimated

glomerularfiltrationrate(eGFR)below30mL/min/1,73m2(14),suggestingthattheoptimal

dose of ertapenem is different for various health conditions. A recent study on

pharmacokineticsofMDR-TBpatientssuggestedthattherewassubstantialpharmacokinetic

variabilityinthesepatients(8).

ForstudiesexploringtheuseofertapenemagainstM.tuberculosis itwouldbevaluableto

assessf40%T>MICinpatients.Tobeabletocalculatethef40%T>MIC,agoodindicationof

the plasma concentration profile is mandatory. However, measurement of the plasma

concentrationovertheentire24hdosingintervalistimeconsuming,expensiveandburdening

111

7

Pharmacokinetic Modelling and Limited Sampling Strategies of Ertapenem

AbstractErtapenem is a broad spectrum carbapenem antibiotic and is being explored against

Mycobacterium tuberculosis. Carbapenems have anti-bacterial activity when the plasma

concentration exceeds the minimal inhibitory concentration at least 40% of the time

(40%T>MIC).Toassess40%T>MIC inmultidrug-resistant tuberculosis (MDR-TB)patients,a

limitedsamplingstrategywasdevelopedusingapopulationpharmacokineticmodelbased

on healthy volunteers. A two-compartment population pharmacokinetic model was

developedfromdatainforty-twohealthyvolunteers,usinganiterativetwo-stageBayesian

method.ExternalvalidationwasperformedbyBayesian fittingof themodeldeveloped in

volunteers to the individual data of MDR-TB patients (AUC0-24h,fit) using the developed

populationmodelforvolunteersasaprior.AMonteCarlosimulation(n=1000)wasusedto

evaluatelimitedsamplingstrategies.Additionally,thefreeertapenemfraction(f)40%T>MIC

for MDR-TB patients was estimated with the population pharmacokinetic model. The

developedpopulationpharmacokineticmodelwasshowntoestimatetheAUC0-24hinMDR-

TBpatientswithanoverestimationof6.8(range:-17.2–30.7)%.Thebestperforminglimited

samplingstrategy,withatime-restrictionof0-6h,wasfoundtobesamplingat1and5h(R2=

0.78,meanpredictionerror=-0.33%andarootmeansquareerror=5.5%).Drugexposure

wasoverestimatedbyameanpercentageof4.2(-15.2–23.6)%.Consideringafreefraction

of5%andtheMICsetat0.5mg/L,9outof12patientswouldhaveexceededaminimumoff

40%T>MIC.Apopulationpharmacokineticmodelandlimitedsamplingstrategy,developed

usingdatafromhealthyvolunteers,showedtobeadequatetopredictertapenemexposure

inMDR-TBpatients.

IntroductionErtapenem isabroad spectrumcarbapenemantibiotic,usedagainsta rangeof infectious

diseases(1).Likeforallotherbeta-lactamantimicrobialproducts,theefficacyofertapenem

ischaracterizedbytime-dependentkilling.Carbapenemshaveanti-bacterialactivitywhenthe

plasmaconcentrationexceedstheminimalinhibitoryconcentrationatleast40%ofthetime

(40%T>MIC)(1,2).Althoughnotyetstudiedintuberculosis(TB)patients,free40%T>MICis

expectedtobeanimportantpharmacodynamicparameter(3).Carbapenemsincombination

withclavulanicacidhascreatedinterestsinceactivitywasshowninamurinemodelofTB(3).

Additionally,arecentstudyshowedthatcarbapenemsefficientlyinactivatedpeptidoglycan

cross-linkinginM.tuberculosis(3,4)andarecentmeropenemamoxicillin/clavulanicacidEBA

studyshowedactivityofcarbapenemsinTB(5).Recentlyanewsusceptibilitytestingmethod

toestimatetheMICofertapenemhasbeenintroduced(6)showingthatertapenemmightbe

morepotent in vitro thanpreviously thoughtbecause its chemicaldegradationhadnever

beenconsidered(7).Todateonlyalimitednumberofmultidrug-resistanttuberculosis(MDR-

TB)patientshavebeentreatedwithertapenemaspartofamultidrugregimen.Basedonthis

data, the drug appeared well tolerated during prolonged treatment (8,9). However,

ertapenemisnotyetaddedtotheWorldHealthOrganization(WHO)listofanti-TBdrugs,in

contrasttoimipenemandmeropenem.

Pharmacokineticsofertapenemhavetypicallybeenstudiedinhealthyvolunteers(10),people

withobesity(11,12),patientswithrenalfailure(13-15)andcriticallyillpatientswithvarious

pathologies (16-18). Lowerdrug exposurewasobserved in obese individuals (12), and an

increaseindoseintervalwasneededinpatientswithrenalinsufficiencywithanestimated

glomerularfiltrationrate(eGFR)below30mL/min/1,73m2(14),suggestingthattheoptimal

dose of ertapenem is different for various health conditions. A recent study on

pharmacokineticsofMDR-TBpatientssuggestedthattherewassubstantialpharmacokinetic

variabilityinthesepatients(8).

ForstudiesexploringtheuseofertapenemagainstM.tuberculosis itwouldbevaluableto

assessf40%T>MICinpatients.Tobeabletocalculatethef40%T>MIC,agoodindicationof

the plasma concentration profile is mandatory. However, measurement of the plasma

concentrationovertheentire24hdosingintervalistimeconsuming,expensiveandburdening

112

Chapter 7

topatients.Alimitedsamplingstrategythroughapopulationpharmacokineticmodelcanbe

usedtopredictthisplasmaconcentrationprofileashasbeendoneforotheranti-TBdrugs

(19-22).

The aim of this studywas to develop a population pharmacokineticmodel and a limited

samplingstrategybasedondatafromhealthyvolunteers,inordertoestimatedrugexposure

ofertapeneminMDR-TBpatients.

Materialsandmethods

This studywas based on two data sets. The first setwas comprised of forty-two healthy

volunteers fromfiveclinicalstudiesreceiving0.25to2g i.v.dosesofertapenem(10).For

populationpharmacokineticmodelcomparisonwithMDR-TBpatientsweonlyusedthedata

ofhealthyvolunteersreceiving1g.Thesecondsetwascomprisedofaretrospectivedataset

ofpatientswithMDR-TB,receiving1gertapenemadministeredoncedailyviaa30-minute

infusionattheTuberculosisCenterBeatrixoord,UniversityMedicalCenterGroningen,The

NetherlandsbetweenDecember1,2010andMarch1,2013(8).Plasmaconcentrationsof

ertapenemwerecollectedatsteadystatebeforeadministrationandat1,2,3,4,5,6,8and

12 hours after administration. Ertapenem plasma concentrations were analyzed by a

validatedliquidchromatography-tandemmass-spectrometry(LC-MS/MS)method(23).Both

datasetsincludeddemographicandmedicaldata,suchasageatstartoftreatment,height

andbodyweightandserumcreatinineatthetimeofpharmacokineticassessment.

Populationpharmacokineticmodel

All pharmacokinetic calculations were performed using MW\Pharm 3.82 (Mediware,

Zuidhorn,TheNetherlands).Basedonpreviousreportsandrecentpharmacokineticstudies

on ertapenem (11-18, 24-26), concentration time-curves were evaluated in a one-

compartmentalandtwo-compartmentalmodel.Thefinalmodelwasselectedbasedonthe

Akaike information criterion (AIC) (27). The drug plasma-concentrations of the forty-two

healthyvolunteerswereusedtodevelopatwo-compartmentalpopulationpharmacokinetic

modelusinganiterativetwo-stageBayesian(ITSB)procedure(moduleKinPopofMW\Pharm)

(28).TheclearancewascalculatedusingCL=CLm(metabolicclearance(L/h/1.85m2))*BSA

(body surface area (m2)) /1.85 + fr (drug clearance – creatinine clearance ratio) * CLcr

(creatinine clearance (L/h)) (24). Pharmacokinetic parameters were assumed to be log-

normallydistributedandtheresidualerrorwasassumedtobenormallydistributedwithSD

=0.1+0.1*C,inwhichCistheobservedplasmaconcentrationofertapenem.Nonparametric

95%confidence intervalsof thepopulationparametersand their inter-individual standard

deviationwereestimatedusingabootstrapanalysis(n=1000).Theareaundertheplasma

concentration-timecurvefrom0upto24hours(AUC0-24h,ref),usedasareferencevalue,was

calculatedusingthelogtrapezoidalrule(KinFitmoduleofMW\Pharm).

Internal validation was performed by leaving three healthy volunteers out of the

pharmacokinetic model development, creating fourteen n-3 sub models, obtained by

randomizationusingMicrosoftExcel2010.TheestimatedAUC0-24h,(AUC0-24h,n-3)wasobtained

byBayesianfittingusingthedataofthethreeleftoutvolunteersinthecorrespondingn-3

submodels.AgreementbetweenAUC0-24h,n-3andAUC0-24h,refwasassessedbyBland-Altman

analysisandPassingandBablokregressionandsubsequentresidualplot.Externalvalidation

wasperformedbyBayesianfittingofthemodeldevelopedinvolunteerstotheindividualdata

ofMDR-TBpatients(AUC0-24h,fit),usingthedevelopedpopulationmodelforvolunteersasa

prior.ForcomparisonofthepharmacokineticsbetweenMDR-TBpatientsandvolunteers,a

similaranalysiswasperformedwiththedataof18volunteerswhoreceived1gofertapenem.

Bland-Altmananalysiswasalsoused toassess theagreementbetweenAUC0-24h,fitand the

AUC0-24h,refofMDR-TBpatients.

Limitedsamplingstrategies

AMonteCarlosimulationwasusedtocalculatelimitedsamplingstrategiesforestimationof

AUC (AUC0-24h,LSS), as implemented inMW\Pharm. This stochastic simulation consisted of

1000randompatientsdrawnfromthepopulationpharmacokineticmodel.Foreachpatient

limitedsamplingstrategieswerecalculatedat1to3samplingtimepointsbyBayesianMAP

procedure.We evaluated limited sampling strategies based on separate calculationswith

time restrictions of 0-6h, 0-12h, and 0-24h. Performance was considered suitable for

applicationinprospectivestudies iftheadjustedrsquared(R2)was>0.95,therootmean

squareerror(RMSE)was<15%,andthemeanpredictionerror(MPE)was<5%.Theprediction

errorswerecalculatedas(AUC0-24h,LSS-AUC0-24h,ref)/AUC0-24h,ref·100%.

113

7


topatients.Alimitedsamplingstrategythroughapopulationpharmacokineticmodelcanbe

usedtopredictthisplasmaconcentrationprofileashasbeendoneforotheranti-TBdrugs

(19-22).

The aim of this studywas to develop a population pharmacokineticmodel and a limited

samplingstrategybasedondatafromhealthyvolunteers,inordertoestimatedrugexposure

ofertapeneminMDR-TBpatients.

Materialsandmethods

This studywas based on two data sets. The first setwas comprised of forty-two healthy

volunteers fromfiveclinicalstudiesreceiving0.25to2g i.v.dosesofertapenem(10).For

populationpharmacokineticmodelcomparisonwithMDR-TBpatientsweonlyusedthedata

ofhealthyvolunteersreceiving1g.Thesecondsetwascomprisedofaretrospectivedataset

ofpatientswithMDR-TB,receiving1gertapenemadministeredoncedailyviaa30-minute

infusionattheTuberculosisCenterBeatrixoord,UniversityMedicalCenterGroningen,The

NetherlandsbetweenDecember1,2010andMarch1,2013(8).Plasmaconcentrationsof

ertapenemwerecollectedatsteadystatebeforeadministrationandat1,2,3,4,5,6,8and

12 hours after administration. Ertapenem plasma concentrations were analyzed by a

validatedliquidchromatography-tandemmass-spectrometry(LC-MS/MS)method(23).Both

datasetsincludeddemographicandmedicaldata,suchasageatstartoftreatment,height

andbodyweightandserumcreatinineatthetimeofpharmacokineticassessment.

Populationpharmacokineticmodel

All pharmacokinetic calculations were performed using MW\Pharm 3.82 (Mediware,

Zuidhorn,TheNetherlands).Basedonpreviousreportsandrecentpharmacokineticstudies

on ertapenem (11-18, 24-26), concentration time-curves were evaluated in a one-

compartmentalandtwo-compartmentalmodel.Thefinalmodelwasselectedbasedonthe

Akaike information criterion (AIC) (27). The drug plasma-concentrations of the forty-two

healthyvolunteerswereusedtodevelopatwo-compartmentalpopulationpharmacokinetic

modelusinganiterativetwo-stageBayesian(ITSB)procedure(moduleKinPopofMW\Pharm)

(28).TheclearancewascalculatedusingCL=CLm(metabolicclearance(L/h/1.85m2))*BSA

(body surface area (m2)) /1.85 + fr (drug clearance – creatinine clearance ratio) * CLcr

(creatinine clearance (L/h)) (24). Pharmacokinetic parameters were assumed to be log-

normallydistributedandtheresidualerrorwasassumedtobenormallydistributedwithSD

=0.1+0.1*C,inwhichCistheobservedplasmaconcentrationofertapenem.Nonparametric

95%confidence intervalsof thepopulationparametersand their inter-individual standard

deviationwereestimatedusingabootstrapanalysis(n=1000).Theareaundertheplasma

concentration-timecurvefrom0upto24hours(AUC0-24h,ref),usedasareferencevalue,was

calculatedusingthelogtrapezoidalrule(KinFitmoduleofMW\Pharm).

Internal validation was performed by leaving three healthy volunteers out of the

pharmacokinetic model development, creating fourteen n-3 sub models, obtained by

randomizationusingMicrosoftExcel2010.TheestimatedAUC0-24h,(AUC0-24h,n-3)wasobtained

byBayesianfittingusingthedataofthethreeleftoutvolunteersinthecorrespondingn-3

submodels.AgreementbetweenAUC0-24h,n-3andAUC0-24h,refwasassessedbyBland-Altman

analysisandPassingandBablokregressionandsubsequentresidualplot.Externalvalidation

wasperformedbyBayesianfittingofthemodeldevelopedinvolunteerstotheindividualdata

ofMDR-TBpatients(AUC0-24h,fit),usingthedevelopedpopulationmodelforvolunteersasa

prior.ForcomparisonofthepharmacokineticsbetweenMDR-TBpatientsandvolunteers,a

similaranalysiswasperformedwiththedataof18volunteerswhoreceived1gofertapenem.

Bland-Altmananalysiswasalsoused toassess theagreementbetweenAUC0-24h,fitand the

AUC0-24h,refofMDR-TBpatients.


AMonteCarlosimulationwasusedtocalculatelimitedsamplingstrategiesforestimationof

AUC (AUC0-24h,LSS), as implemented inMW\Pharm. This stochastic simulation consisted of

1000randompatientsdrawnfromthepopulationpharmacokineticmodel.Foreachpatient

limitedsamplingstrategieswerecalculatedat1to3samplingtimepointsbyBayesianMAP

procedure.We evaluated limited sampling strategies based on separate calculationswith

time restrictions of 0-6h, 0-12h, and 0-24h. Performance was considered suitable for

applicationinprospectivestudies iftheadjustedrsquared(R2)was>0.95,therootmean

squareerror(RMSE)was<15%,andthemeanpredictionerror(MPE)was<5%.Theprediction

errorswerecalculatedas(AUC0-24h,LSS-AUC0-24h,ref)/AUC0-24h,ref·100%.

114

Chapter 7

Predictionoffree40%T>MIC

The ertapenem concentration-time curve of each patient was used to establish if the f

40%T>MICwasreached.Forthiscausethetimethattheconcentration-timecurvewasabove

theMICwasassessedinMW/Pharm.Thepercentageproteinunboundertapenemusedfor

the assessment was 5 (4,7). European Committee on Antimicrobial Susceptibility Testing

(EUCAST)MICvalueforertapenem(non-speciesrelated)of0.5mg·L−1wasusedtocalculate

f40%T>MIC(6,7).Exposurewasconsideredadequateiftheconcentrationwas40%oftime

aboveMIC,whichcorrespondswith9.6hofeach24hintervalasshowninfigure1.

Figure1.Ertapenemplasmaconcentrationtimecurve. 40%oftimeiscorrespondingwith9.6hofeach24hinterval.

Statistics

Differences between the population characteristics and pharmacokinetic parameters of

healthy volunteers and TB patients were calculated using the MannWhitney U test. All

statisticswerecalculatedwithAnalyse-it™forMicrosoftExcel(version2.30).

ResultsDataset

Dataofforty-twohealthyvolunteerswasusedtodevelopthepopulationpharmacokinetic

model.SincebloodsamplesfromMDR-TBpatientswerecollectedforanotherpurpose,no

datawasavailablebetween5and8hours.Thebaselinecharacteristicsofhealthyvolunteers

andMDR-TBpatientswereshowntodiffersignificantly(P<0.05),exceptforage(table1).The

medianageofthevolunteerswas31(23-38)yearsandbodymassindexwas24.5(23.6–

26.2)kg/m2.

Table1.Baselinecharacteristicsofhealthyvolunteersversusmultidrug-resistanttuberculosispatients

Healthyvolunteers MDR-TBpatients p-value n=42 n=12 Sex[n(%)] Male 36(86%) 5(42%) 0.0083aFemale 6(14%) 7(58%) Age(years;median,IQR) 31(23-38) 25(18-30) 0.064bWeight(kg;median,IQR) 78.9(72.2-83.8) 55.5(47.3-70.3) 0.000b

Height(cm;median,IQR) 178(172-183) 167(164-174) 0.004b

BodymassIndex(kg/m2;median,IQR) 24.5(23.6-26.2) 19.2(17.9-23.7) 0.002b Ethnicity Black(%) 5(12%) 7(58%) 0.000cCaucasian(%) 36(86%) 3(25%) Asian(%) 1(2%) 1(8%) Other(%) 0(0%) 1(8%) SerumCreatinine(mg/dl;median,IQR)* 0.9(0.8-1.1)** 0.5(0.4-0.7) 0.000b

Creatinineclearance(ml/min/1.73m2;median,IQR) 91(81–97) 181(122–248) <0.0001b

Dose/weight(mg/kg;median;IQR) 18.0(14.2-21.1)** 12.9(6.0-20.0) 0.044b

Samplesperpatient(median;IQR) 28(16-48) 7(6-8)

IQR=Interquartilerange*Onthedaytheplasmaconcentrationsweredetermined**Somepatientsreceivedmorethanonedosewithsubsequentserumcreatininea.Fisherexacttest,b.Mann-WhitneyUtest,c.PearsonChi-Squaredtest

115

7


Predictionoffree40%T>MIC

The ertapenem concentration-time curve of each patient was used to establish if the f

40%T>MICwasreached.Forthiscausethetimethattheconcentration-timecurvewasabove

theMICwasassessedinMW/Pharm.Thepercentageproteinunboundertapenemusedfor

the assessment was 5 (4,7). European Committee on Antimicrobial Susceptibility Testing

(EUCAST)MICvalueforertapenem(non-speciesrelated)of0.5mg·L−1wasusedtocalculate

f40%T>MIC(6,7).Exposurewasconsideredadequateiftheconcentrationwas40%oftime

aboveMIC,whichcorrespondswith9.6hofeach24hintervalasshowninfigure1.

Figure1.Ertapenemplasmaconcentrationtimecurve. 40%oftimeiscorrespondingwith9.6hofeach24hinterval.

Statistics

Differences between the population characteristics and pharmacokinetic parameters of

healthy volunteers and TB patients were calculated using the MannWhitney U test. All

statisticswerecalculatedwithAnalyse-it™forMicrosoftExcel(version2.30).

ResultsDataset

Dataofforty-twohealthyvolunteerswasusedtodevelopthepopulationpharmacokinetic

model.SincebloodsamplesfromMDR-TBpatientswerecollectedforanotherpurpose,no

datawasavailablebetween5and8hours.Thebaselinecharacteristicsofhealthyvolunteers

andMDR-TBpatientswereshowntodiffersignificantly(P<0.05),exceptforage(table1).The

medianageofthevolunteerswas31(23-38)yearsandbodymassindexwas24.5(23.6–

26.2)kg/m2.

Table1.Baselinecharacteristicsofhealthyvolunteersversusmultidrug-resistanttuberculosispatients

Healthyvolunteers MDR-TBpatients p-value n=42 n=12 Sex[n(%)] Male 36(86%) 5(42%) 0.0083aFemale 6(14%) 7(58%) Age(years;median,IQR) 31(23-38) 25(18-30) 0.064bWeight(kg;median,IQR) 78.9(72.2-83.8) 55.5(47.3-70.3) 0.000b

Height(cm;median,IQR) 178(172-183) 167(164-174) 0.004b

BodymassIndex(kg/m2;median,IQR) 24.5(23.6-26.2) 19.2(17.9-23.7) 0.002b Ethnicity Black(%) 5(12%) 7(58%) 0.000cCaucasian(%) 36(86%) 3(25%) Asian(%) 1(2%) 1(8%) Other(%) 0(0%) 1(8%) SerumCreatinine(mg/dl;median,IQR)* 0.9(0.8-1.1)** 0.5(0.4-0.7) 0.000b

Creatinineclearance(ml/min/1.73m2;median,IQR) 91(81–97) 181(122–248) <0.0001b

Dose/weight(mg/kg;median;IQR) 18.0(14.2-21.1)** 12.9(6.0-20.0) 0.044b

Samplesperpatient(median;IQR) 28(16-48) 7(6-8)

IQR=Interquartilerange*Onthedaytheplasmaconcentrationsweredetermined**Somepatientsreceivedmorethanonedosewithsubsequentserumcreatininea.Fisherexacttest,b.Mann-WhitneyUtest,c.PearsonChi-Squaredtest

116

Chapter 7

Pharmacokineticmodel

Developmentofpharmacokineticmodelinvolunteers

Theselectionofthetwo-compartmentalmodelwasbasedontheAICvaluesfortheone-(AIC

=1280)andtwo-(AIC=-1073)compartmentmodels(28).Finalpopulationpharmacokinetic

modelparametersdevelopedfromdataofhealthyvolunteers(N=42)areshownintable2.

AUC0-24h,n-3valuesestimatedintheinternalvalidationwereunderestimatedbyameanvalue

of0.3%(range:-8.1–7.6),whencomparedwithAUC0-24h,refshowninFigure2.Theobserved

AUC0-24h,refandmodelcalculatedertapenemAUC0-24h,n=3wereassessedforagreement,using

PassingandBablokregressioninfigure3.

Figure 2. Internal validation of the population pharmacokineticmodel. Bland-Altman plotshowing theagreementbetween theareaunder the concentration-time curve for 24hofhealthyvolunteersestimatedwiththepopulationpharmacokineticmodel(AUC0-24h,n=3)andtheAUC0-24h,ref

Table2.Pharmacokineticpopulationmodeldevelopedfromdatainvolunteers.

Parameter Mean(95%CI) SD(95%CI)CLm(L/h/1.85m2) 1.06(0.54–1.34) 0.16(0.09–0.23)fr 0.130(0.073–0.237) 0.039(0.016–0.063)V1(L/kg) 0.0824(0.0789–0.0860) 0.0095(0.0073–0.0119)CL12(L/h/1.85m2) 2.56(2.34–2.85) 0.14(0.11–0.28)V2(L/kg) 0.0543(0.0527–0.0559) 0.0016(0.0013–0.0021)

CLm = metabolic clearance; fr =ertapenem clearance/creatinine clearance ratio; V1 = volume ofdistribution of the central compartment; CL12 = inter-compartmental clearance; V2 = volume ofdistributionintheperipheralcompartment;CI=nonparametricconfidenceintervalfrombootstrapanalysisPopulationPKparametersTBpatientscomparedtohealthyvolunteers

Pharmacokinetic parameters of 18 healthy volunteers, who received 1 g of ertapenem,

comparedtoTBpatientsareshownintable3.AUC0-24h,fitvaluesoftheMDR-TBpatientdata

wereunderestimatedbymean6.8%(range:-17.2–30.7),whencomparedwithAUC0-24h,ref.

AUC0-24h,fit correlatedwell with AUC0-24h,ref , as determinedwith a Bland-Altman analysis -

showninFigure4.

Table3.Pharmacokineticparametersinvolunteerswhoreceiveda1gdoseandMDR-TBpatients.

Healthyvolunteers(n=18) Tuberculosispatients(n=12) p-value*CLm(L/h/1.85m2) 1.06±0.08 0.944±0.132 0.0023fr 0.158±0.051 0.104±0.039 0.0017V1(L/kg) 0.0846±0.0094 0.0876±0.0166 0.4717CL12(L/h/1.85m2) 2.52±0.03 2.55±0.04 0.0023V2(L/kg) 0.0543±0.0005 0.0549±0.0008 0.2885

*Mann-WhitneyUtestClm = metabolic clearance; fr =ertapenem clearance/creatinine clearance ratio; V1 = volume ofdistribution of the central compartment; CL12 = inter-compartmental clearance; V2 = volume ofdistributionintheperipheralcompartment

117

7


Pharmacokineticmodel

Developmentofpharmacokineticmodelinvolunteers

Theselectionofthetwo-compartmentalmodelwasbasedontheAICvaluesfortheone-(AIC

=1280)andtwo-(AIC=-1073)compartmentmodels(28).Finalpopulationpharmacokinetic

modelparametersdevelopedfromdataofhealthyvolunteers(N=42)areshownintable2.

AUC0-24h,n-3valuesestimatedintheinternalvalidationwereunderestimatedbyameanvalue

of0.3%(range:-8.1–7.6),whencomparedwithAUC0-24h,refshowninFigure2.Theobserved

AUC0-24h,refandmodelcalculatedertapenemAUC0-24h,n=3wereassessedforagreement,using

PassingandBablokregressioninfigure3.

Figure 2. Internal validation of the population pharmacokineticmodel. Bland-Altman plotshowing theagreementbetween theareaunder the concentration-time curve for 24hofhealthyvolunteersestimatedwiththepopulationpharmacokineticmodel(AUC0-24h,n=3)andtheAUC0-24h,ref

Table2.Pharmacokineticpopulationmodeldevelopedfromdatainvolunteers.

Parameter Mean(95%CI) SD(95%CI)CLm(L/h/1.85m2) 1.06(0.54–1.34) 0.16(0.09–0.23)fr 0.130(0.073–0.237) 0.039(0.016–0.063)V1(L/kg) 0.0824(0.0789–0.0860) 0.0095(0.0073–0.0119)CL12(L/h/1.85m2) 2.56(2.34–2.85) 0.14(0.11–0.28)V2(L/kg) 0.0543(0.0527–0.0559) 0.0016(0.0013–0.0021)

CLm = metabolic clearance; fr =ertapenem clearance/creatinine clearance ratio; V1 = volume ofdistribution of the central compartment; CL12 = inter-compartmental clearance; V2 = volume ofdistributionintheperipheralcompartment;CI=nonparametricconfidenceintervalfrombootstrapanalysisPopulationPKparametersTBpatientscomparedtohealthyvolunteers

Pharmacokinetic parameters of 18 healthy volunteers, who received 1 g of ertapenem,

comparedtoTBpatientsareshownintable3.AUC0-24h,fitvaluesoftheMDR-TBpatientdata

wereunderestimatedbymean6.8%(range:-17.2–30.7),whencomparedwithAUC0-24h,ref.

AUC0-24h,fit correlatedwell with AUC0-24h,ref , as determinedwith a Bland-Altman analysis -

showninFigure4.

Table3.Pharmacokineticparametersinvolunteerswhoreceiveda1gdoseandMDR-TBpatients.

Healthyvolunteers(n=18) Tuberculosispatients(n=12) p-value*CLm(L/h/1.85m2) 1.06±0.08 0.944±0.132 0.0023fr 0.158±0.051 0.104±0.039 0.0017V1(L/kg) 0.0846±0.0094 0.0876±0.0166 0.4717CL12(L/h/1.85m2) 2.52±0.03 2.55±0.04 0.0023V2(L/kg) 0.0543±0.0005 0.0549±0.0008 0.2885

*Mann-WhitneyUtestClm = metabolic clearance; fr =ertapenem clearance/creatinine clearance ratio; V1 = volume ofdistribution of the central compartment; CL12 = inter-compartmental clearance; V2 = volume ofdistributionintheperipheralcompartment

118

Chapter 7

Figure3.PassingandBablokregression.Plotshowingtheagreementbetweentheareaunderthe concentration-time curve for 24h (AUC0-24h,ref) and theAUC0-24h,n=3 estimatedwith thepopulationpharmacokineticmodel(dottedlines:95%confidenceinterval(CI)).


Usingthepopulationpharmacokineticmodel,limitedsamplingstrategieswereevaluatedfor

0-6h, 0-12h and 0-24h restriction of the dosing intervals. The R2, bias and RMSE were

subsequentlydetermined.Foreachdosingintervalandforone,twoorthreesamplingtime

points,thelimitedsamplingstrategieswiththebestperformanceofRMSEandbiasareshown

inTable4.

Figure4. External validationof thepopulationpharmacokineticmodel.BlandAltmanplotshowing the agreement of the area under the concentration-time curve of multidrug-resistanttuberculosispatients(AUC0-24h,ref)andtheAUC0-24h,fitestimatedwiththepopulationpharmacokineticmodel.MeanofAllisthemeanAUCofbothAUC0-24h,fitandAUC0-24h,ref.

Table4.Best-performinglimitedsamplingstrategies

Firstsamplingtimepoint(h)

Secondsamplingtimepoint(h)

Thirdsamplingtimepoint(h)

Coefficientofdetermination(r2)

Meanpredictionerror(%bias) %RMSEa

0-6h 5 0.57 -0.09 7.40-6h 1 5 0.78 -0.33 5.50-6h 1 3 5 0.83 -0.39 4.90-12h 8 0.72 -0.84 7.10-12h 1 11 0.83 -0.83 5.60-12h 1 4 8 0.87 -0.89 4.90-24h 14 0.82 -0.19 6.90-24h 1 14 0.88 -0.44 5.50-24h 1 9 14 0.92 -0.46 4.7

a. Rootmeansquareerror

119

7


Figure3.PassingandBablokregression.Plotshowingtheagreementbetweentheareaunderthe concentration-time curve for 24h (AUC0-24h,ref) and theAUC0-24h,n=3 estimatedwith thepopulationpharmacokineticmodel(dottedlines:95%confidenceinterval(CI)).


Usingthepopulationpharmacokineticmodel,limitedsamplingstrategieswereevaluatedfor

0-6h, 0-12h and 0-24h restriction of the dosing intervals. The R2, bias and RMSE were

subsequentlydetermined.Foreachdosingintervalandforone,twoorthreesamplingtime

points,thelimitedsamplingstrategieswiththebestperformanceofRMSEandbiasareshown

inTable4.

Figure4. External validationof thepopulationpharmacokineticmodel.BlandAltmanplotshowing the agreement of the area under the concentration-time curve of multidrug-resistanttuberculosispatients(AUC0-24h,ref)andtheAUC0-24h,fitestimatedwiththepopulationpharmacokineticmodel.MeanofAllisthemeanAUCofbothAUC0-24h,fitandAUC0-24h,ref.

Table4.Best-performinglimitedsamplingstrategies

Firstsamplingtimepoint(h)

Secondsamplingtimepoint(h)

Thirdsamplingtimepoint(h)

Coefficientofdetermination(r2)

Meanpredictionerror(%bias) %RMSEa

0-6h 5 0.57 -0.09 7.40-6h 1 5 0.78 -0.33 5.50-6h 1 3 5 0.83 -0.39 4.90-12h 8 0.72 -0.84 7.10-12h 1 11 0.83 -0.83 5.60-12h 1 4 8 0.87 -0.89 4.90-24h 14 0.82 -0.19 6.90-24h 1 14 0.88 -0.44 5.50-24h 1 9 14 0.92 -0.46 4.7

a. Rootmeansquareerror

120

Chapter 7

Figure 5. Validation of the limited sampling strategy. The Bland-Altman plot shows theagreementbetweentheareaundertheconcentration-timecurvefor24hfrommultidrug-resistanttuberculosispatientsobtainedfromthepopulationpharmacokineticmodelapplyingthelimitedsamplingstrategyof1and5h(AUC0-24h,LSS)andtheAUC0-24h,refMeanofAllisthemeanAUCofbothAUC0-24h,fitandAUC0-24h,ref.

AlllimitedsamplingstrategiesmetthebiasandRMSEcriteria.Threesamplingtimepoints,at

1, 4 and 9h, enabled the best prediction of ertapenemexposure reflected byAUC0-24h,LSS,

consideringbias,RMSEandR2(R2=0.92,MPE=—0,46%andRMSE=4.7%).However,dueto

thelackofdatawithinthesetimeintervalsandclinicalrelevanceoftimesamplingswithina

certainamountoftime,itwouldbepreferredtouse1,3and5h(R2=0.83,RMSE=4.7%and

MPE = -0.39%). Based on clinical suitability, using two sampling time-pointswith a time-

restrictionof0-6h,alimitedsamplingstrategyat1and5h,showedthelowestRMSE(5.5%)

andlowMPE(-0.33%).TheAUC0-24h,LSS,estimatedbyapplyingthistwosamplingtime-point

limited sampling strategywas comparedwith theAUC0-24h,ref usingBland-Altmananalysis,

showingabiasinAUC0-24h,LSS,of4.2%(-15.2–23.6)(figure5).

%T>MIC

Consideringafreefractionof5%andtheMICsetat0.5mg/L,9outof12patientswouldhave

exceeded a minimum of f 40% T>MIC (range 6.8h – 19.7h) thereby having sufficient

therapeuticeffectinMDR-TBPatientswithoncedailydosing.

DiscussionThisisthefirststudyshowingthatapopulationpharmacokineticmodelofertapenembased

ondataofhealthyvolunteerscanpredictpharmacokineticsofertapeneminpatientswith

MDR-TB,eventhoughthebaselinecharacteristicsofbothhealthyvolunteersandMDR-TB

patientsdifferedsignificantly(table1).WeshowedthattheAUC0-24hofMDR-TBpatientscould

beestimatedwiththispopulationpharmacokineticmodelwithameanoverestimationof6.8

(range:-17.2–30.7%).

Therobustnessofthispopulationpharmacokineticmodelwasvalidatedusingan-3cross-

validation, showing an underestimation of 0.3%. The limited sampling strategy that we

presentherecanbeusedtoassessindividualdrugexposureofertapeneminTBpatientswith

limitedtreatmentoptions.Moreover,themodelandlimitedsamplingstrategycanbeused

toevaluatedrugexposureofertapeneminphaseIIstudiesstudyingearlybactericidalactivity

ofertapeneminTBpatients.Suchastudyisurgentlyneededtoprovidedataonefficacyof

thepotentialattractiveTBdrug.

Inthepopulationpharmacokineticmodel,multipledosesweretreatedassingledosesonday

onetoavoidduplication,asanearlierstudyhadfoundthattherewasnoaccumulationof

ertapenemfollowingdosingovereightdaysandmeanplasmaconcentrationswerefoundto

beverysimilarondayoneaswellasondayeight(9).

Pharmacokineticmodelingofertapenemhasbeenperformedinpreviousstudies(11-18,24-

26),butithasneverbeenperformedforapplicationinMDR-TBtreatment.Comparinghealthy

volunteersandtheTBpatients,we foundthat there isasmallpharmacokineticvariability

betweenthetwogroupsincontrasttootherantimicrobialdrugs,whichshowhighvariability

inpharmacokineticparameters(19,20).Thismightbeexplainedbytheparenteralrouteof

121

7


Figure 5. Validation of the limited sampling strategy. The Bland-Altman plot shows theagreementbetweentheareaundertheconcentration-timecurvefor24hfrommultidrug-resistanttuberculosispatientsobtainedfromthepopulationpharmacokineticmodelapplyingthelimitedsamplingstrategyof1and5h(AUC0-24h,LSS)andtheAUC0-24h,refMeanofAllisthemeanAUCofbothAUC0-24h,fitandAUC0-24h,ref.

AlllimitedsamplingstrategiesmetthebiasandRMSEcriteria.Threesamplingtimepoints,at

1, 4 and 9h, enabled the best prediction of ertapenemexposure reflected byAUC0-24h,LSS,

consideringbias,RMSEandR2(R2=0.92,MPE=—0,46%andRMSE=4.7%).However,dueto

thelackofdatawithinthesetimeintervalsandclinicalrelevanceoftimesamplingswithina

certainamountoftime,itwouldbepreferredtouse1,3and5h(R2=0.83,RMSE=4.7%and

MPE = -0.39%). Based on clinical suitability, using two sampling time-pointswith a time-

restrictionof0-6h,alimitedsamplingstrategyat1and5h,showedthelowestRMSE(5.5%)

andlowMPE(-0.33%).TheAUC0-24h,LSS,estimatedbyapplyingthistwosamplingtime-point

limited sampling strategywas comparedwith theAUC0-24h,ref usingBland-Altmananalysis,

showingabiasinAUC0-24h,LSS,of4.2%(-15.2–23.6)(figure5).

%T>MIC

Consideringafreefractionof5%andtheMICsetat0.5mg/L,9outof12patientswouldhave

exceeded a minimum of f 40% T>MIC (range 6.8h – 19.7h) thereby having sufficient

therapeuticeffectinMDR-TBPatientswithoncedailydosing.

DiscussionThisisthefirststudyshowingthatapopulationpharmacokineticmodelofertapenembased

ondataofhealthyvolunteerscanpredictpharmacokineticsofertapeneminpatientswith

MDR-TB,eventhoughthebaselinecharacteristicsofbothhealthyvolunteersandMDR-TB

patientsdifferedsignificantly(table1).WeshowedthattheAUC0-24hofMDR-TBpatientscould

beestimatedwiththispopulationpharmacokineticmodelwithameanoverestimationof6.8

(range:-17.2–30.7%).

Therobustnessofthispopulationpharmacokineticmodelwasvalidatedusingan-3cross-

validation, showing an underestimation of 0.3%. The limited sampling strategy that we

presentherecanbeusedtoassessindividualdrugexposureofertapeneminTBpatientswith

limitedtreatmentoptions.Moreover,themodelandlimitedsamplingstrategycanbeused

toevaluatedrugexposureofertapeneminphaseIIstudiesstudyingearlybactericidalactivity

ofertapeneminTBpatients.Suchastudyisurgentlyneededtoprovidedataonefficacyof

thepotentialattractiveTBdrug.

Inthepopulationpharmacokineticmodel,multipledosesweretreatedassingledosesonday

onetoavoidduplication,asanearlierstudyhadfoundthattherewasnoaccumulationof

ertapenemfollowingdosingovereightdaysandmeanplasmaconcentrationswerefoundto

beverysimilarondayoneaswellasondayeight(9).

Pharmacokineticmodelingofertapenemhasbeenperformedinpreviousstudies(11-18,24-

26),butithasneverbeenperformedforapplicationinMDR-TBtreatment.Comparinghealthy

volunteersandtheTBpatients,we foundthat there isasmallpharmacokineticvariability

betweenthetwogroupsincontrasttootherantimicrobialdrugs,whichshowhighvariability

inpharmacokineticparameters(19,20).Thismightbeexplainedbytheparenteralrouteof

122

Chapter 7

administrationofertapenemtherebyhavingnolossofertapenemduetoabsorption.Several

studieslookingattheexposureofertapeneminpatientshaveshownthattheexposurein

patients varies greatly (14,15). There is little pharmacokinetic variability in the MDR-TB

patients(table3).

A limitedsamplingstrategyusingtwosamplingtimepoints is favoredas itwouldgivethe

leastburdentopatientsas it isminimally invasiveand leasttime-consuming.Additionally,

less timebetweensamplingtimepoints ismorefeasible inclinicalpractice,since it is less

pronetosamplingmistakes.Moreover, limitationofthisstudyisthatafter6hsparsedata

wasavailable,thereforeresultsafter6hislesssubstantiatedwithclinicaldata.

AstherearelimitedoptionsfortreatingMDR-TBandresistanceofantibioticsisanemerging

problem,wethink it is timetostartassessingefficacyofertapeneminMDR-Bpatients in

Phase II clinical trial testing theearlybactericidalactivity.Thedeveloped limitedsampling

strategycanbeusedtoevaluatedrugexposureandtherebyreducecostsandburdenforthe

studysubjects.

Conclusion

A pharmacokinetic model and limited sampling strategy bases on data from healthy

volunteerswasabletopredicttheAUC0-24handf40%T>MICinMDR-TBpatients.Thismodel

canbeusedinphaseIIstudies.

Conflictofinterest:nonedeclared

Acknowledgement:TheauthorsthankMerck&Co.Inc.USA,forprovidingdatafromearlier

pharmacokineticsstudiesofertapenem

References1. MoutonJW,TouzwDJ,Horrevorts,VinkAA.2000.Comparativepharmacokineticsofthe

carbapenems:Clinicalimplications.ClinPharmacokinet39:185-201.

2. Craig WA. 1998. Pharmacokinetic/pharmacodynamic parameters: Rationale for

antibacterialdosingofmiceandmen.ClinInfectDis26:1,10;quiz11-2.


clavulanateagainstmurinetuberculosis.AntimicrobAgentsChemother55:2597-600.

4. CordillotM,DubeeV,TribouletS,DubostL,MarieA,HugonnetJE,ARthurM,Mainardi

JL. 2013. In vitro cross-linking of mycobacterium tuberculosis peptidoglycan by L,D-

transpeptidasesandinactivationoftheseenzymesbycarbapenems.AntimicrobAgents

Chemother57:5940-5.

5. DiaconAH, vanderMerweL,BarnardM, vonGroote-Bidlingmaier F, LangeC,García-

BasteiroAL,SeveneE,BallellL,Barros-AguirreD.2016.β-LactamsagainstTuberculosis--

New Trick for an Old Dog? N Engl J Med. Jul 28;375(4):393-4. doi:

10.1056/NEJMc1513236.Epub2016Jul13.

6. SrivastaS,vanRijnSP,WesselsAM,AlffenaarJW,GumboT.2016.Susceptibilitytesting

ofAntibioticsthatdegradefasterthanthedoublingtimeofslow-growingmycobacteria:

Ertapenem sterilizing effect versus Mycobacterium tuberculosis. Antimicrob Agents

Chemother60(5):3193-5


clavulanateagainstmurinetuberculosis.AntimicrobAgentsChemother55(6):2597-600


Kostering JG, van derWerf TS, Alffenaar JW 2016. Pharmacokinetics of ertapenem in

patientswithmultidrug-resistanttuberculosis.EurRespirJ47:1229-34

9. TiberiS,D'AmbrosioL,DeLorenzoSViggianiP,CentisR,Sothiu,AlfenaarJW,MiglioriGB.





2002.Pharmacokineticsof ertapenem inhealthy young volunteers.AntimicrobAgents


123

7


administrationofertapenemtherebyhavingnolossofertapenemduetoabsorption.Several

studieslookingattheexposureofertapeneminpatientshaveshownthattheexposurein

patients varies greatly (14,15). There is little pharmacokinetic variability in the MDR-TB

patients(table3).

A limitedsamplingstrategyusingtwosamplingtimepoints is favoredas itwouldgivethe

leastburdentopatientsas it isminimally invasiveand leasttime-consuming.Additionally,

less timebetweensamplingtimepoints ismorefeasible inclinicalpractice,since it is less

pronetosamplingmistakes.Moreover, limitationofthisstudyisthatafter6hsparsedata

wasavailable,thereforeresultsafter6hislesssubstantiatedwithclinicaldata.

AstherearelimitedoptionsfortreatingMDR-TBandresistanceofantibioticsisanemerging

problem,wethink it is timetostartassessingefficacyofertapeneminMDR-Bpatients in

Phase II clinical trial testing theearlybactericidalactivity.Thedeveloped limitedsampling

strategycanbeusedtoevaluatedrugexposureandtherebyreducecostsandburdenforthe

studysubjects.

Conclusion

A pharmacokinetic model and limited sampling strategy bases on data from healthy

volunteerswasabletopredicttheAUC0-24handf40%T>MICinMDR-TBpatients.Thismodel

canbeusedinphaseIIstudies.

Conflictofinterest:nonedeclared

Acknowledgement:TheauthorsthankMerck&Co.Inc.USA,forprovidingdatafromearlier

pharmacokineticsstudiesofertapenem

References1. MoutonJW,TouzwDJ,Horrevorts,VinkAA.2000.Comparativepharmacokineticsofthe

carbapenems:Clinicalimplications.ClinPharmacokinet39:185-201.

2. Craig WA. 1998. Pharmacokinetic/pharmacodynamic parameters: Rationale for

antibacterialdosingofmiceandmen.ClinInfectDis26:1,10;quiz11-2.


clavulanateagainstmurinetuberculosis.AntimicrobAgentsChemother55:2597-600.

4. CordillotM,DubeeV,TribouletS,DubostL,MarieA,HugonnetJE,ARthurM,Mainardi

JL. 2013. In vitro cross-linking of mycobacterium tuberculosis peptidoglycan by L,D-

transpeptidasesandinactivationoftheseenzymesbycarbapenems.AntimicrobAgents

Chemother57:5940-5.

5. DiaconAH, vanderMerweL,BarnardM, vonGroote-Bidlingmaier F, LangeC,García-

BasteiroAL,SeveneE,BallellL,Barros-AguirreD.2016.β-LactamsagainstTuberculosis--

New Trick for an Old Dog? N Engl J Med. Jul 28;375(4):393-4. doi:

10.1056/NEJMc1513236.Epub2016Jul13.

6. SrivastaS,vanRijnSP,WesselsAM,AlffenaarJW,GumboT.2016.Susceptibilitytesting

ofAntibioticsthatdegradefasterthanthedoublingtimeofslow-growingmycobacteria:

Ertapenem sterilizing effect versus Mycobacterium tuberculosis. Antimicrob Agents

Chemother60(5):3193-5


clavulanateagainstmurinetuberculosis.AntimicrobAgentsChemother55(6):2597-600


Kostering JG, van derWerf TS, Alffenaar JW 2016. Pharmacokinetics of ertapenem in

patientswithmultidrug-resistanttuberculosis.EurRespirJ47:1229-34

9. TiberiS,D'AmbrosioL,DeLorenzoSViggianiP,CentisR,Sothiu,AlfenaarJW,MiglioriGB.





2002.Pharmacokineticsof ertapenem inhealthy young volunteers.AntimicrobAgents


124

Chapter 7

11. BorracciT,AdembriC,AccettaG,BertiJ,CappelliniI,LuccheseM,BiggeriA,DeGaudio

AR,NovelliA.2014.Useoftheparenteralantibioticertapenemasshorttermprophylaxis

inbariatricsurgery:Apharmaco-kinetic-pharmacodynamicstudyinclassIIIobesefemale

patients.MinervaAnestesiol80:1005-11.

12. Chen M, Nafziger AN, Drusano GL, Ma L, Bertino JS Jr. 2006. Comparative


weight,obese,andextremelyobeseadults.AntimicrobAgentsChemother50:1222-7.

13. EylerRF,VilayAM,NaderAM,HeungM,PlevaM,SowinskiKM,DePestelDD,SorgelF,

Kinzig M, Mueller BA. 2014. Pharmacokinetics of ertapenem in critically ill patients

receivingcontinuousvenovenoushemodialysisorhemodiafiltration.AntimicrobAgents

Chemother8:1320-6.

14. MistryGC,Majumdar AK, Swan S, SicaD, Fisher A, Xu Y, HesneyM, Xi L,Wagner JA,

Deutsch PJ. 2006. Pharmacokinetics of ertapenem in patientswith varying degrees of

renalinsufficiencyandinpatientsonhemodialysis.JClinPharmacol46:1128-38.

15. BurkhardtO,HaferC,LanghoffA,KaeverV,KumarV,WelteT,HallerH,FliserD,Kielsteijn

JT.2009.Pharmacokineticsofertapenemincriticallyillpatientswithacuterenalfailure

undergoingextendeddailydialysis.NephrolDialTransplant24:267-71.

16. BrinkAJ,RichardsGA,SchillackV,KiemS,SchentagJ.2009.Pharmacokineticsofonce-

daily dosing of ertapenem in critically ill patients with severe sepsis. Int J Antimicrob

Agents33:432-6.



pneumonia: Pharmacokineticswith special consideration of free-drug concentration. J


18. CardoneKE,GrabeDW,KulawyRW,KulawyRW,DaouiR,RoglieriJ,MeolaS,DrusanoGL,

LodiseTP2012.Ertapenempharmacokineticsandpharmacodynamicsduringcontinuous

ambulatoryperitonealdialysis.AntimicrobAgentsChemother56:725-30.

19. Alffenaar JW, Kosterink JG, vanAltenaR, van derWerf TS,UgesDR, Proost JH. 2010.

Limitedsamplingstrategiesfortherapeuticdrugmonitoringoflinezolidinpatientswith

multidrug-resistanttuberculosis.TherDrugMonit.32:97-101.

20. PrangerAD,KosterinkJG,vanAltenaR,AarnoutseRE,vanderWerfTS,UgesDR,Alffenaar

JW.2011.Limited-samplingstrategiesfortherapeuticdrugmonitoringofmoxifloxacinin

patientswithtuberculosis.TherDrugMonit.33:350-4.

21. Magis-Escurra C, Alffenaar JW, Hoefnagels I, Dekhuijzen PN, BoereeMR, van Ingen J,

Aarnoutse RE. 2013. Pharmacokinetic studies in patients with nontuberculous

mycobacteriallunginfections.IntJAntimicrobAgents42:256-61.

22. Mould DR, Upton RN. 2013. Basic Concepts in Population Modeling, Simulation, and

Model-Based Drug Development—Part 2: Introduction to Pharmacokinetic Modeling

Methods.CPTPharmacometricsSystPharmacol.Apr;2(4):e38.



method.AntimicrobAgentsChemother58(6):3481-4.

24. DaillyE,ArnouldJF,FraissinetF,NauxE,LetarddelaBouraliereMA,BouquieR,Deslandes

G, Jolliet P, Le Floch R. 2013. Pharmacokinetics of ertapenem in burns patients. Int J

AntimicrobAgents42:48-52.

25. LiebchenU,KratzerA,WichaSG,KeesF,KloftC,KeesMG.2014.Unboundfractionof

ertapeneminintensivecareunitpatients.JAntimicrobChemother69(11):3108-11

26. 2Lewis SJ, KaysMB,Mueller BA. 2016. Use ofMonte Carlo simulations to determine

optimalcarbapenemdosingincriticallyillpatientsreceivingprolongedintermittentrenal

replacementtherapy.JClinPharmacol26.doi:10.1002/jcph.727.[Epubaheadofprint]

27. 27.SheinerLB,BealSL.1981.SomeSuggestionsforMeasuringPredictivePerformance.

JournalofPharmacokineticsandBiopharmaceutics9(4):503-512

28. 28.ProostJH,EleveldDJ.2006.Performanceofaniterativetwo-stageBayesiantechnique

for population pharmacokinetic analysis of rich data-sets. Pharm Res 23:2748-2759.

http://dx.doi.org/10.1007/s11095-006-9116-0

125

7


11. BorracciT,AdembriC,AccettaG,BertiJ,CappelliniI,LuccheseM,BiggeriA,DeGaudio

AR,NovelliA.2014.Useoftheparenteralantibioticertapenemasshorttermprophylaxis

inbariatricsurgery:Apharmaco-kinetic-pharmacodynamicstudyinclassIIIobesefemale

patients.MinervaAnestesiol80:1005-11.

12. Chen M, Nafziger AN, Drusano GL, Ma L, Bertino JS Jr. 2006. Comparative


weight,obese,andextremelyobeseadults.AntimicrobAgentsChemother50:1222-7.

13. EylerRF,VilayAM,NaderAM,HeungM,PlevaM,SowinskiKM,DePestelDD,SorgelF,

Kinzig M, Mueller BA. 2014. Pharmacokinetics of ertapenem in critically ill patients

receivingcontinuousvenovenoushemodialysisorhemodiafiltration.AntimicrobAgents

Chemother8:1320-6.

14. MistryGC,Majumdar AK, Swan S, SicaD, Fisher A, Xu Y, HesneyM, Xi L,Wagner JA,

Deutsch PJ. 2006. Pharmacokinetics of ertapenem in patientswith varying degrees of

renalinsufficiencyandinpatientsonhemodialysis.JClinPharmacol46:1128-38.

15. BurkhardtO,HaferC,LanghoffA,KaeverV,KumarV,WelteT,HallerH,FliserD,Kielsteijn

JT.2009.Pharmacokineticsofertapenemincriticallyillpatientswithacuterenalfailure

undergoingextendeddailydialysis.NephrolDialTransplant24:267-71.

16. BrinkAJ,RichardsGA,SchillackV,KiemS,SchentagJ.2009.Pharmacokineticsofonce-

daily dosing of ertapenem in critically ill patients with severe sepsis. Int J Antimicrob

Agents33:432-6.



pneumonia: Pharmacokineticswith special consideration of free-drug concentration. J


18. CardoneKE,GrabeDW,KulawyRW,KulawyRW,DaouiR,RoglieriJ,MeolaS,DrusanoGL,

LodiseTP2012.Ertapenempharmacokineticsandpharmacodynamicsduringcontinuous

ambulatoryperitonealdialysis.AntimicrobAgentsChemother56:725-30.

19. Alffenaar JW, Kosterink JG, vanAltenaR, van derWerf TS,UgesDR, Proost JH. 2010.

Limitedsamplingstrategiesfortherapeuticdrugmonitoringoflinezolidinpatientswith

multidrug-resistanttuberculosis.TherDrugMonit.32:97-101.

20. PrangerAD,KosterinkJG,vanAltenaR,AarnoutseRE,vanderWerfTS,UgesDR,Alffenaar

JW.2011.Limited-samplingstrategiesfortherapeuticdrugmonitoringofmoxifloxacinin

patientswithtuberculosis.TherDrugMonit.33:350-4.

21. Magis-Escurra C, Alffenaar JW, Hoefnagels I, Dekhuijzen PN, BoereeMR, van Ingen J,

Aarnoutse RE. 2013. Pharmacokinetic studies in patients with nontuberculous

mycobacteriallunginfections.IntJAntimicrobAgents42:256-61.

22. Mould DR, Upton RN. 2013. Basic Concepts in Population Modeling, Simulation, and

Model-Based Drug Development—Part 2: Introduction to Pharmacokinetic Modeling

Methods.CPTPharmacometricsSystPharmacol.Apr;2(4):e38.



method.AntimicrobAgentsChemother58(6):3481-4.

24. DaillyE,ArnouldJF,FraissinetF,NauxE,LetarddelaBouraliereMA,BouquieR,Deslandes

G, Jolliet P, Le Floch R. 2013. Pharmacokinetics of ertapenem in burns patients. Int J

AntimicrobAgents42:48-52.

25. LiebchenU,KratzerA,WichaSG,KeesF,KloftC,KeesMG.2014.Unboundfractionof

ertapeneminintensivecareunitpatients.JAntimicrobChemother69(11):3108-11

26. 2Lewis SJ, KaysMB,Mueller BA. 2016. Use ofMonte Carlo simulations to determine

optimalcarbapenemdosingincriticallyillpatientsreceivingprolongedintermittentrenal

replacementtherapy.JClinPharmacol26.doi:10.1002/jcph.727.[Epubaheadofprint]

27. 27.SheinerLB,BealSL.1981.SomeSuggestionsforMeasuringPredictivePerformance.

JournalofPharmacokineticsandBiopharmaceutics9(4):503-512

28. 28.ProostJH,EleveldDJ.2006.Performanceofaniterativetwo-stageBayesiantechnique

for population pharmacokinetic analysis of rich data-sets. Pharm Res 23:2748-2759.

http://dx.doi.org/10.1007/s11095-006-9116-0

CHAPTER 8

General Discussion and Future Perspectives

128

Chapter 8

GeneralDiscussionThelandscapeofMulti-andExtensivedrugresistant(M/XDR)treatmentofTBiscontinuously

changing.M/XDR-TBmustbetreatedwithmultipledrugstoachieveefficacyandtoprevent

development of further resistance [1].Multiple trials for different regimenswithminimal

resistanceareongoingtoassessshorter,affordable,fullyoralandoptimizedTBtreatment.

Recently, an individual patient data meta-analysis was performed of observational and

experimental studies, published between January 2009 and April 2016, reporting end of

treatmentoutcomes(completion,failureorrelapse)of12,030patientsfrom25countriesin

50 studies. Treatment success of MDR-TB was positively associated with the use of

bedaquiline,latergenerationfluoroquinolones,linezolid,clofazimineandcarbapenems.This

meta-analysis of predominantly observational studies had a large impact on setting new

standardsofcareforM/XDRtreatment.InthenewguidelinesissuedbyWHO[2],theroleof

2nd line injectable agents changed entirely. These compoundswere no longer considered

primordialtoobtainsuccessfuloutcomes;twooftheseagents-kanamycinandcapreomycin

-wereevenassociatedwithapooroutcome,andarenolongerrecommendedintherevised

treatment[2].FortheinitialphaseofMDR-TBtreatment,atleastfoureffectivedrugs;andin

thecontinuationphase,atleastfiveagentsarerecommended.Bedaquilline,moxifloxacinand

linezolidhavebeenlistedasbackboneandstandardofcareinlongerMDR-TBregimens[1-

2].Imipenem-cilastatinandmeropenemhavebeenlistedasGroupCdrugsforuseinlonger

MDR-TBregimensguidelinestocompleteregimens,whenothermedicinescannotbeused

[2].Whileertapenemhasnotyetbeen included in theWHOregimen, ithasa favourable

advantageovertheothercarbapenemsduetoitsonce-dailydosingitsactivityinMDR-TBhas

been demonstrated. This thesis provides additional data on ertapenem as potentially

advantageouscarbapenemcompoundintreatmentofMDR-TB.

DrugSusceptibilityTesting

Effective management of M/XDR-TB relies on rapid diagnosis and treatment. Currently,

phenotypicDrugSusceptibilityTesting(DST)isthegoldstandardfordrugresistancedetection

[3].ThisDSTmethodologyhasbeeninuse inmedicalmicrobiologybuthas inherentflaws

whenitcomestopredictingwhathappensinvivo.Thedrugthatistobetestedisaddedto

theculturemediumatafixedconcentration;inanimalmodelsandinhumanstreatedforTB,

however,drugconcentrationstypicallyfollowapharmacokineticconcentration-timecurve.

Moreover,with every new drug dosage every day, in vivo systems are not vulnerable to

chemicaldegradationwhichisaninherentflawintheinvitrostandardDSTassay.Oneway

toovercomethesemethodologicalflawswouldbetousethehollowfiberinfectionmodel

(HF).HFisaninvitrosystemthatusesapumpsystemwithculturemediawithfluctuating

drugconcentrationsovertime,mimickingmuchmorecloselythe invivoexposuretodrug

concentrationsovertime,correctingforchemicaldegradationofthecompoundunderstudy

aswellasmanyotherinherentflawsintheclassicalDSTassay.

InChapter2,wediscussinvitrostudiesreportingMICsofimipenemandmeropenemalone

orcombinedwithclavulanicacidatarangebetween0.16–32mg/L[4].Thishighvariability

of results between in vitro studiesmight be best explained by the chemical instability of

carbapenemsingrowthmediaattemperaturestypicallyusedininvitrostudies.Carbapenems

areheat-sensitiveandasM.tuberculosisgrowthneedsanincubationat37°Cfor15daysin

liquidmediaand4weeksinsolidmedia,carbapenemshavetowithstandahightemperature

foralongperiodoftime.Ertapenem,thatisunstableat37°C,islikelytobedegradedbefore

killingorinhibitingslow-growingM.tuberculosis,suggestingthaterroneouslyhighMICsfor

Mtbmighthavebeenreportedforcarbapenems[5].Itiscomprehensiblethatthisstability

issueofertapenemhaslikelynotoccurredbefore,sinceertapenemhasonlybeenprescribed

forawiderangeofbacterialinfections,consistingmostlyofawiderangeofgram-positive,

gram-negativeandanaerobicbacteriawithatypicalgenerationtimeof20min.DSTforthese

micro-organismsareavailablewithindays(maximumof48h),comparedtoslowgrowingM.

tuberculosiswithgenerationtimearound24h-resultinginDSTreportingtimeinweeks.

InChapter5,wedemonstrateanewstrategythatshowshowperformDSTsandmeasure

MICsforsuchunstablecompounds[6].Wefirstshowthatertapenemdegradesconsiderably

and secondly,we show thatertapenemsupplementation - to compensate for lossdue to

chemicaldegradation-bringsitwellwithintheEUCASTsusceptibilityrangeof0.5–1mg/L

[6]. However, only one reference strain, M. tuberculosis H37Ra, was used in our MIC

experiment.TodayantimicrobialDSTisbasedoncriticalconcentration-alsoreferredtoas

thebreakpoint -definedas ‘the lowestconcentrationofdrugthatwill inhibit95%ofwild

strainsofM.tuberculosisthathaveneverbeenexposedtodrugs,whileatthesametimenot

inhibitingclinicalstrainsofM.tuberculosisthatareconsideredtoberesistant’[3].Anextstep

129

8


GeneralDiscussionThelandscapeofMulti-andExtensivedrugresistant(M/XDR)treatmentofTBiscontinuously

changing.M/XDR-TBmustbetreatedwithmultipledrugstoachieveefficacyandtoprevent

development of further resistance [1].Multiple trials for different regimenswithminimal

resistanceareongoingtoassessshorter,affordable,fullyoralandoptimizedTBtreatment.

Recently, an individual patient data meta-analysis was performed of observational and

experimental studies, published between January 2009 and April 2016, reporting end of

treatmentoutcomes(completion,failureorrelapse)of12,030patientsfrom25countriesin

50 studies. Treatment success of MDR-TB was positively associated with the use of

bedaquiline,latergenerationfluoroquinolones,linezolid,clofazimineandcarbapenems.This

meta-analysis of predominantly observational studies had a large impact on setting new

standardsofcareforM/XDRtreatment.InthenewguidelinesissuedbyWHO[2],theroleof

2nd line injectable agents changed entirely. These compoundswere no longer considered

primordialtoobtainsuccessfuloutcomes;twooftheseagents-kanamycinandcapreomycin

-wereevenassociatedwithapooroutcome,andarenolongerrecommendedintherevised

treatment[2].FortheinitialphaseofMDR-TBtreatment,atleastfoureffectivedrugs;andin

thecontinuationphase,atleastfiveagentsarerecommended.Bedaquilline,moxifloxacinand

linezolidhavebeenlistedasbackboneandstandardofcareinlongerMDR-TBregimens[1-

2].Imipenem-cilastatinandmeropenemhavebeenlistedasGroupCdrugsforuseinlonger

MDR-TBregimensguidelinestocompleteregimens,whenothermedicinescannotbeused

[2].Whileertapenemhasnotyetbeen included in theWHOregimen, ithasa favourable

advantageovertheothercarbapenemsduetoitsonce-dailydosingitsactivityinMDR-TBhas

been demonstrated. This thesis provides additional data on ertapenem as potentially

advantageouscarbapenemcompoundintreatmentofMDR-TB.

DrugSusceptibilityTesting

Effective management of M/XDR-TB relies on rapid diagnosis and treatment. Currently,

phenotypicDrugSusceptibilityTesting(DST)isthegoldstandardfordrugresistancedetection

[3].ThisDSTmethodologyhasbeeninuse inmedicalmicrobiologybuthas inherentflaws

whenitcomestopredictingwhathappensinvivo.Thedrugthatistobetestedisaddedto

theculturemediumatafixedconcentration;inanimalmodelsandinhumanstreatedforTB,

however,drugconcentrationstypicallyfollowapharmacokineticconcentration-timecurve.

Moreover,with every new drug dosage every day, in vivo systems are not vulnerable to

chemicaldegradationwhichisaninherentflawintheinvitrostandardDSTassay.Oneway

toovercomethesemethodologicalflawswouldbetousethehollowfiberinfectionmodel

(HF).HFisaninvitrosystemthatusesapumpsystemwithculturemediawithfluctuating

drugconcentrationsovertime,mimickingmuchmorecloselythe invivoexposuretodrug

concentrationsovertime,correctingforchemicaldegradationofthecompoundunderstudy

aswellasmanyotherinherentflawsintheclassicalDSTassay.

InChapter2,wediscussinvitrostudiesreportingMICsofimipenemandmeropenemalone

orcombinedwithclavulanicacidatarangebetween0.16–32mg/L[4].Thishighvariability

of results between in vitro studiesmight be best explained by the chemical instability of

carbapenemsingrowthmediaattemperaturestypicallyusedininvitrostudies.Carbapenems

areheat-sensitiveandasM.tuberculosisgrowthneedsanincubationat37°Cfor15daysin

liquidmediaand4weeksinsolidmedia,carbapenemshavetowithstandahightemperature

foralongperiodoftime.Ertapenem,thatisunstableat37°C,islikelytobedegradedbefore

killingorinhibitingslow-growingM.tuberculosis,suggestingthaterroneouslyhighMICsfor

Mtbmighthavebeenreportedforcarbapenems[5].Itiscomprehensiblethatthisstability

issueofertapenemhaslikelynotoccurredbefore,sinceertapenemhasonlybeenprescribed

forawiderangeofbacterialinfections,consistingmostlyofawiderangeofgram-positive,

gram-negativeandanaerobicbacteriawithatypicalgenerationtimeof20min.DSTforthese

micro-organismsareavailablewithindays(maximumof48h),comparedtoslowgrowingM.

tuberculosiswithgenerationtimearound24h-resultinginDSTreportingtimeinweeks.

InChapter5,wedemonstrateanewstrategythatshowshowperformDSTsandmeasure

MICsforsuchunstablecompounds[6].Wefirstshowthatertapenemdegradesconsiderably

and secondly,we show thatertapenemsupplementation - to compensate for lossdue to

chemicaldegradation-bringsitwellwithintheEUCASTsusceptibilityrangeof0.5–1mg/L

[6]. However, only one reference strain, M. tuberculosis H37Ra, was used in our MIC

experiment.TodayantimicrobialDSTisbasedoncriticalconcentration-alsoreferredtoas

thebreakpoint -definedas ‘the lowestconcentrationofdrugthatwill inhibit95%ofwild

strainsofM.tuberculosisthathaveneverbeenexposedtodrugs,whileatthesametimenot

inhibitingclinicalstrainsofM.tuberculosisthatareconsideredtoberesistant’[3].Anextstep

130

Chapter 8

shouldbetostudythewildtypepopulationdistributionMICofcarbapenemswithaminimum

rangeofatleast15putativewild-typeisolatesandwithanoveralltotalofatleast100isolates,

preferably using the HF model. Subsequently, the critical concentration and the

epidemiologicalcutoff(ECOFF)canthenbedefined[7].

TreatmentofMDR-TB

InChapter2,weshowthatthereisapaucityofstudiesontheuseofcarbapenemsinM/XDR

TB.Fewinvivostudieshavebeencarriedouttodate,andonlytwolargeretrospectivestudies

withM/XDRTBpatientsusingimipenemandmeropenemhavebeenperformed;wefound

noclearevidencetoselectoneparticularcarbapenem[4].Noevidenceisavailabletolabel

ertapenemaspotentialGroupCdrugamongthecarbapenemsinthetreatmentofM/XDR

TB. Our focus of this thesis was therefore, to explore ertapenem treatment in order to

performaphaseIIstudyforertapenemtosubstantiateitspotentialasrepurposeddrug.

For studies exploring the clinical efficacy of ertapenem, a pharmacokinetic profile of

ertapenem needs to be established andwe therefore need a procedure to quantify and

validateertapenem inhumanplasmaandhumanserum.Asmentionedabove,stabilityof

ertapenemisanissue,thereforeertapenemneedstobetestedindifferenttestconditions,

including storage stability and at different storage temperatures as samples need to be

movedfromclinicaltrialsitestolaboratories.Wehavethereforedevelopedandvalidateda

liquid chromatography tandem-mass spectrometry (LS-MS/MS) method to quantify

ertapeneminhumanplasma,describedinChapter3ofthisthesis[8].

Inchapter4,weperformedaretrospectivestudytoevaluatesafetyandpharmacokineticsfor

allpatientswithsuspectedMDR-TBwhoreceived1gertapenemonadailybasisaspartof

theirtreatmentregimen[9].Itisinterestingtonotethatpatientsweregivenertapenemfor

aprolongedtime,upto9months;thisisincontrastwiththetreatmentofGram-negativeor

Gram-positivebacterialinfectionswheretheon-labelusetypicallylaststwoweeks.Inastudy

onsafetyandefficacyoflong-termoutpatientertapenemtreatment,ertapenemwasgiven

consecutivelyforaprolongedperiodoftime,8to16weeks,forconsecutivelyskinandsoft

tissueinfections,intra-abdominalinfectionsandosteomyelitis[10].

Ingeneral,ertapenemtreatmentappearswelltoleratedinlong-termtherapyinabroadrange

of bacterial infections. Ertapenem also showed a favorable pharmacokinetic/

pharmacodynamicprofile,withoutanyconstraintsinMDR-TBpatients[9].

Likeallbeta-lactamantimicrobials,ertapenemexhibitstime-dependantkilling.Assessment

of drug exposure of ertapenem should aim at assessing cumulative time above theMIC.

Obviously,andideally,theassessmentshouldaddressthefulldrugcombinationusedtotreat

MDR-TB[11].Usinglimitedsamplingstrategiesmightfacilitatetrialsinlessaffluentsettings.

Inchapter7of this thesis,wedescribea two-compartmentpharmacokineticmodelanda

limitedsamplingstrategythatwedevelopedtosupporttherapeuticdrugmonitoringforan

ertapenemdosageof1gdaily.Weshowthattwowell-timedsamples-at1and5h-was

adequatetopredictertapenemexposureinMDR-TBpatientstoreducetheburdenimposed

onpatientsandthehealthsystembymoreextensivemonitoring[12].Typically,twoorthree

samples are needed to estimate exposure.Other limited sampling strategieswith one to

threesamplingtimepointspost-dosehavealreadybeendevelopedforothersecond-lineanti-

TBdrugs,suchaslinezolid,levofloxacinandamikacin.Resultsshowedthatsamplingpointsat

1and5hpost-dosingwouldbesufficientforamikacin,0and5hforlevofloxacinandat0and

12h for linezolid.Asonlybest limitedsamplingstrategieswereshownfortheseTBdrugs

individually,itwouldbeimportantasanextsteptodeveloponelimitedsamplingstrategyfor

allanti-TBdrugs,usedasbackbonetherapyinthetreatmentofMDR-TB[13-15].

Dosefinding

Correctdosingisacriticalfirststepduringclinicaldevelopment.Thoughourpatientswere

treatedwithadosageof1g,thisdosewasnotclinicallyexploredinaprospectivephase2

(dose-finding)study.Pharmacokinetics/pharmacodynamicmodellingisessentialtooptimise

dosing.DosefractionationisdeemednecessarytodeterminewhichPK/PDparameterismost

importantforclinicalefficacy.Theoptimaltherapeuticdoserangecanbeselectedinadose-

findingstudyusingaHFinfectionmodelstudytomimicpharmacokineticconcentration-time

curvesofantibioticsobservedinTBpatients.ResultsofthisHFsystemcanbeusedinaMonte

Carlosimulationtoidentifytheoptimaldoseofertapenemandthesusceptibilitybreakpoint

basedonMICabovewhichtherapybyertapenemwillfail[16-17].

131

8


shouldbetostudythewildtypepopulationdistributionMICofcarbapenemswithaminimum

rangeofatleast15putativewild-typeisolatesandwithanoveralltotalofatleast100isolates,

preferably using the HF model. Subsequently, the critical concentration and the

epidemiologicalcutoff(ECOFF)canthenbedefined[7].

TreatmentofMDR-TB

InChapter2,weshowthatthereisapaucityofstudiesontheuseofcarbapenemsinM/XDR

TB.Fewinvivostudieshavebeencarriedouttodate,andonlytwolargeretrospectivestudies

withM/XDRTBpatientsusingimipenemandmeropenemhavebeenperformed;wefound

noclearevidencetoselectoneparticularcarbapenem[4].Noevidenceisavailabletolabel

ertapenemaspotentialGroupCdrugamongthecarbapenemsinthetreatmentofM/XDR

TB. Our focus of this thesis was therefore, to explore ertapenem treatment in order to

performaphaseIIstudyforertapenemtosubstantiateitspotentialasrepurposeddrug.

For studies exploring the clinical efficacy of ertapenem, a pharmacokinetic profile of

ertapenem needs to be established andwe therefore need a procedure to quantify and

validateertapenem inhumanplasmaandhumanserum.Asmentionedabove,stabilityof

ertapenemisanissue,thereforeertapenemneedstobetestedindifferenttestconditions,

including storage stability and at different storage temperatures as samples need to be

movedfromclinicaltrialsitestolaboratories.Wehavethereforedevelopedandvalidateda

liquid chromatography tandem-mass spectrometry (LS-MS/MS) method to quantify

ertapeneminhumanplasma,describedinChapter3ofthisthesis[8].

Inchapter4,weperformedaretrospectivestudytoevaluatesafetyandpharmacokineticsfor

allpatientswithsuspectedMDR-TBwhoreceived1gertapenemonadailybasisaspartof

theirtreatmentregimen[9].Itisinterestingtonotethatpatientsweregivenertapenemfor

aprolongedtime,upto9months;thisisincontrastwiththetreatmentofGram-negativeor

Gram-positivebacterialinfectionswheretheon-labelusetypicallylaststwoweeks.Inastudy

onsafetyandefficacyoflong-termoutpatientertapenemtreatment,ertapenemwasgiven

consecutivelyforaprolongedperiodoftime,8to16weeks,forconsecutivelyskinandsoft

tissueinfections,intra-abdominalinfectionsandosteomyelitis[10].

Ingeneral,ertapenemtreatmentappearswelltoleratedinlong-termtherapyinabroadrange

of bacterial infections. Ertapenem also showed a favorable pharmacokinetic/

pharmacodynamicprofile,withoutanyconstraintsinMDR-TBpatients[9].

Likeallbeta-lactamantimicrobials,ertapenemexhibitstime-dependantkilling.Assessment

of drug exposure of ertapenem should aim at assessing cumulative time above theMIC.

Obviously,andideally,theassessmentshouldaddressthefulldrugcombinationusedtotreat

MDR-TB[11].Usinglimitedsamplingstrategiesmightfacilitatetrialsinlessaffluentsettings.

Inchapter7of this thesis,wedescribea two-compartmentpharmacokineticmodelanda

limitedsamplingstrategythatwedevelopedtosupporttherapeuticdrugmonitoringforan

ertapenemdosageof1gdaily.Weshowthattwowell-timedsamples-at1and5h-was

adequatetopredictertapenemexposureinMDR-TBpatientstoreducetheburdenimposed

onpatientsandthehealthsystembymoreextensivemonitoring[12].Typically,twoorthree

samples are needed to estimate exposure.Other limited sampling strategieswith one to

threesamplingtimepointspost-dosehavealreadybeendevelopedforothersecond-lineanti-

TBdrugs,suchaslinezolid,levofloxacinandamikacin.Resultsshowedthatsamplingpointsat

1and5hpost-dosingwouldbesufficientforamikacin,0and5hforlevofloxacinandat0and

12h for linezolid.Asonlybest limitedsamplingstrategieswereshownfortheseTBdrugs

individually,itwouldbeimportantasanextsteptodeveloponelimitedsamplingstrategyfor

allanti-TBdrugs,usedasbackbonetherapyinthetreatmentofMDR-TB[13-15].

Dosefinding

Correctdosingisacriticalfirststepduringclinicaldevelopment.Thoughourpatientswere

treatedwithadosageof1g,thisdosewasnotclinicallyexploredinaprospectivephase2

(dose-finding)study.Pharmacokinetics/pharmacodynamicmodellingisessentialtooptimise

dosing.DosefractionationisdeemednecessarytodeterminewhichPK/PDparameterismost

importantforclinicalefficacy.Theoptimaltherapeuticdoserangecanbeselectedinadose-

findingstudyusingaHFinfectionmodelstudytomimicpharmacokineticconcentration-time

curvesofantibioticsobservedinTBpatients.ResultsofthisHFsystemcanbeusedinaMonte

Carlosimulationtoidentifytheoptimaldoseofertapenemandthesusceptibilitybreakpoint

basedonMICabovewhichtherapybyertapenemwillfail[16-17].

132

Chapter 8

Inchapter6,wepresentaHFinfectionmodelfordosefindingontheuseofertapenem.We

testeddifferentertapenemexposures,basedonhumanequivalentdosesinarangeof0.25–

10gertapenem.Dosefractionationshowedthatertapenemwaslinkedtothepercentageof

the 24 h dosing interval of ertapenem concentration persisting aboveMIC (%T/MIC). An

intravenousdosageof2gonceperdaywasidentifiedasmosteffectiveforsterilizingeffect.

This dosage can be used as a once-a-day dose for the treatment of MDR-TB.

AnertapenemsusceptibilitybreakpointMICof2mg/lwasidentifiedforthatdose[18].

The best possibleway to establish efficacy in a proof-of-principle studywould be to test

ertapeneminanearlybactericidalactivity(EBA)studyof2-weeksdurationintreatment-naive

patientswithdrug-susceptibleTB.Therearetwotechniques thatcanbeusedtomeasure

EBA;inliquidandsolidmedia.Insolidmedia,theEBAismeasuredtothefallinlog10CFUof

Mycobacteriumtuberculosispermlsputumperdayoverthefirst14daysoftreatment. In

liquidmediatheEBAisdeterminedbymeasuringthedailyprolongationoftimetopositivity

(TTP) frombaseline. ForTBdrugs,anEBAstudyhasbeenestablishedas thebestway to

establishefficacy[19-20].Datainthisthesiscanbeusedasastartingpointforawell-designed

prospective phase 2 EBA study to substantiate efficacy and safety of 2 g ertapenem in

combinationwithclavulanicacidontopofanoptimizedbackgroundregimenversusstandard

ofcareinpatientswithdrug-resistantTB.Apopulationmodelandlimitedsamplingstrategy

wasdesignedtosupporttherapeuticdrugmonitoringfor2gertapenem[21].

Futureperspectives

CurrentstandardDSTsystemscannotovercomerapiddecreaseofinitialdrugconcentration

over time due to chemical instability of ertapenem in standard-agar based MIC assays.

Althoughconceptuallysuperior,HFinfectionmodelsareexpensiveforroutineDST.Genotypic

testing might be an option to monitor for resistance to carbapenems. In other bacteria

unrelated to M. tuberculosis, it has already been shown that changes in membrane

permeability andpresenceof effluxpumpsmight lead to resistance tobeta lactams [22].

Recently,a singlenucleotidepolymorphism (SNP) indeRv2421c-Rv2422 intergenic region

wasfoundtobecommonamongM.tuberculosismutants,namedascarbapenemresistance

factor A (CrfA) [22].Whole-genome sequencingwas compelling to attribute carbapenem

resistance to thismutation. Incontrast to time-consumingMIC testing,utilizinggenotypic

testingforthesemutationswouldaccelerateknowledgeoncarbapenemresistance,thereby

preventingpoortreatmentoutcomes.Wholegenomesequencing(WGS)hasthepotentialto

rapidly enable insight into resistance profiles of Mycobacterium tuberculosis strains and

improveindividualizedtreatmentonalargescale[23].Therefore,furthergeneticstudiesand

explorationofthiscarbapenemresistancefactorareessentialtounderstanditspotentialfor

detectionofcarbapenemresistancetoM.tuberculosis.

Ahigh-qualityindividualpatientdatameta-analysisreportedalackofbenefitofcommonly

prescribed second-line injectable (kanamycin, capreomycin) andoraldrugs (ethionamide);

indeed,theirusewasassociatedwithpooroutcomes[1].Itwascommunicatedthatitisnot

expedient or desirable to give these second-line injectables. Moreover, FDA black box

warning for aminoglycosides appeared on the package insert due to side effects such as

hearinglossandkidneydamage[24].Wehypothesizethatcarbapenemsmightbepreferred

abovethese injectabledrugs.Afterefficacyandsafetyofertapenemhasbeenshown ina

phase 2 EBA study, a phase 3 study could help for evaluating the different TB injectable

classes. Amikacin could be comparedwith the carbapenem that shows best efficacy in a

comparativeEBAstudy;suchphase3trialmighthelptoestablishwhichinjectableshouldbe

preferredinMDR-TBtreatment.

Beta-LactamaseC(BLaC)inhibitors,forexampleclavulanate,tazobactamandsulbactam,are

currentlynotavailableonthemarketassingleagentorincombinationwithacarbapenem.

Clinicalstudiesareconfinedsincecarbapenemsneedcombinedtreatment incombination

with amoxicillin/clavulanate. Unfortunately, gastrointestinal side effects are common,

complicating prolonged treatment. Addition of amoxicillin in combination showed a

synergisticeffectinvitro,butitsusewasassociatedwithsignificantlylesssuccessandgreat

mortality[1,25].Therefore,amoxicillinisnotrecommendedasaseparateagentagainstMDR-

TB[2].Itwouldbringaddedvaluewhenfiguringouthowmanypatientswouldbenefitfrom

acombinationofacarbapenemwithabeta-lactamaseinhibitortocreateabusinesscasefor

genericpharmaceuticalcompanies.Choosingacombinationofacarbapenemwithabeta-

lactamaseinhibitor,thebestpartnerbeta-lactamantibioticneedstobeidentified.Clavulanic

acid, tazobactam and sulbactam all have a half-life of 1h, however pharmacokinetic

propertiesas invitroactivity incombinationwithcarbapenemsareconsiderablydifferent

133

8


Inchapter6,wepresentaHFinfectionmodelfordosefindingontheuseofertapenem.We

testeddifferentertapenemexposures,basedonhumanequivalentdosesinarangeof0.25–

10gertapenem.Dosefractionationshowedthatertapenemwaslinkedtothepercentageof

the 24 h dosing interval of ertapenem concentration persisting aboveMIC (%T/MIC). An

intravenousdosageof2gonceperdaywasidentifiedasmosteffectiveforsterilizingeffect.

This dosage can be used as a once-a-day dose for the treatment of MDR-TB.

AnertapenemsusceptibilitybreakpointMICof2mg/lwasidentifiedforthatdose[18].

The best possibleway to establish efficacy in a proof-of-principle studywould be to test

ertapeneminanearlybactericidalactivity(EBA)studyof2-weeksdurationintreatment-naive

patientswithdrug-susceptibleTB.Therearetwotechniques thatcanbeusedtomeasure

EBA;inliquidandsolidmedia.Insolidmedia,theEBAismeasuredtothefallinlog10CFUof

Mycobacteriumtuberculosispermlsputumperdayoverthefirst14daysoftreatment. In

liquidmediatheEBAisdeterminedbymeasuringthedailyprolongationoftimetopositivity

(TTP) frombaseline. ForTBdrugs,anEBAstudyhasbeenestablishedas thebestway to

establishefficacy[19-20].Datainthisthesiscanbeusedasastartingpointforawell-designed

prospective phase 2 EBA study to substantiate efficacy and safety of 2 g ertapenem in

combinationwithclavulanicacidontopofanoptimizedbackgroundregimenversusstandard

ofcareinpatientswithdrug-resistantTB.Apopulationmodelandlimitedsamplingstrategy

wasdesignedtosupporttherapeuticdrugmonitoringfor2gertapenem[21].

Futureperspectives

CurrentstandardDSTsystemscannotovercomerapiddecreaseofinitialdrugconcentration

over time due to chemical instability of ertapenem in standard-agar based MIC assays.

Althoughconceptuallysuperior,HFinfectionmodelsareexpensiveforroutineDST.Genotypic

testing might be an option to monitor for resistance to carbapenems. In other bacteria

unrelated to M. tuberculosis, it has already been shown that changes in membrane

permeability andpresenceof effluxpumpsmight lead to resistance tobeta lactams [22].

Recently,a singlenucleotidepolymorphism (SNP) indeRv2421c-Rv2422 intergenic region

wasfoundtobecommonamongM.tuberculosismutants,namedascarbapenemresistance

factor A (CrfA) [22].Whole-genome sequencingwas compelling to attribute carbapenem

resistance to thismutation. Incontrast to time-consumingMIC testing,utilizinggenotypic

testingforthesemutationswouldaccelerateknowledgeoncarbapenemresistance,thereby

preventingpoortreatmentoutcomes.Wholegenomesequencing(WGS)hasthepotentialto

rapidly enable insight into resistance profiles of Mycobacterium tuberculosis strains and

improveindividualizedtreatmentonalargescale[23].Therefore,furthergeneticstudiesand

explorationofthiscarbapenemresistancefactorareessentialtounderstanditspotentialfor

detectionofcarbapenemresistancetoM.tuberculosis.

Ahigh-qualityindividualpatientdatameta-analysisreportedalackofbenefitofcommonly

prescribed second-line injectable (kanamycin, capreomycin) andoraldrugs (ethionamide);

indeed,theirusewasassociatedwithpooroutcomes[1].Itwascommunicatedthatitisnot

expedient or desirable to give these second-line injectables. Moreover, FDA black box

warning for aminoglycosides appeared on the package insert due to side effects such as

hearinglossandkidneydamage[24].Wehypothesizethatcarbapenemsmightbepreferred

abovethese injectabledrugs.Afterefficacyandsafetyofertapenemhasbeenshown ina

phase 2 EBA study, a phase 3 study could help for evaluating the different TB injectable

classes. Amikacin could be comparedwith the carbapenem that shows best efficacy in a

comparativeEBAstudy;suchphase3trialmighthelptoestablishwhichinjectableshouldbe

preferredinMDR-TBtreatment.

Beta-LactamaseC(BLaC)inhibitors,forexampleclavulanate,tazobactamandsulbactam,are

currentlynotavailableonthemarketassingleagentorincombinationwithacarbapenem.

Clinicalstudiesareconfinedsincecarbapenemsneedcombinedtreatment incombination

with amoxicillin/clavulanate. Unfortunately, gastrointestinal side effects are common,

complicating prolonged treatment. Addition of amoxicillin in combination showed a

synergisticeffectinvitro,butitsusewasassociatedwithsignificantlylesssuccessandgreat

mortality[1,25].Therefore,amoxicillinisnotrecommendedasaseparateagentagainstMDR-

TB[2].Itwouldbringaddedvaluewhenfiguringouthowmanypatientswouldbenefitfrom

acombinationofacarbapenemwithabeta-lactamaseinhibitortocreateabusinesscasefor

genericpharmaceuticalcompanies.Choosingacombinationofacarbapenemwithabeta-

lactamaseinhibitor,thebestpartnerbeta-lactamantibioticneedstobeidentified.Clavulanic

acid, tazobactam and sulbactam all have a half-life of 1h, however pharmacokinetic

propertiesas invitroactivity incombinationwithcarbapenemsareconsiderablydifferent

134

Chapter 8

between these compounds [26]. Sulbactamwas shown to have themost potent activity

againstMDR-TBisolates[27].Meanwhile,ceftazidime-avibactam,acephalosporinisavailable

onthemarket incombinationwithaBLaC inhibitorasparentalagentandshowedpotent

sterilizingactivityagainstdrug-resistantTB[28].

TheWorldHealthOrganizationhasnow rankedTBmedicationwith apreference fororal

agentsoverinjectabledrugs[2].Therefore,weanticipatethatTBprogramswillstartusing

injectables less frequently. In order for ertapenem to have a place in an oral treatment

regimen,anextstepwouldbetoalterthephysical-chemicalpropertiesofertapeneminsuch

amatterthatitcanbedesignedasanoraldrug.Asoftoday,twocarbapenems,faropenem-

medoxomilandtebipenem-pivoxil,arealreadyavailableasoralpro-drugs,butbothhaveyet

tobeapprovedinEurope(EMA)andtheUnitedstates(FDA).Tebipenem-pivoxil isnowin

clinicaldevelopmentasfirstoralcarbapenemfortreatment inbacterial infections inadult

patients[29].Likeertapenemabetterunderstandingofsafety,tolerability,pharmacokinetics

andadose-findingstudyforbothdrugsinMDR-TBpatientsshouldbeperformedtoexplore

thefeasibilityasanti-TBoralagents.

Recently, inhaled antibiotic therapy has attracted increased attention and is becoming a

promising alternative for parental administration. A formulation of colistin improved the

aerolization of meropenem and showed synergistic bacterial killing against multi-drug

resistant gram-negative pathogens [30]. Some of the potential benefits of inhalation

antibiotics are that the inhaled drug doses are delivered directly to the target areas in

pulmonaryTB;andthattopicaldeliverymightreducesystemicsideeffects.Therefore,dry

powderinhalationantibiotics in low-costgeneric inhalers,whichcandelivermultiplehigh-

dosedrypowdercapsulesinresource-lowsettingsmightbethelifesavereveryoneiswaiting

for.

Conclusion

Inconclusion,ertapenemshouldbefurtherdevelopedandstudiedtoexploreitspotentialas

avaluableassetinthetreatmentofMDR-TB.Itistimetostudyitsmeritsinaphase2EBA

studyandnext,labelertapenemasgroupCdrugamongsttheothercarbapenems.

References1. WHO report: Rapid Communication: Key changes to treatment of multidrug- and




treatment–2017,AhmadN,AhujaSD,AkkermanOW,AlffenaarJC,AndersonLF,Baghaei

P,BangD,BarryPM,BastosML,BeheraD,BenedettiA,BissonGP,BoereeMJ,BonnetM,

BrodeSK,BrustJCM,CaiY,CaumesE,CegielskiJP,CentisR,ChanPC,ChanED,ChangKC,

CharlesM,CiruleA,DalcolmoMP,D'AmbrosioL,deVriesG,DhedaK,EsmailA,FloodJ,

FoxGJ,Fréchet-JachymM,FregonaG,GayosoR,GegiaM,GlerMT,GuS,GuglielmettiL,

HoltzTH,HughesJ,IsaakidisP,JarlsbergL,KempkerRR,KeshavjeeS,KhanFA,KipianiM,

KoenigSP,KohWJ,KritskiA,KuksaL,KvasnovskyCL,KwakN,LanZ,LangeC,Laniado-

LaborínR,LeeM,LeimaneV,LeungCC,LeungEC,LiPZ,LowenthalP,MacielEL,Marks

SM,MaseS,MbuagbawL,MiglioriGB,MilanovV,MillerAC,MitnickCD,ModongoC,

MohrE,MonederoI,NahidP,NdjekaN,O'DonnellMR,PadayatchiN,PalmeroD,Pape

JW,PodewilsLJ,Reynolds I,RiekstinaV,Robert J,RodriguezM,SeaworthB,SeungKJ,

Schnippel K, Shim TS, Singla R, Smith SE, Sotgiu G, Sukhbaatar G, Tabarsi P, Tiberi S,

TrajmanA,TrieuL,UdwadiaZF,vanderWerfTS,VezirisN,ViikleppP,VilbrunSC,Walsh

K,WestenhouseJ,YewWW,YimJJ,ZetolaNM,ZignolM,MenziesD.Treatmentcorrelates

of successful outcomes in pulmonary multidrug-resistant tuberculosis: an individual

patientdatameta-analysis.Lancet.2018Sep8;392(10150):821-834.doi:10.1016/S0140-

6736(18)31644-1.Review.

3. WHO report 2018: Technical report on critical concentrations for drug susceptibility

testingofmedicinesused in the treatmentofdrug-resistant tuberculosis.Availableat:

www.who.int/tb/publications/2018/WHO_technical_report_concentrations_TB_drug_s

usceptibility/en/

4. SanderP.vanRijn,MarlankaA.Zuur,RichardAnthony,BobWilffert,RichardvanAltena,

OnnoW.Akkerman,WielC.M.deLange,TjipS.vanderWerf,JosG.W.Kosterink,Jan-

Willem C. Alffenaar. Evaluation of carbapenems for multi/extensive-drug resistant

Mycobacterium tuberculosis treatment. Antimicrob. Agents Chemother. [To be

published].

135

8


between these compounds [26]. Sulbactamwas shown to have themost potent activity

againstMDR-TBisolates[27].Meanwhile,ceftazidime-avibactam,acephalosporinisavailable

onthemarket incombinationwithaBLaC inhibitorasparentalagentandshowedpotent

sterilizingactivityagainstdrug-resistantTB[28].

TheWorldHealthOrganizationhasnow rankedTBmedicationwith apreference fororal

agentsoverinjectabledrugs[2].Therefore,weanticipatethatTBprogramswillstartusing

injectables less frequently. In order for ertapenem to have a place in an oral treatment

regimen,anextstepwouldbetoalterthephysical-chemicalpropertiesofertapeneminsuch

amatterthatitcanbedesignedasanoraldrug.Asoftoday,twocarbapenems,faropenem-

medoxomilandtebipenem-pivoxil,arealreadyavailableasoralpro-drugs,butbothhaveyet

tobeapprovedinEurope(EMA)andtheUnitedstates(FDA).Tebipenem-pivoxil isnowin

clinicaldevelopmentasfirstoralcarbapenemfortreatment inbacterial infections inadult

patients[29].Likeertapenemabetterunderstandingofsafety,tolerability,pharmacokinetics

andadose-findingstudyforbothdrugsinMDR-TBpatientsshouldbeperformedtoexplore

thefeasibilityasanti-TBoralagents.

Recently, inhaled antibiotic therapy has attracted increased attention and is becoming a

promising alternative for parental administration. A formulation of colistin improved the

aerolization of meropenem and showed synergistic bacterial killing against multi-drug

resistant gram-negative pathogens [30]. Some of the potential benefits of inhalation

antibiotics are that the inhaled drug doses are delivered directly to the target areas in

pulmonaryTB;andthattopicaldeliverymightreducesystemicsideeffects.Therefore,dry

powderinhalationantibiotics in low-costgeneric inhalers,whichcandelivermultiplehigh-

dosedrypowdercapsulesinresource-lowsettingsmightbethelifesavereveryoneiswaiting

for.

Conclusion

Inconclusion,ertapenemshouldbefurtherdevelopedandstudiedtoexploreitspotentialas

avaluableassetinthetreatmentofMDR-TB.Itistimetostudyitsmeritsinaphase2EBA

studyandnext,labelertapenemasgroupCdrugamongsttheothercarbapenems.

References1. WHO report: Rapid Communication: Key changes to treatment of multidrug- and




treatment–2017,AhmadN,AhujaSD,AkkermanOW,AlffenaarJC,AndersonLF,Baghaei

P,BangD,BarryPM,BastosML,BeheraD,BenedettiA,BissonGP,BoereeMJ,BonnetM,

BrodeSK,BrustJCM,CaiY,CaumesE,CegielskiJP,CentisR,ChanPC,ChanED,ChangKC,

CharlesM,CiruleA,DalcolmoMP,D'AmbrosioL,deVriesG,DhedaK,EsmailA,FloodJ,

FoxGJ,Fréchet-JachymM,FregonaG,GayosoR,GegiaM,GlerMT,GuS,GuglielmettiL,

HoltzTH,HughesJ,IsaakidisP,JarlsbergL,KempkerRR,KeshavjeeS,KhanFA,KipianiM,

KoenigSP,KohWJ,KritskiA,KuksaL,KvasnovskyCL,KwakN,LanZ,LangeC,Laniado-

LaborínR,LeeM,LeimaneV,LeungCC,LeungEC,LiPZ,LowenthalP,MacielEL,Marks

SM,MaseS,MbuagbawL,MiglioriGB,MilanovV,MillerAC,MitnickCD,ModongoC,

MohrE,MonederoI,NahidP,NdjekaN,O'DonnellMR,PadayatchiN,PalmeroD,Pape

JW,PodewilsLJ,Reynolds I,RiekstinaV,Robert J,RodriguezM,SeaworthB,SeungKJ,

Schnippel K, Shim TS, Singla R, Smith SE, Sotgiu G, Sukhbaatar G, Tabarsi P, Tiberi S,

TrajmanA,TrieuL,UdwadiaZF,vanderWerfTS,VezirisN,ViikleppP,VilbrunSC,Walsh

K,WestenhouseJ,YewWW,YimJJ,ZetolaNM,ZignolM,MenziesD.Treatmentcorrelates

of successful outcomes in pulmonary multidrug-resistant tuberculosis: an individual

patientdatameta-analysis.Lancet.2018Sep8;392(10150):821-834.doi:10.1016/S0140-

6736(18)31644-1.Review.

3. WHO report 2018: Technical report on critical concentrations for drug susceptibility

testingofmedicinesused in the treatmentofdrug-resistant tuberculosis.Availableat:

www.who.int/tb/publications/2018/WHO_technical_report_concentrations_TB_drug_s

usceptibility/en/

4. SanderP.vanRijn,MarlankaA.Zuur,RichardAnthony,BobWilffert,RichardvanAltena,


Willem C. Alffenaar. Evaluation of carbapenems for multi/extensive-drug resistant

Mycobacterium tuberculosis treatment. Antimicrob. Agents Chemother. [To be

published].

136

Chapter 8



55:2597-2600.doi:10.1128/AAC.01824-10;10.1128/AAC.01824-10.





7. EUCAST: Standard operating procedure MIC distributions and the setting of

epidemiological cut-off (ECOFF) values. 2017. Available at:

http://www.eucast.org/mic_distributions_and_ecoffs/

8. vanRijnSP,WesselsAM,GreijdanusB,etal.Quantificationandvalidationofertapenem

usinga liquidchromatography-tandemmassspectrometrymethod.AntimicrobAgents

Chemother2014;58:3481–3484.




Doi:10.1183/13993003.01654-2015.

10. QureshiZA,SyedA,DoiY.2014.Safetyandefficacyoflong-termoutpatientertapenem

therapy.AntimicrobAgentsChemother.Jun;58(6)

11. NicolauDP.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.ClinInfect

Dis2008;47:Suppl.1,S32–S40.






13. KampJ,BolhuisMS,TiberiS,AkkermanOW,CentisR,deLangeWC,KosterinkJG,vander

WerfTS,MiglioriGB,AlffenaarJC.Simplestrategytoassesslinezolidexposureinpatients

with multi-drug-resistant and extensively-drug-resistant tuberculosis. Int J Antimicrob

Agents.2017Jun;49(6):688-694.doi:10.1016/j.ijantimicag.2017.01.017.Epub2017Apr4

14. vandenElsenSHJ,SturkenboomMGG,Van'tBoveneind-VrubleuskayaN,SkrahinaA,van

derWerfTS,Heysell SK,MpagamaS,MiglioriGB,PeloquinCA,TouwDJ,Alffenaar JC.

2018. Population Pharmacokinetic Model and Limited Sampling Strategies for

Personalized Dosing of Levofloxacin in Tuberculosis Patients. Antimicrob Agents

Chemother.Nov26;62(12).

15. Dijkstra JA, van Altena R, Akkerman OW, de LangeWC, Proost JH, van derWerf TS,

KosterinkJG,AlffenaarJW.Limitedsamplingstrategiesfortherapeuticdrugmonitoring

of amikacin and kanamycin in patients with multidrug-resistant tuberculosis. Int J

AntimicrobAgents.2015Sep;46(3):332-7.doi:10.1016/j.ijantimicag.2015.06.008.Epub

2015Jul15.

16. GumboT,Angulo-BarturenI,Ferrer-BazagaS.2015.Pharmacokinetic-Pharmacodynamic

and Dose-Response Relationships of Antituberculosis Drugs: Recommendations and

StandardsforIndustryandAcademia.Jinfectdis.211(S3):S96-106

17. EMA Guideline on Hollow fiber system. 2018: Available at:

https://www.ema.europa.eu/documents/regulatory-procedural-guideline/qualification-

opinion-vitro-hollow-fibre-system-model-tuberculosis-hfs-tb_en.pdf




19. DiaconAH,DawsonR,vonGroote-BidlingmaierF,SymonsG,VenterA,DonaldPR,van

Niekerk C, Everitt D, Winter H, Becker P, Mendel CM, Spigelman MK. 2012. 14-day

bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin

combinations:arandomisedtrial.Lancet.Sep15;380(8846):986-93.

20. Diacon AH, Donald PR. 2014. The early bactericidal acitivity of antituberculosis drugs.

ExpertRev.AntiInfect.Ther.12(2):223-37.




Chemother.

137

8




55:2597-2600.doi:10.1128/AAC.01824-10;10.1128/AAC.01824-10.





7. EUCAST: Standard operating procedure MIC distributions and the setting of

epidemiological cut-off (ECOFF) values. 2017. Available at:

http://www.eucast.org/mic_distributions_and_ecoffs/

8. vanRijnSP,WesselsAM,GreijdanusB,etal.Quantificationandvalidationofertapenem

usinga liquidchromatography-tandemmassspectrometrymethod.AntimicrobAgents

Chemother2014;58:3481–3484.




Doi:10.1183/13993003.01654-2015.

10. QureshiZA,SyedA,DoiY.2014.Safetyandefficacyoflong-termoutpatientertapenem

therapy.AntimicrobAgentsChemother.Jun;58(6)

11. NicolauDP.Pharmacokineticandpharmacodynamicpropertiesofmeropenem.ClinInfect

Dis2008;47:Suppl.1,S32–S40.






13. KampJ,BolhuisMS,TiberiS,AkkermanOW,CentisR,deLangeWC,KosterinkJG,vander

WerfTS,MiglioriGB,AlffenaarJC.Simplestrategytoassesslinezolidexposureinpatients

with multi-drug-resistant and extensively-drug-resistant tuberculosis. Int J Antimicrob

Agents.2017Jun;49(6):688-694.doi:10.1016/j.ijantimicag.2017.01.017.Epub2017Apr4

14. vandenElsenSHJ,SturkenboomMGG,Van'tBoveneind-VrubleuskayaN,SkrahinaA,van

derWerfTS,Heysell SK,MpagamaS,MiglioriGB,PeloquinCA,TouwDJ,Alffenaar JC.

2018. Population Pharmacokinetic Model and Limited Sampling Strategies for

Personalized Dosing of Levofloxacin in Tuberculosis Patients. Antimicrob Agents

Chemother.Nov26;62(12).

15. Dijkstra JA, van Altena R, Akkerman OW, de LangeWC, Proost JH, van derWerf TS,

KosterinkJG,AlffenaarJW.Limitedsamplingstrategiesfortherapeuticdrugmonitoring

of amikacin and kanamycin in patients with multidrug-resistant tuberculosis. Int J

AntimicrobAgents.2015Sep;46(3):332-7.doi:10.1016/j.ijantimicag.2015.06.008.Epub

2015Jul15.

16. GumboT,Angulo-BarturenI,Ferrer-BazagaS.2015.Pharmacokinetic-Pharmacodynamic

and Dose-Response Relationships of Antituberculosis Drugs: Recommendations and

StandardsforIndustryandAcademia.Jinfectdis.211(S3):S96-106

17. EMA Guideline on Hollow fiber system. 2018: Available at:

https://www.ema.europa.eu/documents/regulatory-procedural-guideline/qualification-

opinion-vitro-hollow-fibre-system-model-tuberculosis-hfs-tb_en.pdf




19. DiaconAH,DawsonR,vonGroote-BidlingmaierF,SymonsG,VenterA,DonaldPR,van

Niekerk C, Everitt D, Winter H, Becker P, Mendel CM, Spigelman MK. 2012. 14-day

bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin

combinations:arandomisedtrial.Lancet.Sep15;380(8846):986-93.

20. Diacon AH, Donald PR. 2014. The early bactericidal acitivity of antituberculosis drugs.

ExpertRev.AntiInfect.Ther.12(2):223-37.




Chemother.

138

Chapter 8

22. KumarP,KaushikA,BellDT,ChauhanV,XiaF,StevensRL,LamichhaneG.2017.Mutation

inanunannotatedproteinconferscarbapenemresistanceinmycobacteriumtuberculosis.

Antimicrobagentschemother.Feb23;61(3).

23. GröshelMI,WalkerTM,vanderWerfTS,LangeC,NiemannS,MerkerM.2018.Pathogen-

basedprecisionmedicinefordrug-resistanttuberculosis.PLoSPathog12(10):e1007297.

24. vanAltenaR,DijkstraJA,vanderMeerME,BorjasHowardJF,KosterinkJG,vanSoolingen

D, van der Werf TS, Alffenaar JW. Reduced Chance of Hearing Loss Associated with

TherapeuticDrugMonitoringofAminoglycosidesintheTreatmentofMultidrug-Resistant

Tuberculosis. Antimicrob Agents Chemother. 2017 Feb 23;61(3). pii: e01400-16. doi:

10.1128/AAC.01400-16.Print2017Mar.




10.1093/jac/dks395;10.1093/jac/dks395.

26. delaPenaA,DerendorfH.1999.Pharmacokineticpropertiesofbeta-lactamaseinhibitors.

IntJClinPharmacolTher.Feb;37(2):63-75

27. ZhangD,WangY,LuJ,PangY.2015.InVitroActivityofβ-LactamsinCombinationwithβ-

Lactamase Inhibitors againstMultidrug-ResistantMycobacterium tuberculosis Isolates.

AntimicrobAgentsChemother.Nov2;60(1):393-9.


PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactam has potent sterilizing

activityagainsthighlydrug-resistanttuberculosis.SciAdv.2017Aug30;3(8)

29. JainA,UtleyL,ParrTR,ZabawaT,PucciMJ.2018.Tebipenem,thefirstoralcarbapenem

antibiotic.ExpertRevAntiInfectTher.Jul;16)7:513-522

30. MangalS,ParkH,ZengL,YuHH,LinYW,VelkovT,DenmanJA,ZemlyanovD,LiJ,Zhou

QT.Compositeparticleformulationsofcolistinandmeropenemwithimproved in-vitro

bacterialkillingandaerosolizationforinhalation.IntJPharm.2018Sep5;548(1):443-453.

22. KumarP,KaushikA,BellDT,ChauhanV,XiaF,StevensRL,LamichhaneG.2017.Mutation

inanunannotatedproteinconferscarbapenemresistanceinmycobacteriumtuberculosis.

Antimicrobagentschemother.Feb23;61(3).

23. GröshelMI,WalkerTM,vanderWerfTS,LangeC,NiemannS,MerkerM.2018.Pathogen-

basedprecisionmedicinefordrug-resistanttuberculosis.PLoSPathog12(10):e1007297.

24. vanAltenaR,DijkstraJA,vanderMeerME,BorjasHowardJF,KosterinkJG,vanSoolingen

D, van der Werf TS, Alffenaar JW. Reduced Chance of Hearing Loss Associated with

TherapeuticDrugMonitoringofAminoglycosidesintheTreatmentofMultidrug-Resistant

Tuberculosis. Antimicrob Agents Chemother. 2017 Feb 23;61(3). pii: e01400-16. doi:

10.1128/AAC.01400-16.Print2017Mar.




10.1093/jac/dks395;10.1093/jac/dks395.

26. delaPenaA,DerendorfH.1999.Pharmacokineticpropertiesofbeta-lactamaseinhibitors.

IntJClinPharmacolTher.Feb;37(2):63-75

27. ZhangD,WangY,LuJ,PangY.2015.InVitroActivityofβ-LactamsinCombinationwithβ-

Lactamase Inhibitors againstMultidrug-ResistantMycobacterium tuberculosis Isolates.

AntimicrobAgentsChemother.Nov2;60(1):393-9.


PS, Koeuth T, Dheda K, Gumbo T. 2017. Ceftazidime-avibactam has potent sterilizing

activityagainsthighlydrug-resistanttuberculosis.SciAdv.2017Aug30;3(8)

29. JainA,UtleyL,ParrTR,ZabawaT,PucciMJ.2018.Tebipenem,thefirstoralcarbapenem

antibiotic.ExpertRevAntiInfectTher.Jul;16)7:513-522

30. MangalS,ParkH,ZengL,YuHH,LinYW,VelkovT,DenmanJA,ZemlyanovD,LiJ,Zhou

QT.Compositeparticleformulationsofcolistinandmeropenemwithimprovedin-vitro

bacterialkillingandaerosolizationforinhalation.IntJPharm.2018Sep5;548(1):443-453.

CHAPTER 9

Summary

142

Chapter 9

SummaryTuberculosis (TB) is caused byMycobacterium tuberculosis and is the deadliest infectious

diseaseworldwide.OurworldisfacingapublichealthcrisisastreatmentofTBhasbecome

morechallengingwiththeemergenceofresistancetofirst-linedrugs,makingitdifficultto

eradicate TBby 2030. Therefore, it is urgent to focus on improving current treatment by

developing more active – sterilizing- anti TB drugs. One particularly effective strategy is

rediscovery of old drugs as new agents for treatment against in multidrug resistant

tuberculosis.Beta-lactamantimicrobialdrugsarewidelyuseddrugsforthetreatmentofa

rangeofinfections.Ertapenem,approvedin2001bytheFDAandwidelyusedagainstgram

positiveandnegativebacteria,hasshowntobeactiveinMDRTB.Tobetterunderstandthe

potential roleofertapenemfor the treatmentofM/XDR-TB, theaimof this thesiswas to

evaluatecurrentliterature,invitroactivity,andpharmacokineticsandsafetyinTBpatients.


Inchapter2weevaluatedthepotentialofcarbapenemsforthetreatmentofM/XDR-TB.The

aimofthisreviewwastoevaluatetheliteratureoncurrentlyavailableinvitro,invivoand

clinicaldataoncarbapenemsinthetreatmentofM.tuberculosisanddetectionofknowledge

gaps, inordertotargetfutureresearch.Overalltheresultsofthestudies identifiedinthis

review are consistent. Carbapenems in combination with clavulanate showed increased

activity in vitro. Few in vivo studies have been performed. Ten clinical studies to assess


and ertapenem) were performed. No clear evidence was found to select one particular

carbapenemtodesignaneffectiveM/XDR-TBregimen.Moreclinicalevidenceisneededto

supportrepurposingcarbapenemsforthetreatmentofM/XDR-TB.

In chapter 3 a new simple and robust LC-MS/MS method using a quadruple mass

spectrometerwasdevelopedforanalysisofertapeneminhumanplasma,usingdeuterated

ertapenemasinternalstandard.Thecalibrationcurvewaslinearoverarangeof0.1(LLOQ)

to125mg/L.Thecalculatedaccuracyrangedfrom-2.4%to10.3%.Within-runCVranged

from2.7%to11.8%andbetween-runCVranged from0%to8.4%.Freeze-thawstability

biasedbetween-3.3%and0.1%.StorageofQCsamplesfor96hat4°Cdiffered-4.3to5.6%,

storage at room temperature for 24h, biased from -10.7% to -14.8% and storage in the

autosamplerbiasedbetween-2.9%and-10.0%.

Inchapter4,aretrospectivestudywasperformedforallMDR-TBsuspectedpatientsatthe

Tuberculosis Center Beatrixoord of University Medical Center Groningen (Haren, The

Netherlands)whoreceivedertapenemaspartoftheirtreatmentregimenbetweenthefirst

ofDecember2010andthefirstofMarch2013.Safetyandpharmacokineticswereevaluated.

Eighteenpatientsweretreatedwith1000mgertapenemforameanof77days(range5-210).

Sputumsmearandculturewereconvertedinallpatients.Drugexposurewasevaluatedin12

patients. ThemeanAUC0-24was544,9 (range309–1130)mg*h/L. ThemeanCmaxwas

127.5(73.9–277.9)mg/L.Ingeneral,ertapenemtreatmentwaswelltoleratedduringMDR-

TBtreatmentandshowedafavourablePK/PDprofileinMDR-TBpatients.

Inchapter5wepresentedtherapiddeclineofertapenemduringDSTandwehavethereby

developedanewstrategytoperformDSTandMICsforsuchunstablecompounds.Wehave

shownthatertapenemsupplementationbringsitwellwithinthesusceptibilityrangeandis

likelytohavegoodsterilizingeffectintuberculosis.Thissuggeststhatmostofthepublished

MICsforthisdrugarelikelyfalselyhighandratesofresistancearelikelyfalselyelevated.

Inchapter6ourobjectivewastoidentifytheertapenemexposureassociatedwithoptimal





bacterialburden.Ertapenem-clavulanatecombinationdemonstratedgoodmicrobialkilland

sterilizingeffect.Inadose-fractionationhollowfiberstudy,efficacywaslinkedtopercentage

of the 24-hour dosing interval of ertapenem concentration persisting above MIC. We

identifiedan intravenousdoseof2gramsonceperdayasachieving the target in96%of

patients.

143

9

Summary

SummaryTuberculosis (TB) is caused byMycobacterium tuberculosis and is the deadliest infectious

diseaseworldwide.OurworldisfacingapublichealthcrisisastreatmentofTBhasbecome

morechallengingwiththeemergenceofresistancetofirst-linedrugs,makingitdifficultto

eradicate TBby 2030. Therefore, it is urgent to focus on improving current treatment by

developing more active – sterilizing- anti TB drugs. One particularly effective strategy is

rediscovery of old drugs as new agents for treatment against in multidrug resistant

tuberculosis.Beta-lactamantimicrobialdrugsarewidelyuseddrugsforthetreatmentofa

rangeofinfections.Ertapenem,approvedin2001bytheFDAandwidelyusedagainstgram

positiveandnegativebacteria,hasshowntobeactiveinMDRTB.Tobetterunderstandthe

potential roleofertapenemfor the treatmentofM/XDR-TB, theaimof this thesiswas to

evaluatecurrentliterature,invitroactivity,andpharmacokineticsandsafetyinTBpatients.


Inchapter2weevaluatedthepotentialofcarbapenemsforthetreatmentofM/XDR-TB.The

aimofthisreviewwastoevaluatetheliteratureoncurrentlyavailableinvitro,invivoand

clinicaldataoncarbapenemsinthetreatmentofM.tuberculosisanddetectionofknowledge

gaps, inordertotargetfutureresearch.Overalltheresultsofthestudies identifiedinthis

review are consistent. Carbapenems in combination with clavulanate showed increased

activity in vitro. Few in vivo studies have been performed. Ten clinical studies to assess


and ertapenem) were performed. No clear evidence was found to select one particular

carbapenemtodesignaneffectiveM/XDR-TBregimen.Moreclinicalevidenceisneededto

supportrepurposingcarbapenemsforthetreatmentofM/XDR-TB.

In chapter 3 a new simple and robust LC-MS/MS method using a quadruple mass

spectrometerwasdevelopedforanalysisofertapeneminhumanplasma,usingdeuterated

ertapenemasinternalstandard.Thecalibrationcurvewaslinearoverarangeof0.1(LLOQ)

to125mg/L.Thecalculatedaccuracyrangedfrom-2.4%to10.3%.Within-runCVranged

from2.7%to11.8%andbetween-runCVranged from0%to8.4%.Freeze-thawstability

biasedbetween-3.3%and0.1%.StorageofQCsamplesfor96hat4°Cdiffered-4.3to5.6%,

storage at room temperature for 24h, biased from -10.7% to -14.8% and storage in the

autosamplerbiasedbetween-2.9%and-10.0%.

Inchapter4,aretrospectivestudywasperformedforallMDR-TBsuspectedpatientsatthe

Tuberculosis Center Beatrixoord of University Medical Center Groningen (Haren, The

Netherlands)whoreceivedertapenemaspartoftheirtreatmentregimenbetweenthefirst

ofDecember2010andthefirstofMarch2013.Safetyandpharmacokineticswereevaluated.

Eighteenpatientsweretreatedwith1000mgertapenemforameanof77days(range5-210).

Sputumsmearandculturewereconvertedinallpatients.Drugexposurewasevaluatedin12

patients. ThemeanAUC0-24was544,9 (range309–1130)mg*h/L. ThemeanCmaxwas

127.5(73.9–277.9)mg/L.Ingeneral,ertapenemtreatmentwaswelltoleratedduringMDR-

TBtreatmentandshowedafavourablePK/PDprofileinMDR-TBpatients.

Inchapter5wepresentedtherapiddeclineofertapenemduringDSTandwehavethereby

developedanewstrategytoperformDSTandMICsforsuchunstablecompounds.Wehave

shownthatertapenemsupplementationbringsitwellwithinthesusceptibilityrangeandis

likelytohavegoodsterilizingeffectintuberculosis.Thissuggeststhatmostofthepublished

MICsforthisdrugarelikelyfalselyhighandratesofresistancearelikelyfalselyelevated.

Inchapter6ourobjectivewastoidentifytheertapenemexposureassociatedwithoptimal





bacterialburden.Ertapenem-clavulanatecombinationdemonstratedgoodmicrobialkilland

sterilizingeffect.Inadose-fractionationhollowfiberstudy,efficacywaslinkedtopercentage

of the 24-hour dosing interval of ertapenem concentration persisting above MIC. We

identifiedan intravenousdoseof2gramsonceperdayasachieving the target in96%of

patients.

144

Chapter 9

Inchapter7alimitedsamplingstrategywasdevelopedusingapopulationpharmacokinetic

model based, using an iterative two-stage Bayesian method, on healthy volunteers and

showedtobeadequatetopredictertapenemexposureinMDR-TBpatients.

ExternalvalidationwasperformedbyBayesianfittingofthemodeldevelopedinvolunteers

to the individual data of MDR-TB patients using the developed population model for

volunteers as a prior. A Monte Carlo simulation (n=1000) was used to evaluate limited

samplingstrategies.Thebestperforminglimitedsamplingstrategy,withatime-restrictionof

0-6h,wasfoundtobesamplingat1and5h(R2=0.78,meanpredictionerror=-0.33%anda

rootmeansquareerror=5.5%).Drugexposurewasoverestimatedbyameanpercentageof

4.2(-15.2–23.6%).Consideringafreefractionof5%andtheMICsetat0.5mg/L,9outof12

patientswouldhaveexceededaminimumoff40%T>MIC.

In thegeneral discussionwediscussed insight, tools and understanding of ertapenem as

potentialintreatmentofmultidrug-resistantTuberculosis.Wediscusseddrugsusceptibility

testingofcarbapenemsandthatthehighvariabilityofresultsofinvitrostudiesmightbebest

explained by the chemical instability of carbapenems in growth media at temperatures

typicallyusedininvitrostudies.Wenoticedthatertapenemtreatmentseemswelltolerated

inlong-termtherapyinbroadrangeofbacterialinfectionsasitwasshownretrospectivelyfor

MDR-TBtreatment.Aspatientsweretreatedwithadosageof1gram,itwasnotclinically

substantiated in a prospective phase 2 study. Therefore, we discussed that a dose

fractionationwasdeemednecessarytodeterminewhichPK/PDparameterismostimportant

forclinicalefficacy.Toattaintheoptimaltherapeuticdoserange,ahollowfiberstudywas

performedtomimicpharmacokineticconcentrationprofilesofertapeneminTBpatients.As

2gonceperdaywasidentifiedasmosteffectiveforsterilizingeffect,wesuggestthatdatain

thisthesiscanbeusedasstartingpointforthedesignofawell-designedprospectivephase2

study.Thisphase2studycansubstantiateefficacyandsafetyof2gertapenemcombined

withclavulanicacidontopofanoptimizedbackgroundregimenversusstandardofcarein

patientswithMDR-TB.

In the future perspectives we elaborate on genotypic testing as option to anticipate

resistancetocarbapenemsashollowfibermodelsareexpensiveanddrugsusceptibility is

complicateddue to chemical instability of ertapenem.Wehypothesize that carbapenems

mightbepreferredabovetheseinjectabledrugsasanindividualpatientdatameta-analysis,

reportedalackofbenefitofcommonlyprescribedsecond-lineinjectabledrugs.Furthermore,

weanticipatethatinjectableswillbeusedlessfrequentlybyTBprogramsupcomingyears.In

orderforertapenemtohaveaplaceinanoraltreatmentregimen,nextstepwouldbetoalter

thephysical-chemicalpropertiesofertapeneminsuchamatterthatitcanbedesignedasan

oraldrugoradrypowderinhalationantibiotic.

Weconcludethatthat2gertapenemincombinationwithclavulanicacidmightbeavaluable

assetinthetreatmentofmultidrugresistantTB.

145

9

Summary

Inchapter7alimitedsamplingstrategywasdevelopedusingapopulationpharmacokinetic

model based, using an iterative two-stage Bayesian method, on healthy volunteers and

showedtobeadequatetopredictertapenemexposureinMDR-TBpatients.

ExternalvalidationwasperformedbyBayesianfittingofthemodeldevelopedinvolunteers

to the individual data of MDR-TB patients using the developed population model for

volunteers as a prior. A Monte Carlo simulation (n=1000) was used to evaluate limited

samplingstrategies.Thebestperforminglimitedsamplingstrategy,withatime-restrictionof

0-6h,wasfoundtobesamplingat1and5h(R2=0.78,meanpredictionerror=-0.33%anda

rootmeansquareerror=5.5%).Drugexposurewasoverestimatedbyameanpercentageof

4.2(-15.2–23.6%).Consideringafreefractionof5%andtheMICsetat0.5mg/L,9outof12

patientswouldhaveexceededaminimumoff40%T>MIC.

In thegeneral discussionwediscussed insight, tools and understanding of ertapenem as

potentialintreatmentofmultidrug-resistantTuberculosis.Wediscusseddrugsusceptibility

testingofcarbapenemsandthatthehighvariabilityofresultsofinvitrostudiesmightbebest

explained by the chemical instability of carbapenems in growth media at temperatures

typicallyusedininvitrostudies.Wenoticedthatertapenemtreatmentseemswelltolerated

inlong-termtherapyinbroadrangeofbacterialinfectionsasitwasshownretrospectivelyfor

MDR-TBtreatment.Aspatientsweretreatedwithadosageof1gram,itwasnotclinically

substantiated in a prospective phase 2 study. Therefore, we discussed that a dose

fractionationwasdeemednecessarytodeterminewhichPK/PDparameterismostimportant

forclinicalefficacy.Toattaintheoptimaltherapeuticdoserange,ahollowfiberstudywas

performedtomimicpharmacokineticconcentrationprofilesofertapeneminTBpatients.As

2gonceperdaywasidentifiedasmosteffectiveforsterilizingeffect,wesuggestthatdatain

thisthesiscanbeusedasstartingpointforthedesignofawell-designedprospectivephase2

study.Thisphase2studycansubstantiateefficacyandsafetyof2gertapenemcombined

withclavulanicacidontopofanoptimizedbackgroundregimenversusstandardofcarein

patientswithMDR-TB.

In the future perspectives we elaborate on genotypic testing as option to anticipate

resistancetocarbapenemsashollowfibermodelsareexpensiveanddrugsusceptibility is

complicateddue to chemical instability of ertapenem.Wehypothesize that carbapenems

mightbepreferredabovetheseinjectabledrugsasanindividualpatientdatameta-analysis,

reportedalackofbenefitofcommonlyprescribedsecond-lineinjectabledrugs.Furthermore,

weanticipatethatinjectableswillbeusedlessfrequentlybyTBprogramsupcomingyears.In

orderforertapenemtohaveaplaceinanoraltreatmentregimen,nextstepwouldbetoalter

thephysical-chemicalpropertiesofertapeneminsuchamatterthatitcanbedesignedasan

oraldrugoradrypowderinhalationantibiotic.

Weconcludethatthat2gertapenemincombinationwithclavulanicacidmightbeavaluable

assetinthetreatmentofmultidrugresistantTB.

CHAPTER 10

SamenvattingDankwoord

About the AuthorPublication List

148

Chapter 10

SamenvattingTuberculose(tbc)wordtveroorzaaktdoorMycobacteriumtuberculosiseniswereldwijdde

dodelijkste infectieziekte. Onze wereld wordt geconfronteerd met een crisis voor de

volksgezondheiddoordatdebehandelingvantuberculoseeengrotereuitdagingisgeworden

metdeopkomstvanresistentietegeneerstelijnsgeneesmiddelen.Hetisnoodzakelijkomde

huidigebehandelingteverbeterendoormeeractieve-steriliserende-tbc-geneesmiddelen

teontwikkelen.Eenbijzondereffectievestrategieisherontdekkingvanoudegeneesmiddelen

alsnieuwemiddelenvoordebehandelingvanmultiresistentetuberculose(MDR-tbc).Bèta-

lactam antimicrobiële geneesmiddelen zijn veel gebruikte geneesmiddelen voor de

behandelingvaneenreeksinfecties.Ertapenemisgoedgekeurdin2001doordeFDAenwordt

veelgebruikttegengrampositieveengramnegatievebacteriën.Ertapenemlijktactieftezijn

tegenMDR-tbc.OmdepotentiëlerolvanertapenemvoordebehandelingvanM/XDR-tbc

beter te begrijpen, was het doel van dit proefschrift om de huidige literatuur, in vitro

activiteit,farmacokinetiekenveiligheidbijtbc-patiëntenteevalueren.

Carbapenemszijngeoormerktalspotentieelactievegeneesmiddelenvoordebehandeling

vanM. tuberculosis. In hoofdstuk 2 hebbenwehet potentieel van carbapenems voorde

behandelingvanM/XDR-tbconderzocht.Hetdoelvandezereviewwasomdeliteratuurte

evalueren over de momenteel beschikbare in vitro, in vivo en klinische gegevens over

carbapenemsbijdebehandelingvanM.tuberculosisenhetopsporenvankennislacunes,om

zich te richten op toekomstig onderzoek. Over het algemeen zijn de resultaten van de

onderzoeken die in deze beoordeling zijn geïdentificeerd consistent. Carbapenems in

combinatiemetclavulanaatvertoondenverhoogdeactiviteit invitro.Erzijnweinig invivo

studies uitgevoerd. Er zijn tien klinische onderzoeken uitgevoerd om de werkzaamheid,

veiligheidenverdraagbaarheidvandrieverschillendecarbapenems(imipenem,meropenem

en ertapenem) te beoordelen. Er werd geen duidelijk bewijs gevonden om een bepaald

carbapenemteselecterenomeeneffectiefM/XDR-tbc-regimeteontwerpen.Meerklinisch

bewijsisnodigterondersteuningvanherbestemmingvancarbapenemsvoordebehandeling

vanM/XDR-tbc.

Inhoofdstuk3werdeennieuweeenvoudigeenrobuusteLC-MS/MS-methodeontwikkeld

met behulp van een viervoudige massaspectrometer voor de analyse van ertapenem in

menselijkplasma,waarbijgedeutereerdeertapenemalsinternestandaardwerdgebruikt.De

kalibratiecurve was lineair over een bereik van 0,1 (LLOQ) tot 125mg/ L. De berekende

nauwkeurigheidvarieerdevan-2,4%tot10,3%.DeCVbinnenhetbedrijfvarieerdevan2,7%

tot 11,8% en de CV tussen de runs varieerde van 0% tot 8,4%. Bevries-dooi-stabiliteit

vooroordeel tussen -3,3% en 0,1%. Opslag van QC-monsters gedurende 96 uur bij 4 ° C

verschilde-4,3tot5,6%,opslagbijkamertemperatuurgedurende24uur,vooringesteldvan-

10,7%tot-14,8%enopslagindeautosamplervooringesteldtussen-2,9%en-10,0%.

Inhoofdstuk4werdeenretrospectievestudieuitgevoerdvoorallevermoedelijkeMDR-tbc-

patiënten in het tuberculosecentrum Beatrixoord van het Universitair Medisch Centrum

Groningen (Haren, Nederland) die ertapenem kregen als onderdeel van hun

behandelingsregimetussen1december2010en1maart2013.Veiligheidenfarmacokinetiek

werden geëvalueerd. Achttien patiënten werden behandeld met 1000 mg ertapenem

gedurende een gemiddelde van 77 dagen (bereik 5-210). Sputumuitstrijkje en -cultuur

werdenbijallepatiëntenomgezet.Blootstellingaangeneesmiddelenwerdgeëvalueerdbij

12patiënten.DegemiddeldeAUC0-24was544,9(bereik309-1130)mg*h/L.Degemiddelde

Cmaxwas127,5(73,9-277,9)mg/L.Overhetalgemeenwerddebehandelingmetertapenem

goedverdragentijdensMDR-tbc-behandelingenvertoondeertapenemeengunstigPK/PD-

profielbijMDR-tbc-patiënten.

In hoofdstuk 5 hebben we de snelle afname van ertapenem tijdens een geneesmiddel

gevoeligheidstest(DST)latenzien.Hiervoorhebbenweeennieuwestrategieontwikkeldom

DSTenMIC'suittevoerenvoordergelijkeonstabieleverbindingen.Wehebbenaangetoond

datertapenem-suppletiehetgoedbinnenhetgevoeligheidsbereikbrengtenwaarschijnlijk

een goed steriliserend effect bij tuberculose heeft. Dit suggereert dat de meeste

gepubliceerde MIC's voor dit medicijn waarschijnlijk onjuist hoog zijn en dat de

resistentiepercentageswaarschijnlijklagerzijndanwijnuvermoeden.

Inhoofdstuk6wasonsdoelhetidentificerenvandeertapenem-blootstellinggeassocieerd

metoptimaalsterilisatie-effectenvervolgenseeneenmaaldaagsedosisteontwerpenvoor

149

10

Samenvatting

SamenvattingTuberculose(tbc)wordtveroorzaaktdoorMycobacteriumtuberculosiseniswereldwijdde

dodelijkste infectieziekte. Onze wereld wordt geconfronteerd met een crisis voor de

volksgezondheiddoordatdebehandelingvantuberculoseeengrotereuitdagingisgeworden

metdeopkomstvanresistentietegeneerstelijnsgeneesmiddelen.Hetisnoodzakelijkomde

huidigebehandelingteverbeterendoormeeractieve-steriliserende-tbc-geneesmiddelen

teontwikkelen.Eenbijzondereffectievestrategieisherontdekkingvanoudegeneesmiddelen

alsnieuwemiddelenvoordebehandelingvanmultiresistentetuberculose(MDR-tbc).Bèta-

lactam antimicrobiële geneesmiddelen zijn veel gebruikte geneesmiddelen voor de

behandelingvaneenreeksinfecties.Ertapenemisgoedgekeurdin2001doordeFDAenwordt

veelgebruikttegengrampositieveengramnegatievebacteriën.Ertapenemlijktactieftezijn

tegenMDR-tbc.OmdepotentiëlerolvanertapenemvoordebehandelingvanM/XDR-tbc

beter te begrijpen, was het doel van dit proefschrift om de huidige literatuur, in vitro

activiteit,farmacokinetiekenveiligheidbijtbc-patiëntenteevalueren.

Carbapenemszijngeoormerktalspotentieelactievegeneesmiddelenvoordebehandeling

vanM. tuberculosis. In hoofdstuk 2 hebbenwehet potentieel van carbapenems voorde

behandelingvanM/XDR-tbconderzocht.Hetdoelvandezereviewwasomdeliteratuurte

evalueren over de momenteel beschikbare in vitro, in vivo en klinische gegevens over

carbapenemsbijdebehandelingvanM.tuberculosisenhetopsporenvankennislacunes,om

zich te richten op toekomstig onderzoek. Over het algemeen zijn de resultaten van de

onderzoeken die in deze beoordeling zijn geïdentificeerd consistent. Carbapenems in

combinatiemetclavulanaatvertoondenverhoogdeactiviteit invitro.Erzijnweinig invivo

studies uitgevoerd. Er zijn tien klinische onderzoeken uitgevoerd om de werkzaamheid,

veiligheidenverdraagbaarheidvandrieverschillendecarbapenems(imipenem,meropenem

en ertapenem) te beoordelen. Er werd geen duidelijk bewijs gevonden om een bepaald

carbapenemteselecterenomeeneffectiefM/XDR-tbc-regimeteontwerpen.Meerklinisch

bewijsisnodigterondersteuningvanherbestemmingvancarbapenemsvoordebehandeling

vanM/XDR-tbc.

Inhoofdstuk3werdeennieuweeenvoudigeenrobuusteLC-MS/MS-methodeontwikkeld

met behulp van een viervoudige massaspectrometer voor de analyse van ertapenem in

menselijkplasma,waarbijgedeutereerdeertapenemalsinternestandaardwerdgebruikt.De

kalibratiecurve was lineair over een bereik van 0,1 (LLOQ) tot 125mg/ L. De berekende

nauwkeurigheidvarieerdevan-2,4%tot10,3%.DeCVbinnenhetbedrijfvarieerdevan2,7%

tot 11,8% en de CV tussen de runs varieerde van 0% tot 8,4%. Bevries-dooi-stabiliteit

vooroordeel tussen -3,3% en 0,1%. Opslag van QC-monsters gedurende 96 uur bij 4 ° C

verschilde-4,3tot5,6%,opslagbijkamertemperatuurgedurende24uur,vooringesteldvan-

10,7%tot-14,8%enopslagindeautosamplervooringesteldtussen-2,9%en-10,0%.

Inhoofdstuk4werdeenretrospectievestudieuitgevoerdvoorallevermoedelijkeMDR-tbc-

patiënten in het tuberculosecentrum Beatrixoord van het Universitair Medisch Centrum

Groningen (Haren, Nederland) die ertapenem kregen als onderdeel van hun

behandelingsregimetussen1december2010en1maart2013.Veiligheidenfarmacokinetiek

werden geëvalueerd. Achttien patiënten werden behandeld met 1000 mg ertapenem

gedurende een gemiddelde van 77 dagen (bereik 5-210). Sputumuitstrijkje en -cultuur

werdenbijallepatiëntenomgezet.Blootstellingaangeneesmiddelenwerdgeëvalueerdbij

12patiënten.DegemiddeldeAUC0-24was544,9(bereik309-1130)mg*h/L.Degemiddelde

Cmaxwas127,5(73,9-277,9)mg/L.Overhetalgemeenwerddebehandelingmetertapenem

goedverdragentijdensMDR-tbc-behandelingenvertoondeertapenemeengunstigPK/PD-

profielbijMDR-tbc-patiënten.

In hoofdstuk 5 hebben we de snelle afname van ertapenem tijdens een geneesmiddel

gevoeligheidstest(DST)latenzien.Hiervoorhebbenweeennieuwestrategieontwikkeldom

DSTenMIC'suittevoerenvoordergelijkeonstabieleverbindingen.Wehebbenaangetoond

datertapenem-suppletiehetgoedbinnenhetgevoeligheidsbereikbrengtenwaarschijnlijk

een goed steriliserend effect bij tuberculose heeft. Dit suggereert dat de meeste

gepubliceerde MIC's voor dit medicijn waarschijnlijk onjuist hoog zijn en dat de

resistentiepercentageswaarschijnlijklagerzijndanwijnuvermoeden.

Inhoofdstuk6wasonsdoelhetidentificerenvandeertapenem-blootstellinggeassocieerd

metoptimaalsterilisatie-effectenvervolgenseeneenmaaldaagsedosisteontwerpenvoor

150

Chapter 10

klinischgebruik.WegebruiktenhetHollow-Fiber-systeemmodelvantuberculoseineen28-

dagenblootstellingsreactieonderzoekvan8verschillendeertapenem-doses in combinatie

metclavulanaat.Desystemenwerdenbemonsterdopvoorafbepaaldetijdstippenomhet

concentratie-tijdprofielteverifiërenendetotalebacteriëlelastteidentificeren.Ertapenem-

clavulanaat-combinatievertoondeeengoedmicrobieeldodings-ensteriliserendeffect. In

een dosis-fractioneringHollow-Fiber onderzoekwas dewerkzaamheid gekoppeld aan het

percentage van het 24-uurs doseringsinterval van ertapenem concentratie dat aanhoudt

bovenMIC.Weidentificeerdeneenintraveneuzedosisvan2grameenmaalperdagomhet

doelwittebereikenbij96%vandepatiënten.

Inhoofdstuk7werdeenLimitedsamplingstrategieontwikkeldmetbehulpvaneenpopulatie

farmacokinetischmodelopgezondevrijwilligersentoondevoldoendetezijnomertapenem

blootstellingbijMDR-tbc-patiëntentevoorspellen.Externevalidatiewerduitgevoerddoor

BayesiaanseaanpassingvanhetmodelaandeindividuelegegevensvanMDR-tbc-patiënten

metbehulpvanhetontwikkeldepopulatiemodelvoorvrijwilligersalseenvoorafgaande.Een

Monte Carlo-simulatie (n = 1000) werd gebruikt om limited sampling strategieën te

evalueren.Debestpresterendestrategievoorlimitedsampling,meteentijdbeperkingvan0-

6uur,bleektezijnop1en5uur(R2=0,78,gemiddeldevoorspellingsfout=-0,33%eneen

RSME = 5,5%). De blootstelling aan geneesmiddelen werd overschat met een gemiddeld

percentagevan4,2(-15,2-23,6%).Alsweeenvrijefractievan5%endeMICingesteldop0,5

mg/Lbeschouwen,zouden9vande12patiënteneenminimumvanf40%T>MIChebben

overschreden.

In de algemene discussie bespraken we het inzicht, hulpmiddelen en het begrip van

ertapenemalspotentieelindebehandelingvanmultiresistentetuberculose.Webespraken

het testen van de gevoeligheid voor geneesmiddelen van carbapenems en dat de hoge

variabiliteitvanderesultatenvanin-vitro-onderzoekenhetbestkanwordenverklaarddoor

dechemischeinstabiliteitvancarbapenemsingroeimediamettemperaturendiedoorgaans

wordengebruiktinin-vitro-onderzoeken.Wehebbengemerktdatertapenem-behandeling,

netzoalsretrospectiefwerdaangetoondvoorMDR-tbc-behandeling,goedverdragenlijktte

worden gedurende lange termijn therapie bij een breed scala aan bacteriële infecties.

Patiënten werden behandeld met een dosering van 1 gram, echter is dit niet klinisch

onderbouwdineenprospectieffase2-onderzoek.Daaromhebbenwebediscussieerddateen

dosis-fractionering noodzakelijkwerd geacht om te bepalenwelke PK/ PD-parameter het

belangrijkstisvoorklinischewerkzaamheid.Omhetoptimaletherapeutischedosisbereikte

bereiken, werd een Hollow-Fiber studie uitgevoerd om de farmacokinetische

concentratieprofielenvanertapenembijtbc-patiëntennatebootsen.Omdat2geenmaalper

dagwerdgeïdentificeerdalshetmeesteffectiefvoorhetsteriliserendeeffect,stellenwevoor

datdegegevensinditproefschriftkunnenwordengebruiktalsstartpuntvoorhetontwerp

van een goed ontworpen prospectief fase 2-onderzoek. Deze fase 2-studie kan de

werkzaamheidenveiligheidvan2gertapenemincombinatiemetclavulaanzuurbevestigen

bovenopeengeoptimaliseerdachtergrondregimeversusstandaardbehandelingbijpatiënten

metMDR-tbc.

In de toekomstperspectieven zijn we ingegaan op genotypische testen als optie om te

anticiperenopresistentietegencarbapenems,omdatHollow-Fibermodellenduurzijnende

gevoeligheidvangeneesmiddelengecompliceerdisalsgevolgvanchemischeinstabiliteitvan

ertapenem. Sinds een individuelepatiëntgegevens-meta-analyseeengebrek aan voordeel

van algemeen voorgeschreven tweedelijns injecteerbare geneesmiddelen gemeld heeft,

veronderstellenwedatcarbapenemsmogelijkdevoorkeurhebbenbovendezeinjecteerbare

geneesmiddelen.Verderverwachtenwedatinjecteerbaregeneesmiddelenindekomende

jarenmindervaakzullenwordengebruiktdoortbc-programma's.Omertapenemeenplaats

tegevenineenregimevoororalebehandeling,zoudevolgendestaperinbestaandefysisch-

chemischeeigenschappenvanertapenemteveranderenineendergelijkezaakdathetkan

worden ontworpen als een oraal geneesmiddel of een antibacterieel middel voor het

inhalerenvandroogpoeder.

We concluderen dat 2 g ertapenem in combinatie met clavulaanzuur een waardevolle

aanwinstkanzijnbijdebehandelingvanmultiresistentetbc.

151

10

Samenvatting

klinischgebruik.WegebruiktenhetHollow-Fiber-systeemmodelvantuberculoseineen28-

dagenblootstellingsreactieonderzoekvan8verschillendeertapenem-doses in combinatie

metclavulanaat.Desystemenwerdenbemonsterdopvoorafbepaaldetijdstippenomhet

concentratie-tijdprofielteverifiërenendetotalebacteriëlelastteidentificeren.Ertapenem-

clavulanaat-combinatievertoondeeengoedmicrobieeldodings-ensteriliserendeffect. In

een dosis-fractioneringHollow-Fiber onderzoekwas dewerkzaamheid gekoppeld aan het

percentage van het 24-uurs doseringsinterval van ertapenem concentratie dat aanhoudt

bovenMIC.Weidentificeerdeneenintraveneuzedosisvan2grameenmaalperdagomhet

doelwittebereikenbij96%vandepatiënten.

Inhoofdstuk7werdeenLimitedsamplingstrategieontwikkeldmetbehulpvaneenpopulatie

farmacokinetischmodelopgezondevrijwilligersentoondevoldoendetezijnomertapenem

blootstellingbijMDR-tbc-patiëntentevoorspellen.Externevalidatiewerduitgevoerddoor

BayesiaanseaanpassingvanhetmodelaandeindividuelegegevensvanMDR-tbc-patiënten

metbehulpvanhetontwikkeldepopulatiemodelvoorvrijwilligersalseenvoorafgaande.Een

Monte Carlo-simulatie (n = 1000) werd gebruikt om limited sampling strategieën te

evalueren.Debestpresterendestrategievoorlimitedsampling,meteentijdbeperkingvan0-

6uur,bleektezijnop1en5uur(R2=0,78,gemiddeldevoorspellingsfout=-0,33%eneen

RSME = 5,5%). De blootstelling aan geneesmiddelen werd overschat met een gemiddeld

percentagevan4,2(-15,2-23,6%).Alsweeenvrijefractievan5%endeMICingesteldop0,5

mg/Lbeschouwen,zouden9vande12patiënteneenminimumvanf40%T>MIChebben

overschreden.

In de algemene discussie bespraken we het inzicht, hulpmiddelen en het begrip van

ertapenemalspotentieelindebehandelingvanmultiresistentetuberculose.Webespraken

het testen van de gevoeligheid voor geneesmiddelen van carbapenems en dat de hoge

variabiliteitvanderesultatenvanin-vitro-onderzoekenhetbestkanwordenverklaarddoor

dechemischeinstabiliteitvancarbapenemsingroeimediamettemperaturendiedoorgaans

wordengebruiktinin-vitro-onderzoeken.Wehebbengemerktdatertapenem-behandeling,

netzoalsretrospectiefwerdaangetoondvoorMDR-tbc-behandeling,goedverdragenlijktte

worden gedurende lange termijn therapie bij een breed scala aan bacteriële infecties.

Patiënten werden behandeld met een dosering van 1 gram, echter is dit niet klinisch

onderbouwdineenprospectieffase2-onderzoek.Daaromhebbenwebediscussieerddateen

dosis-fractionering noodzakelijkwerd geacht om te bepalenwelke PK/ PD-parameter het

belangrijkstisvoorklinischewerkzaamheid.Omhetoptimaletherapeutischedosisbereikte

bereiken, werd een Hollow-Fiber studie uitgevoerd om de farmacokinetische

concentratieprofielenvanertapenembijtbc-patiëntennatebootsen.Omdat2geenmaalper

dagwerdgeïdentificeerdalshetmeesteffectiefvoorhetsteriliserendeeffect,stellenwevoor

datdegegevensinditproefschriftkunnenwordengebruiktalsstartpuntvoorhetontwerp

van een goed ontworpen prospectief fase 2-onderzoek. Deze fase 2-studie kan de

werkzaamheidenveiligheidvan2gertapenemincombinatiemetclavulaanzuurbevestigen

bovenopeengeoptimaliseerdachtergrondregimeversusstandaardbehandelingbijpatiënten

metMDR-tbc.

In de toekomstperspectieven zijn we ingegaan op genotypische testen als optie om te

anticiperenopresistentietegencarbapenems,omdatHollow-Fibermodellenduurzijnende

gevoeligheidvangeneesmiddelengecompliceerdisalsgevolgvanchemischeinstabiliteitvan

ertapenem. Sinds een individuelepatiëntgegevens-meta-analyseeengebrek aan voordeel

van algemeen voorgeschreven tweedelijns injecteerbare geneesmiddelen gemeld heeft,

veronderstellenwedatcarbapenemsmogelijkdevoorkeurhebbenbovendezeinjecteerbare

geneesmiddelen.Verderverwachtenwedatinjecteerbaregeneesmiddelenindekomende

jarenmindervaakzullenwordengebruiktdoortbc-programma's.Omertapenemeenplaats

tegevenineenregimevoororalebehandeling,zoudevolgendestaperinbestaandefysisch-

chemischeeigenschappenvanertapenemteveranderenineendergelijkezaakdathetkan

worden ontworpen als een oraal geneesmiddel of een antibacterieel middel voor het

inhalerenvandroogpoeder.

We concluderen dat 2 g ertapenem in combinatie met clavulaanzuur een waardevolle

aanwinstkanzijnbijdebehandelingvanmultiresistentetbc.

152

Chapter 10

Dankwoord

Beste Jan-Willem, ik begin direct met jou. Ik kan bijna geen woorden vinden om jou te

bedanken.Wateenontzettendmooiereishebikmogenmakendankzijjou.Hiereindigthet

helaas.Ikbenjewaanzinnigdankbaardatjealtijdnaastmijhebtgestaan,hetvertrouwenin

mijhebtgehad,ookopdemomentendat ik ineendiepdalzatofevenheteindevande

tunnelnietmeerzag.Ikziejealseenmentordieontzettendveelheeftbijgedragenaanmijn

ontwikkeling, zowel professioneel als persoon. Dankjewel. Ik wens jou en je familie

ontzettendveelplezierenhetallermooisteavontuurinSydney.Genietvanallemomenten.

Wieweetkruistonspadelkaarweerindetoekomst.

BesteTjipen Jos, ikwil jullieenormbedankenvoordemogelijkheiddie julliemijhebben

gegevenomeenpromotietrajectintegaan.Hetwaseenbijzondertrajectwaarinik,naast

mijnstudieentegelijkertijdnaastmijnwerk,wetenschappelijkeartikelenmochtschrijven.Ik

ben jullie ontzettend dankbaar voor jullie begeleiding en tegelijkertijd het geloof en het

vertrouwendatdittrajecttoteengoedeindekonkomen.

Ookwilikgraagdebeoordelingscommissie;prof.dr.B.Wilffert,prof.dr.R.vanCrevel,prof.

dr.J.Rossen,bedankenvoorhetbeoordelenvanmijnmanuscript.

DearTawandaGumbo,ShashikantSrivastavaandCarletonSherman,thankyouforhavingme

inDallas,TexasandtoworkwiththeHollowFibermodelbackin2013.Itwasanhonour.

Graagwilikalleco-auteursvanmijnartikelenbedankenvooralhuntijdenenergie;Richard

vanAltena,MarlankaZuur,OnnoAkkerman,MireilleWessels,RichardAnthony,BobWilffert,

WieldeLange,BenGreijdanus,DaanTouw,DickvanSoolingen,TridiavanderLaan,Johannes

Proost,HuibKerstjens,ShashikantSrivastava,TawandaGumbo,JosKosterink,Tjipvander

WerfenJan-WillemAlffenaar.

Natuurlijk wil ik ook alle tuberculosepatiënten in mijn studies bedanken; zonder jullie

betrokkenheidenjulliebijdrageaandewetenschapwashetnietgelukt.

Beste Alper, Koos, Leonie, Anne-Fleur, Samiksha en Marlanka; ik wil jullie bedanken

gedurendemijnreisenalleliefenleeddatwijopverschillendemomentensamenhebben

gedeeld.Dr.Alper,voorjoueenextraeervollevermelding,geweldighoejemijinhetlaatste

deelvanhettrajecthebtgeholpen.Dankje.

Paranimfen.Toppers.PhillipenAlper.Ikbenblijdatjulliemijindezespannendetijdbijstaan.

Lieve pap enmam, ik prijs mij gelukkigmet jullie onvoorwaardelijke steun en liefde. De

vanzelfsprekendheiddat julliealtijdklaarstaanvoormijenerwarenbijdebelangrijkeen

minderebelangrijkemomentendeafgelopen30jaar,maaktmijeenheelrijkmens.Ikben

heelblijdatikditmetjulliemagdelen.Ditisvoorjullie.

LieveSteven,Lotte,Pieter,PhillipenKarin.Ikbenontzettendtrotsopjullie.Watjullieook

doeninhetlevenendekeuzesdiejulliemaken.Ikstaachterjullie.

LieveHilda,ikbenontzettendblijdatikjouheblerenkenneninhetFeithhuis.Ikgenietecht

vanelkmomentdatwesamenzijn.Wezijnalweertweejaarbijelkaarenikkijkuitnaaralle

momentendiewijnoggaandelenmethetsamenwonenalshoogtepuntditjaar.

Als laatste,wil ikalmijnvrienden,collega’senallendieopverzoekgraaganoniemwillen

blijven, die mij gedurende mijn reis in de wonderbaarlijke wereld van de wetenschap

gesteundhebben,bedanken.

153

10

Dankwoord

Dankwoord

Beste Jan-Willem, ik begin direct met jou. Ik kan bijna geen woorden vinden om jou te

bedanken.Wateenontzettendmooiereishebikmogenmakendankzijjou.Hiereindigthet

helaas.Ikbenjewaanzinnigdankbaardatjealtijdnaastmijhebtgestaan,hetvertrouwenin

mijhebtgehad,ookopdemomentendat ik ineendiepdalzatofevenheteindevande

tunnelnietmeerzag.Ikziejealseenmentordieontzettendveelheeftbijgedragenaanmijn

ontwikkeling, zowel professioneel als persoon. Dankjewel. Ik wens jou en je familie

ontzettendveelplezierenhetallermooisteavontuurinSydney.Genietvanallemomenten.

Wieweetkruistonspadelkaarweerindetoekomst.

BesteTjipen Jos, ikwil jullieenormbedankenvoordemogelijkheiddie julliemijhebben

gegevenomeenpromotietrajectintegaan.Hetwaseenbijzondertrajectwaarinik,naast

mijnstudieentegelijkertijdnaastmijnwerk,wetenschappelijkeartikelenmochtschrijven.Ik

ben jullie ontzettend dankbaar voor jullie begeleiding en tegelijkertijd het geloof en het

vertrouwendatdittrajecttoteengoedeindekonkomen.

Ookwilikgraagdebeoordelingscommissie;prof.dr.B.Wilffert,prof.dr.R.vanCrevel,prof.

dr.J.Rossen,bedankenvoorhetbeoordelenvanmijnmanuscript.

DearTawandaGumbo,ShashikantSrivastavaandCarletonSherman,thankyouforhavingme

inDallas,TexasandtoworkwiththeHollowFibermodelbackin2013.Itwasanhonour.

Graagwilikalleco-auteursvanmijnartikelenbedankenvooralhuntijdenenergie;Richard

vanAltena,MarlankaZuur,OnnoAkkerman,MireilleWessels,RichardAnthony,BobWilffert,

WieldeLange,BenGreijdanus,DaanTouw,DickvanSoolingen,TridiavanderLaan,Johannes

Proost,HuibKerstjens,ShashikantSrivastava,TawandaGumbo,JosKosterink,Tjipvander

WerfenJan-WillemAlffenaar.

Natuurlijk wil ik ook alle tuberculosepatiënten in mijn studies bedanken; zonder jullie

betrokkenheidenjulliebijdrageaandewetenschapwashetnietgelukt.

Beste Alper, Koos, Leonie, Anne-Fleur, Samiksha en Marlanka; ik wil jullie bedanken

gedurendemijnreisenalleliefenleeddatwijopverschillendemomentensamenhebben

gedeeld.Dr.Alper,voorjoueenextraeervollevermelding,geweldighoejemijinhetlaatste

deelvanhettrajecthebtgeholpen.Dankje.

Paranimfen.Toppers.PhillipenAlper.Ikbenblijdatjulliemijindezespannendetijdbijstaan.

Lieve pap enmam, ik prijs mij gelukkigmet jullie onvoorwaardelijke steun en liefde. De

vanzelfsprekendheiddat julliealtijdklaarstaanvoormijenerwarenbijdebelangrijkeen

minderebelangrijkemomentendeafgelopen30jaar,maaktmijeenheelrijkmens.Ikben

heelblijdatikditmetjulliemagdelen.Ditisvoorjullie.

LieveSteven,Lotte,Pieter,PhillipenKarin.Ikbenontzettendtrotsopjullie.Watjullieook

doeninhetlevenendekeuzesdiejulliemaken.Ikstaachterjullie.

LieveHilda,ikbenontzettendblijdatikjouheblerenkenneninhetFeithhuis.Ikgenietecht

vanelkmomentdatwesamenzijn.Wezijnalweertweejaarbijelkaarenikkijkuitnaaralle

momentendiewijnoggaandelenmethetsamenwonenalshoogtepuntditjaar.

Als laatste,wil ikalmijnvrienden,collega’senallendieopverzoekgraaganoniemwillen

blijven, die mij gedurende mijn reis in de wonderbaarlijke wereld van de wetenschap

gesteundhebben,bedanken.

154

Chapter 10

AbouttheAuthorSanderPascalvanRijnwasbornonFebruary12th,1988inBochum(BRD)astheelderofan

identical twin. After attending high School at Johan-de-Witt gymnasium in Dordrecht, he

began studying Pharmacy at the University of Groningen in 2006. In 2008, he was

CommissionerofGeneralAffairs of theRoyalDutchPharmaceutical Students’Association

(KNPSV)and LiaisonSecretaryandDutch representative for theEuropeanPharmaceutical

Students’Association(EPSA).Throughoutthesecondhalveofhismaster’sprogramme,he

startedhisresearchonclinicalpharmacologyofertapenemundersupervisionofJan-Willem

Alffenaar, finally resulting in a PharmD/PhD program at the University Medical Center

Groningen.Duringthisprogram,hehadthehonourtovisitDallas,Texas,UnitedStatesof

America and to evaluate ertapenem in a Hollow-Fiber Infection model against M.

Tuberculosis. At the end of 2014, he earned his Master’s degree of Pharmacy, and

jumpstartedhiscareerdirectlyasGlobalManagementTraineeatFagron.After2yearsand

severalassignmentsbothathomeandabroad,heswitchedtoaninsurancecompanytostart

as ‘beleidsadviseur intramuralegeneesmiddelen’.Duringhis stayatZilverenKruis,hewas

responsible for the affordability, accessibility and quality of expensive medicines within

inpatient hospital care. In addition, he helped designing the ‘gezamenlijke inkoop dure

geneesmiddelenvoorzorgverzekeraarsNederland’.In2018,hestartedasconsultantatTer

Welle&Associés (TW&A), a consultancy that specializes in innovativemarket access and

healthcare pathways. In addition, he started as relations manager for PharmIntel, an

independent big data firm, providing management information and insights in the

(appropriate)useandoutcomeofexpensivemedicineformosthospitalsintheNetherlands.

Hisfutureperspectiveistocreatevaluebyhelpinghospitalsandpharmaceuticalcompanies

tobringaffordableandaccessiblemedicinestopatientsintheNetherlandsandtherestof

theworld.

Publicationlist1. SanderP.vanRijn,MarlankaA.Zuur,RichardAnthony,BobWilffert,RichardvanAltena,


Willem C. Alffenaar. 2019. Evaluation of carbapenems for treatment of multi- and

extensivelydrug-resistantMycobacteriumtuberculosis.Antimicrob.AgentsChemother.









4. Srivastava S, van Rijn SP,Wessels AMA,Alffenaar JWC,Gumbo T.2016. Susceptibility







Doi:10.1183/13993003.01654-2015.

6. vanRijnSP,WesselsAM,GreijdanusB,TouwDJ,AlffenaarJWC.2014.Quantificationand


method.AntimicrobAgentsChemother;58:3481–3484.

155

10

Publication List

AbouttheAuthorSanderPascalvanRijnwasbornonFebruary12th,1988inBochum(BRD)astheelderofan

identical twin. After attending high School at Johan-de-Witt gymnasium in Dordrecht, he

began studying Pharmacy at the University of Groningen in 2006. In 2008, he was

CommissionerofGeneralAffairs of theRoyalDutchPharmaceutical Students’Association

(KNPSV)and LiaisonSecretaryandDutch representative for theEuropeanPharmaceutical

Students’Association(EPSA).Throughoutthesecondhalveofhismaster’sprogramme,he

startedhisresearchonclinicalpharmacologyofertapenemundersupervisionofJan-Willem

Alffenaar, finally resulting in a PharmD/PhD program at the University Medical Center

Groningen.Duringthisprogram,hehadthehonourtovisitDallas,Texas,UnitedStatesof

America and to evaluate ertapenem in a Hollow-Fiber Infection model against M.

Tuberculosis. At the end of 2014, he earned his Master’s degree of Pharmacy, and

jumpstartedhiscareerdirectlyasGlobalManagementTraineeatFagron.After2yearsand

severalassignmentsbothathomeandabroad,heswitchedtoaninsurancecompanytostart

as ‘beleidsadviseur intramuralegeneesmiddelen’.Duringhis stayatZilverenKruis,hewas

responsible for the affordability, accessibility and quality of expensive medicines within

inpatient hospital care. In addition, he helped designing the ‘gezamenlijke inkoop dure

geneesmiddelenvoorzorgverzekeraarsNederland’.In2018,hestartedasconsultantatTer

Welle&Associés (TW&A), a consultancy that specializes in innovativemarket access and

healthcare pathways. In addition, he started as relations manager for PharmIntel, an

independent big data firm, providing management information and insights in the

(appropriate)useandoutcomeofexpensivemedicineformosthospitalsintheNetherlands.

Hisfutureperspectiveistocreatevaluebyhelpinghospitalsandpharmaceuticalcompanies

tobringaffordableandaccessiblemedicinestopatientsintheNetherlandsandtherestof

theworld.

Publicationlist1. SanderP.vanRijn,MarlankaA.Zuur,RichardAnthony,BobWilffert,RichardvanAltena,


Willem C. Alffenaar. 2019. Evaluation of carbapenems for treatment of multi- and

extensivelydrug-resistantMycobacteriumtuberculosis.Antimicrob.AgentsChemother.







sterilizingeffectofertapenem-clavulanate inahollow fibermodelof tuberculosisand


4. Srivastava S, van Rijn SP,Wessels AMA,Alffenaar JWC,Gumbo T.2016. Susceptibility







Doi:10.1183/13993003.01654-2015.

6. vanRijnSP,WesselsAM,GreijdanusB,TouwDJ,AlffenaarJWC.2014.Quantificationand


method.AntimicrobAgentsChemother;58:3481–3484.

university of groningen clinical pharmacology of ertapenem ...8 chapter 1 general introduction...

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