reactive metabolites pbutler

8/8/2019 Reactive Metabolites PButler

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Identification of reactive metabolites in

early drug discoveryPhil Butler Ph.D. Senior Research Scientist, Cyprotex


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Overview

- Impact of reactive metabolites on drug safety/discovery

- Assessing reactive metabolite formation

- High throughput methods of trapping reactive metabolites


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- Most frequently formed via oxidation reactions with P450 enzymes

being predominantly involved in the catalysis.

- Specific chemical substituents (structural alerts/toxicophores) mayundergo oxidative metabolism leading to reactive metaboliteformation.

- Not solely restricted to phase I metabolic pathways. Phase IImetabolism (e.g. glucuronidation) may lead to reactive metabolitegeneration.

Reactive metabolite formation


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Bioactivation

Reactivemetabolites

DRUGPhase I/II

Stable metabolites

Excretion

Toxicity

Covalent modification of cellularmacromolecules

Altered cellular function

Bioinactivation

Defencemechanisms

Reactive metabolite formation


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Adverse drug reactions (ADRs)

- Any unwanted effect of a drug aside from its expected therapeuticactions.

- Major cause of patient morbidity and mortality.

- Major impediment to process of drug development.

- Drug withdrawal from market?

A. W. Asscher et al., (1995) Bmj. 311, 1003-1006J. Lazarou et al., (2005) Jama. 279, 1200-1205

M. Pirmohamed et al., (1998) Bmj. 316, 1295-1298S. Michelson & K. Joho, (2000) Curr Opin Mol Ther. 2, 651-654

In 1998 >$20 billion was spent on identification and development of drugs.>20% spent on screening methods and toxicity tests.


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Type A (augmented):

- predictable- dose-dependent- exaggeration of pharmacology of drug

B. K. Park et al., (1998) Chem Res Toxicol. 11, 969-988

Classification of ADRs

Type C (chemical):

- predictable from chemical structure- e.g. acetaminophen

Type B (idiosyncratic):- unpredictable- more frequently life-threatening

- less common- seemingly dose-independent


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- Liver is common target due to its major role in metabolism ofxenobiotics.

- >800 drugs have been implicated in causing hepatic injury orhepatotoxicity.

- Drug-induced liver injury is most frequent reason for removal of an

approved drug from the market.

- Drug-induced liver injury accounts for more than 50% of cases ofacute liver failure in USA.

M. Dossing & J. Sonne, (1993) Drug Saf. 9, 441-449

W. M. Lee, (2003) Semin LiverDis. 23, 217-226

Effect of ADRs on the liver


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Drugs

withdrawn for

hepatotoxicity:

5 of 6 havereactive

metabolites

BenoxaprofenIproniazidNefazodoneTienilic acidTroglitazone

Bromfenac (notdetermined)

Drugs with Black Box

warnings for

hepatotoxicity:

8 of 15 have reactivemetabolites

Dacarbazine, DantroleneFelbamate, FlutamideIsoniazid, KetoconazoleTolcapone, Valproic acid

Reactive metabolites notreported for :

Acitretin, Bosentan,Gemtuzumab,Ozogamicin, Naltrexone,Nevirapine, Pemoline,Trovafloxacin

Drugs with a warning

of precaution for

hepatotoxicity:

AcetaminophenCarbamazepineClozapine, DiclofenacDisulfiram, HalothaneLeflunomide,Methyldopa, RifampinTacrine, TamoxifenTerbinafine, TiclopidineZileuton

Drugs associated

with hepatotoxicity

& never approved

in US:

Alpidem, AmineptineAmodiaquineCinchophenDihydralazineDilevaolo, EbrotidineGlafenine, IbufenacIsoxanine,Niperotidien

Perhexiline,PirprofenTilbroquinol

62% involve metabolism

and reactive products

J. L.Walgren et al., (2005) Crit. Rev. Toxicol. 35, 325-361

Impact of reactive metaboliteson drug safety


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- Elucidation of the role of a particular reactive metabolite in ADRs is difficult.

- Unable to predict the potential of a new drug to cause ADRs.

Prevent reactive metabolite formation

Improve drug safety Reduce chance of ADRs

Reactive metabolites


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- Chemical manipulation.- Avoiding structural alerts/toxicophores.

- Avoid structure-based risk- Greater scholarship regarding potential metabolic routes anddownstream consequences.

Improving drug safety

- Screening out metabolic risk- Covalent binding studies- Reactive metabolite screens

- Greater efficacy.- Lower dose.


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- Avoid structure-based risk- Greater scholarship regarding potential metabolic routes and

downstream consequences.





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Y.-J. Wu et al., (2003) J Med Chem. 46, 3778-3781

Potassium channel opener

Equipotent potassium channel opener

Irreversible CYP3A4 inhibitor

No CYP3A4 inhibition

O

NH

N

O

O

NH

F F

N

O

Designing around metabolic risk


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- Greater efficacy.

- Lower dose.


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- High doses commonly associated with adverse events.

- However, exposure/biologically effective dose (e.g. AUC) is far better indicator ofactual amount of a drug that a patient is exposed to due to ADME/protein

binding etc.

Reactive metabolite formation in vitro.1 % incidence of agranulocytosis.Dose: 300 mg/day.

0.1-0.5 % glucuronidation.

Reactive metabolite formation in vitro.No in vivo manifestation.Dose: 10 mg/day.

21-25 % glucuronidation.

J. M. Alvir & J. A. Lieberman, (1994) J Clin Psychiatry. 55, 137-138I. Gardner et al., (1998) Mol Pharmacol. 53, 991-1008

J. P. Uetrecht, (2000) CurrDrug Metab. 1, 133-141

Cl

NH

N

N

N

CH3

NH

N

N

N

CH3

CH3

Role of dose in drug-induced toxicity

Clozapine Olanzapine


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- Screening out metabolic risk

- Covalent binding studies

- Reactive metabolite screens



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- Greater chemical flexibility.

- Less quantitative, higher-throughput screening.

- Rank ordering of compounds

Later stage screening may be more quantitative, detailed and definitive, but

what options do you have when detecting a reactive metabolite at a later

stage?

Screening for reactive metabolites in early drugdiscovery


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Radiolabeled Studies

covalent binding of radiolabeled compound (10M)

in vitro using liver preparations (HLM and RLM; 1mg/ml; NADPH)

in vivo studies dose to rat (20mg/kg)

assess adducts to liver and plasma proteins

D.C. Evans et al., (2004) Chem. Res. Toxicol. 17, 3-16

Merck approach for assessingreactive metabolite formation

Biomarker Studies

covalent binding to a model nucleophile (e.g. glutathione (GSH), cyanide)

uses liver microsomes

trap and characterise reactive metabolites

stable adducts formed

surrogate marker of covalent binding potential


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D.C. Evans et al., (2004) Chem. Res. Toxicol. 17, 3-16

Merck approach for assessingreactive metabolite formation

Radiolabeled Studies

>50pmol eq/mg protein?

- Potential structural modification?

- Availability of existing treatments?

- Daily dose


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Radiolabeled Method

Advantages

- In vitro studies - species differences can be explored

- In vivo studies - related to safety studies

- extrahepatic bioactivation

Advantages and disadvantages ofradiolabeled method

Disadvantages

- Requires radiolabeled compound

- Not suitable for early stage studies


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Biomarker Method

Disadvantages

- No single small molecule serves as universal surrogate- May require follow up study in hepatocytes if positive in microsomes

Advantages and disadvantagesof biomarker method

Advantages

-Amenable to HTS and early stage studies

- Characterisation by LC-MS/MS

- Prioritisation of compounds for radiolabeling

- Indirect information on structure of reactive species


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- Most commonly used biomarker for covalent binding

- Endogenous tripeptide thiol.

- Serves several functions including:- Detoxifying electrophiles- Maintaining essential thiol status of proteins- Modulating critical cellular processes

- Soft nucleophile forms conjugates either spontaneously orenzymatically in reactions catalyzed by GSH S-transferases (GSTs).

- Found in most cells but particularly abundant in liver (5mM).

Physiological role of GSH


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-To clarify the relationship between in vitro formation rate of GSHconjugates and covalent binding to protein.

N. Masubuchi et al., (2007) Chem. Res. Toxicol. 20, 455-464

1- Tienilic acid2- Furosemide3- Clozapine4- Imipramine5- Acetaminophen6- Indomethacin7- Diclofenac8- Carbamazepine

Relationship between covalent binding and GSHconjugate formation


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Microsomal incubation + NADPH + GSH

Detection by LC-MS/MS

Monitoring for a constant neutral loss (CNL) of 129(loss of glutamic acid of GSH)

Independent of drug structure

Amenable to HTS

CNL of 129 is not exclusive for GSH adducts

- Issues with sensitivity and selectivity

- False positives

HS

HN

O

NH2

COOHO

NH

HOOC

Standard method for analysis of GSH conjugates


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Exact mass neutral loss of 129.046 Da.- Endogenous structures in biological matrices may have samenominal mass of 129 Da but less likely to have same exact mass

- Excludes false positives

Improving selectivity

Negative ion MS/MS affords common fragment at m/z 272- Not all GSH conjugates may undergo CNL 129.- MS/MS of GSH adducts in negative mode mostly fragments ofGSH- Precursor ion scan of m/z 272 & positive ion CNL 129?

J. Castro-Perez et al., (2005) Rapid Commun Mass Spectrom. 19, 798C. M. Dieckhaus et al., (2005) Chem Res Toxicol. 18, 630

Sensitivity?


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Better MS signal intensity

- Improves detection of reactive metabolites

Enables use of lower substrate concentration

- Reduces compound solubility issues

- Minimises chance of enzyme saturation

- Decreases compound use

J. R. Soglia et al., (2004)J Pharm Biomed Anal. 36

, 105-116

Use of GSH ethyl ester as reactive metabolitetrapping agent


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J. Gan et al., (2005) Chem Res Toxicol. 18, 896-903

- Synthesised fluorescent trapping agent .

- Comparative chemical reactivity vs GSH.

- NOT a co-factor for GST-mediated adduct formation.

- Some separation issues from dGSH itself (30 min HPLC gradient).

- No characteristic loss of 129.

- Issues with compounds that causefluorescent interference.

Use of dansyl GSH as a trapping agent


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Semi-quantitative method for determining reactive metabolite levels usingquaternary ammonium GSH analogue (QA-GSH)

- Fixed positive charge significantly increased limit of detection.

- Equalized MS response from equimolar amounts of different GSHconjugates.

- m/z of QA-GSH conjugate determined for M+ or MH2+ ion.

- Response factor for IS with same charge state determined (peakarea/conc.)

- Peak area of QA-GSH conjugate/response factor of IS providessemi-quantification.

J. R. Soglia et al., (2006) Chem Res Toxicol. 19, 480-490

Quantifying conjugation?


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GSH

Drug

Reactive metabolite

GSH conjugate (MH++305) Triply labeled GSH adduct (MH++308)

GSH*

Z. Yan & G. W. Caldwell, (2004)Anal Chem. 76, 6835-6847Z. Yan et al., (2005) Drug Metab Dispos. 33, 706-713Z. Yan et al., (2005) Rapid Commun Mass Spectrom. 19, 3322-3330

NH

O

HS

NH

O

NH2

COOH

CH2HOOC

13

13

15

NH

O

HS

NH

O

NH2

COOH

CH2HOOC

13

13

15NH

O

HS

NH

O

NH2

COOH

CH2HOOC

Use of stable-isotope labeled GSH in high-throughput screenings of reactive metabolites


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GSH Triply-labeled GSH

1:1 mixture

Microsomal incubation

Solid-phase extraction Neutral loss scanning(129 Da)

M-SG*M-SG


3 Da



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- Unique MS signature of isotopic doublet that differs in mass by 3 Da.

- Isotopic doublet exhibits approximately same intensity.

- Halogenated compounds (e.g. diclofenac, bromobenzene) form > 1doublet providing further confirmation of conjugation to GSH.

- High confidence by subsequent MS/MS analysis of neutral losses of 75and 129 Da for GSH adducts and 78 and 129 Da for isotopic GSH*

adducts.

- Weak intensity of [MH+129] and [MH+75]?- Supporting data Rt and peak area ratio of isotopic adducts.

Z. Yan & G. W. Caldwell, (2004)Anal Chem. 76, 6835-6847Z. Yan et al., (2005) Drug Metab Dispos. 33, 706-713

Z. Yan et al., (2005) Rapid Commun Mass Spectrom. 19, 3322-3330A. Mutlib et al., (2005) Rapid Commun Mass Spectrom. 19, 3482-3492



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Positive controls

BromobenzeneCarbamazepineClozapinep-cresolDiclofenac-estradiol17--ethynylestradiolFelbamate4-hydroxyestrone3-methylindoleOmeprazolePhenacetin

DextromethorphanFluoxetineKetoconazoleMidazolamTerfenadineTestosteroneTolbutamide

Negative controls

Felbamate requires both esterase and aldehyde dehydrogenase.




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Trapping Agents Functional Group

Glutathione (ester), mercaptoethanol,cysteine

Quinones, enones

Cyanide Iminium ionsSemicarbazideMethoxylamine

Aldehydes

Lysine Imides, aryl halides

Lysine + cysteine Furan, epoxide

TEMPO

(Tetramethylpiperidin-N-oxyl)

Free radical trap

Trapping soft and hard reactive metabolites

Standard screens may detect soft electrophiles such as quinones,quinone imines, epoxides etc

Detection of hard electrophiles may require separate trappingexperiments


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Z. Yan et al., (2007)Anal Chem. 79, 4206-4214

Trapping soft and hard reactive metabolites

- Glycine of GSH replaced by lysine residue.

- Isotopic analogue used for stable isotope trapping experiments

- All adducts undergo CNL of 129 Da.

- Both natural and labeled agents added to incubations distinct isotopicdoublet with mass difference of 8 Da.

-Glu Cys 13C6-15N2-Lys

-129 DaS-R N=R


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- Not all reactive intermediates form adducts with GSH.

- Presence of GSH adduct in vitro does not mean pathwaypredominates in vivo.

- Risk of false negatives enzymes involved in bioactivation.

- Covalent binding does not always result in toxicity.

- Absence of GSH adduct does not guarantee safety.

- Liability screens.

Interpreting GSH conjugation screens


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Points to consider

- Quantification of reactive metabolite formation?

- In vitro to in vivo relationship?

- What does it mean?


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LaterStage

< 50 pmol eq/mg

Advance compound

> 50 pmol eq/mg

Qualifying considerations

Suggested screening strategy forreactive metabolite assessment

Designing out metabolic weaknesses?

Prioritization of drug candidates

Radiolabeling for covalent binding

Assess bioactivation potential

Optimize lead compounds

Early

Stage


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Cyprotex Cyprotex

15 Beech Lane 12 Alfred Street [email protected]

Macclesfield Suite 300 www.cyprotex.com

Cheshire Woburn

SK10 2DR MA 01801

UK USA

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