the 2012 fda draft guidance on drug-drug interactions:drug ...apr 26, 2012 · cyp inhibition...
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The 2012 FDA Draft Guidance on Drug-drug Interactions:Drug drug Interactions:
Enzyme Induction and Beyond
Michael Sinz, Ph.D.
Research FellowResearch and DevelopmentResearch and DevelopmentBristol-Myers Squibb Wallingford, CT USA
NJDMDG Symposium April 26, 2012
The “Guidance for Industry”
Reaction phenotypingDDI (inhibition & induction)
In vivo clinical studieso c ca stud esLabeling
CYP3A4 mRNATranscription
Promoter (CYP3A4) Gene (CYP3A4)
rug CYP3A4
( ) ( )
Con
c. o
f Dr
loss ofefficacy
Drug Drug-OH
Slide 2
Time (hr)
C efficacy
General Scheme of Model-based Prediction: Investigational Drug Interacting with CYP Enzymes
CYP inhibition(reversible and time-dependent)
CYP induction
• Measure mRNA change by investigational • Measure enzyme activity in g y gdrug in cultured human hepatocytes from 3 or more donors• Estimate DDI parameters
I i i RNA d fi d th h ld?
Measure enzyme activity in human liver microsomes• Estimate DDI parameters
Basic Models
Is increase in mRNA > a predefined threshold?Or
Is the calculated R value < 0.9?R3 = 1/(1 + d x Emax x [I] / (EC50 + [I]))
Is the calculated R value >1.1Reversible inhibition, R1=1+[I]/Ki
TDE, R2=(Kobs+Kdeg) & Kobs=kinactx[I]/(KI+[I])
Is AUCR>1.25 (inhibition) or <0.8 (induction)Mechanistic Static Model
1 1Mechanistic
OR, Dynamic Model (PBPK)
AUCR= 1
(AgxBgxCg)x(1-Fg)+Fg
1
(AhxBhxCh)x Fm+(1-Fm)xModels
Slide 3
Conduct a clinical study using an appropriate probe substrate
Yes
Basic Induction Model
1 + d x Emax x [I] 1
R3 = Is increase in mRNA > a predefined threshold?
or
d = 1
EC50 + [I]or
Is the calculated R<0.9
[I] = maximum total (free+unbound) systemic concentration
E i h i i d iEmax is the maximum induction responseEC50 is the concentration causing half maximal effect
What if due to solubility or cytotoxicity issues th E d EC t b d t i d?
AUC/F2 Method ?
Options ?
Basic equation is designed to eliminate false negatives,
the Emax and EC50 can not be determined? Method ?
Slide 4
q g g ,but unfortunately leads to increased false positives,hence the need to move to the mechanistic models
Mechanistic Static Induction Model
AUCR = 1
(AgxBgxCg)x(1-Fg)+Fg
1(AhxBhxCh)xFm+(1-Fm)x( g g g) ( g) g ( h h h) m ( m)
Gut Liver
C = 1 + d x Emax x [I]
[I] + EC50
[I]g = Fa x Ka x Dose/Qen
[I]h = fu x (Cmax + (Fa x Ka x Dose/Qh))
A – reversible inhibitionB – irreversible inhibitiond = 1 (?), likely to be < 1 in the mechanistic model (0.3-0.8; ~0.5)d 1 (?), likely to be 1 in the mechanistic model (0.3 0.8; 0.5)Fa = 1 (if unknown)Ka = 0.1/min (if unknown), a value of 0.03/min is probably more reasonableFg – fraction available after intestinal metabolismF f ti f t i l f b t t
Slide 5
Fh – fraction of systemic clearance of substrate
Interpretation: Is AUCR>1.25 (inhibition) or <0.8 (induction)
Basic to Mechanistic Model
d = 1[I] = 2.3 uM
Basic Model
R 0 12 InductionEmax = 25EC50 = 5.3 uM
R3 = 0.12(R3< 0.9 implies induction)
InductionExpected
d = 0.5Fa = 1Fa 1Ka = 0.03/minDose = 81 uM[I] = 2.3 uMF 0 012
Mechanistic Static Model
AUCR = 0 21InductionE t dFu = 0.012
Emax = 25EC50 = 5.3 uMKdeg,g = 0.00032/min
(AUCR<0.8 implies induction)AUCR = 0.21 Expected
‘Moderate Inducer’
Slide 6
deg,gKdeg,h = 0.00048/minFg,h (midazolam)
Model System for Evaluating Induction
• Primary cultures of human hepatocytes (fresh or cryopreserved)– What about immortalized hepatocytes or NHR assays?
“At present, data generated from other in vitro systems are considered complementary and may be reviewed along with data
d i h l d h ”generated with cultured hepatocyte systems.”– EMA, “very well justified” immortalized cell lines acceptable
• Evaluate 3+ donors to account for interindividual• Evaluate 3+ donors to account for interindividual variability– If one donor is positive in Basic model, the drug is considered an
inducer and “follow-up evaluation is needed” (ie. Mechanistic)
• Need to determine performance of hepatocytes in identifying enzyme induction potential with ‘sufficient’identifying enzyme induction potential with sufficient number of clinical inducers (how many?)
Slide 7
Model System for Evaluating Induction
• “The changes in the mRNA level of the target gene should be used as an endpoint”– Not consistent with EMA guidance (activity & RNA)– Activity should be an option in cases when the test compound is
not an inhibitornot an inhibitor
• Inclusion of negative controls– Could provide value in distinguishing real induction from the ‘noise’ p g g
of the assay, but no guidance given on what to use or at what concentration.
• Evaluate CYP1A2 2B6 and 3A• Evaluate CYP1A2, 2B6, and 3A– If CYP3A is positive, then evaluate CYP2Cs (2C8, 2C9, and 2C19)
– Generally, CYP2C induction is less than CYP3A and mediated by factors be ond PXR and CAR (Fahmi et al DMD 2010 & Chen/Goldstein CDM 2009)beyond PXR and CAR (Fahmi et al, DMD 2010 & Chen/Goldstein, CDM 2009)
Slide 8
In Vitro CYP Inducers (Table 2)
CYP In vitro inducer as positive controls
Recommended concentration (uM) of
positive controls
Reported fold induction in
enzyme activityp y y1A2 Omeprazole
Lansoprazole25-100
1014-24
102B6 Phenobarbital 500-1000 5-10
(CITCO-EMA) (<100 nM)2C8 Rifampin 10 2-42C9 Rifampin 10 42C9 Rifampin 10 42C19 Rifampin 10 202D6 None identified3A4 Rif i 10 50 4 313A4 Rifampin 10-50 4-31
No information on ‘reported fold induction in mRNA response’
Slide 9
Classification of In Vivo Inducers (Table 4)CYP Strong Inducers
>80% decrease in AUC
Moderate Inducers50-80% decrease
in AUC
Weak Inducers20-50% decrease
in AUC1A2 Montelukast, phenytoin,
smokersMoricizine, omeprazole,
phenobarbital2B6 Efavirenz, rifampin Nevirapine
2C8 Rif i2C8 Rifampin
2C9 Carbamazepine, rifampin Aprepitant, bosentan, phenobarbital, St. John’s
wortwort2C19 Rifampin Artemisinin
3A4 Avasimibe, carbamazepine,
Bosentan, efavirenz,etravirine, modafinil,
Amprenavir, aprepitant, armodafinil, p ,
phenytoin,rifampin, St. John’s wort
, ,nafcillin
,clobazamechinacea,
pioglitazone, prednisone, rufinamide, vemurafenib
2D6 None known None known None known
Slide 10
2D6 None known None known None known
Change in Clinical Section
“… it is critical to evaluate the time it takes for the enzyme activities to return to normal when induction or TDI is involved so that
a third crossover period in which the interacting drug is removed”a third crossover period in which the interacting drug is removed”
osur
e
baseline
recovery period
MD
Z ex
po
induced
recovery period
Time (days)
Slide 11
……AND BEYOND
A Novel Mechanism of Enzyme Induction:Non Classical PXR or CAR-Mediated Induction
CYP3A4 Gene Activation by PXR and CAR
CYP3A4 mRNATranscription
D
PXR DPXR
RIFHPF
RXR
TranslationBinding
Promoter (CYP3A4) Gene (CYP3A4)PXR
CARD
RXR
RXR
D D OH
CYP3A4
CAR
DPHTNVP
ug
Drug Drug-OHCAR
Both PXR and CAR can cause
onc.
of D
ru
loss of
Both PXR and CAR can cause CYP3A4 gene activation andenzyme induction leading
to significant drug interactions
Slide 13Time (hr)
Co efficacy
to significant drug interactions
Comparative PXR Binding and Transactivation Results
PXR Binding Assay
BMS-536924 binds to PXR: BMS-665351 does not bind to PXR:BMS-536924 binds to PXR: IC50 = 1.0 uM
BMS-665351 does not bind to PXR: IC50 = 30 uM
SAR
se e
PXR Transactivation Assay
nt R
espo
ns
t Res
pons
EC50=5 uM
Perc
en
Perc
en
Slide 14
Conc. uM Conc. uM
PXR and Hepatocyte Data on Both Sets of Compounds
Binds to PXRActivates PXR
BMS-536924Do Not Bind to PXRDo Not Activate PXR
BMS-665351
Activates PXR
Induces CYP3A4 in PrimaryHuman Hepatocytes
Do Not Activate PXR
Induces CYP3A4 in PrimaryHuman HepatocytesHuman Hepatocytes Human Hepatocytes
Slide 15
Rifampicin-positive control for PXR
Two Mechanisms of CAR-Mediated Enzyme Induction
CA
R
D
ConstitutiveAndrostane
Receptor Mechanism 2
CAR
R
D
PB
CARD
CAR CYP3A4
Mechanism 1
CAR
CARD
CYP3A4
CYP3A4
CYP3A4CYP3A4mRNA and
proteinnucleus
Transit of CAR from the
Slide 16
PB-phenobarbital
Transit of CAR from the cytoplasm to the nucleus by any mechanism is termed Translocation
Activation of CAR and PXR in HepG2 Cells
HepG24hCAR3/CYP3A4 (PXRE/XREM)
HepG26
CAR3 Expression Assay PXR Expression Assay
uc A
ctiv
ity
2
3
hCAR3/CYP3A4 (PXRE/XREM)
uc A
ctiv
ity
3
4
5hPXR/CYP3A4 (PXRE/XREM)
** **
Rel
ativ
e Lu
1
2
Rel
ativ
e Lu
1
2
3
0.1%DMSOCITCO 1uM 1uM 5uM
0
0.1% DMSORIF 10uM 1uM 5uM
0
BMS-665351BMS-665351
BMS-665351 does not activate human CAR or PXR
Slide 17
CITCO-positive control for CAR
CAR Translocation Assay: BMS-665351
Human hepatocytes infected with adenovirusfluorescently tagged with CAR (AD/EYFP-hCAR)
CTL (0.1%DMSO) PB 1 mM hCAR1 Localization in HL-#37100 C
ount
(%)
60
80
00 CC=NN
BMS-665351 (1 µM) BMS-665351 (5 µM) Rel
ativ
e C
20
40
CTL PB 1mM 1uM 5uM0
BMS-665351
C-cytoplasmN-nucleus
BMS-665351 does not translocate CAR
Does the Induction Have Anything to do with CAR?
CAR3 Over Expression System (HepG2)on
NA
expr
essi
oYP
3A4
mR
NC
CAR is involved in the CYP3A4i d ti BMS 665351 10 M
Slide 19Li, Sinz, et al. JPET Dec. 2011
PK11195 – a selective CAR deactivator
induction response BMS-665351: 10 uMCITCO: 1 uMPK11195: 10 uM
Does BMS-665351 Induce the Expression of CAR?tio
n 6
7HepG2Huh7
tion
4
5HL-#16HL-#17
****
**
Human Hepatocytes
NA
Fold
Indu
ct
3
4
5
NA
Fold
Indu
ct
2
3
**
***
**
hCAR
mR
N
1
2
3
hCAR
mR
N
1
2
*
0.1% DMSORIF 10uM
CITCO 1uM 1uM 5uM0
0.1% DMSO RIF 10uM
PB 1mM 1uM 5uM0
BMS-665351 BMS-665351
BMS-665351 induces the expression of CARin cell lines and human hepatocytes
D t i d th i f PXRDoes not induce the expression of PXR
Summary
CYP3A4 mRNATranscription
PXR
GC
VDR
TranslationPromoter (CYP3A4) Gene (CYP3A4)D
CARRXR
D D OH
CYP3A4
CARCAR ?
BMS-665351IGF1R Inhibitor Drug Drug-OH
CAR?
IGF1R Inhibitor
• Further studies necessary to link the increase in CAR expression to the increased expression of CYP3A4– CAR promoter-reporter assay and siRNA CAR knock-down– Does this mechanism of CYP3A4 induction translate to an in vivo DDI?
Slide 21
Acknowledgements
Bristol Myers Squibb University of BaltimoreBristol Myers SquibbSean KimMark Wittman
University of BaltimoreLinhao LiHongbing Wang
Kurt ZimmermannJoan CarboniBMS IGF1R TeamBMS IGF1R Team
Slide 22Slide 22