Mechanisms of Hepatic Enzyme Induction in Humans and How to

Assess It In Vitro

Edward L. LeCluyse, Ph.D.Chief Scientific Officer, CellzDirect, Inc.

November 2004

Pertinent Questions

If a NME’s induction effect on CYP3A4 in vitro is NEGATIVE, then is it acceptable to NOT recommend any in vivo studies with substrates of CYP3A, CYP2C9, CYP2B6 and CYP2C19.

– YES or NO?

If the in vitro induction (increase in enzyme activity) is more than 40% of the positive control (e.g., rifampin), then there IS a need to recommend an in vivo induction study.

– YES or NO?

Induced PlasmaInduced PlasmaDrugDrug CYPsCYPs Conc (µm)Conc (µm) NRNR Drugs affected Drugs affected in vivoin vivo

CarbamazepineCarbamazepine 3A, 2B 20-40CAR Praziquantel, Itraconazole, Cyclosporin A

SJWSJW 3A, 2C 0.2 AhR, PXR Indinavir, Digoxin, Cyclosporin, Theophylline

PhenytoinPhenytoin 2C, 3A, 2B >10 CAR Warfarin, Praziquantel, Quinidine, Cyclosporin A

PhenobarbitalPhenobarbital 2C, 3A, 2B 40-130 CAR, PXR Warfarin, Cyclosporin A

RifampinRifampin 2C, 3A, 2B 14 PXR Tolbutamide, WarfarinCyclosporin, Oral contraceptives

TroglitazoneTroglitazone 3A, 2B 7 PXR Cyclosporin A, Terfenadine

AvasimibeAvasimibe 2C, 3A, 2B 1-6 PXR Midazolam, Warfarin, Digoxin

Enzyme Induction in Humans

Inducible P450 Enzymes in Human Liver

P450 Enzyme InducersCYP1A2 Aromatic hydrocarbons, cigarette smoke,

cruciferous vegetables, charbroiled meat

CYP2A6 Anticonvulsants, DEX

CYP2B6 phenytoin, PB, RIF

CYP2C8 Anticonvulsants, rifampin

CYP2C9 Rifampin, Anticonvulsants

CYP2C19 Rifampin, Anticonvulsants

CYP3A4 CLZ, Rifampin, Anticonvulsants

CYP2E1 Isoniazid, alcohol

CYP2D6 Non-inducibleCYP4A11 Non-inducible (?)

CYP2B, CYP3A, CYP1A, CYP2A, CYP2Cs, OATP2UGT1A1, MRP2, SULT1A1

CYP1A, UGT1A1, SULT1A1

CAR RXR

XREM / PBREM

AhR ARNT

XRE / DRE

PXR RXR

XREM / PXRE

CYP3A, CYP2B, CYP2Cs,CYP7A, MDR1, MRP2, OATP2, GSTA-2, UGT1A1,AldHs, Carboxyesterase 2, 3

Strong Mod Weak

CLZ CMZ DTBARIF SPZRITPHNPB

Strong Mod Weak

CLZ PB SDMRIF SPZ DEXRIT* CMZ PHN

DTBA

Faucette S et al. Drug Metab Dispos 2004

0

50

100

150

200

250

300

350

400

450

CT

L

CM

Z

CL

Z

DE

X

DT

BA

MT

X

PA

X

PB

PH

N

PR

OB

RIF

RIT

SD

M

SP

Z

TAO

CY

P2

B6

Ac

tiv

ity

(p

mo

l/m

in/m

g p

rote

in)

2 uM (or 50 uM for PB)

10 uM (or 150 uM for PB)

20 uM (or 250 uM PB)

0

2000

4000

6000

8000

10000

12000

14000

CT

L

CM

Z

CL

Z

DE

X

DT

BA

MT

X

PA

X

PB

PH

N

PR

OB

RIF

RIT

SD

M

SP

Z

TAO

CY

P3A

4 A

ctiv

ity

(pm

ol/

min

/mg

pro

tein

)

Co-regulation of CYP2C9 and CYP3A4 by Avasimibe

HH 1 (CYP2C9)

0

20

40

60

80

100

120

0.001 0.01 0.1 1 10 100

Log Avasimibe Concentration

CY

P2C

9 A

ctiv

ity

(% o

f M

axim

um

) PredictedObserved

HH 1 (CYP3A4)

0

20

40

60

80

100

120

0.001 0.01 0.1 1 10 100

Log (Avasimibe Concentration)

CY

P3A

4 A

ctiv

ity

(% o

f M

axim

um

) Predicted

Observed

HH 2 (CYP2C9)

0

20

40

60

80

100

120

0.001 0.01 0.1 1 10 100

Log Avasimibe Concentration

CY

P2C

9 A

ctiv

ity

(% o

f M

axim

um

) PredictedObserved

HH 2 (CYP3A4)

0

20

40

60

80

100

120

0.001 0.01 0.1 1 10 100

Log (Avasimibe Concentration)

CY

P3A

4 A

ctiv

ity

(% o

f M

axim

um

) Predicted

Observed

Induction of CYP2C8 RNA

0

1

2

3

4

5

CMZ 2 CMZ 10 CMZ 50 PHY 50 CLZ 10 RIF 10 PB 1000 CITCO 1

Fo

ld o

ver

con

tro

lInduction of CYP2C9 RNA

0.0

0.5

1.0

1.5

2.0

2.5

3.0

CMZ 2 CMZ 10 CMZ 50 PHY 50 CLZ 10 RIF 10 PB 1000 CITCO1

Fo

ld o

ver

con

tro

l

Induction of CYP2C19 RNA

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

CMZ 2 CMZ 10 CMZ 50 PHY 50 CLZ 10 RIF 10 PB 1000 CITCO1

Fo

ld o

ve

r c

on

tro

l

Mechanism-based Screening Strategy

Goal is to screen for efficacious activators of AhR, PXR, and CAR

Propose screening protocol using sensitive endpoint for each nuclear receptor

Potent activators of individual NR’s will induce a number of target genes, but differentially:– Potent hPXR activators will induce CYP3A4, CYP2B6,

CYP2C8/9/19, UGT1A1, MDR1 etc., but CYP3A4 is the most sensitive.

– Potent hCAR activators will induce CYP2B6, CYP2C8/9/19, UGT1A1 etc., but CYP2B6 is the most sensitive.

– Potent AhR agonists will induce CYP1A2, UGT1A1, GST’s etc., but CYP1A2 is the most sensitive.

In Vitro Protocol for Enzyme Induction

Cultured HepatocytesCultured Hepatocytes

Treat with NME at 3-4 Treat with NME at 3-4 conc. for 1-3 daysconc. for 1-3 days

Include Positive Include Positive Controls:Controls:3-MC/BNF3-MC/BNF

Phenobarbital/PhenytoinPhenobarbital/PhenytoinRifampinRifampin

mRNA ContentRT/PCR (TaqMan)

Protein ContentWestern Immunoblotting

Enzyme ActivityLC-MS/MS assayHPLC assayRadiometric assay

Major CYP target gene for each nuclear receptor:Major CYP target gene for each nuclear receptor:CYP1A2 CYP1A2 ((AhRAhR)), 2B6 , 2B6 (CAR(CAR)),, 3A4 3A4 ((PXRPXR))

-4000

-2000

0

2000

4000

6000

8000

Probe

necid

Rifam

pin

Ritona

vir

Sulfin

pyra

zone

Carba

mez

apin

e

Clotri

maz

ole

Dexam

etha

sone

Pheny

toin

Treatment Group

No

rmal

ized

sp

ecif

ic a

ctiv

ity

(pm

ol/m

in/m

g p

rote

in)

2 uM

10 uM

20 uM

CYP3A4 Induction in Human Hepatocytes

0

5

10

15

20

25

Probenecid Rifampin Ritonavir Sulfinpyrazone

Treatment Group

No

rmal

ized

fo

ld in

du

ctio

no

ver

con

tro

l

2 uM

10 uM

Effects of Inducers on CYP3A4mRNA Expression

Important Factors to Consider

Inter-donor differences in control/basal activity

Relevant concentration range (plasma vs. tissue)

Appropriate choice and concentration of positive controls

Major species differences exist (e.g., RIF vs. PCN)

Expression of data and relevant endpoints

Exposure time important (# days)

Solvent effects on CYP450 expression and activity

Summary of Key Points

Our mechanistic understanding of enzyme induction in human liver has increased markedly in the past decade.

Most inducible human P450’s, UGT’s and transporters involved in DDI’s are regulated by a few receptors (i.e., PXR, AhR and CAR).

Screening for potential inducers during drug development can be achieved using a single selective and sensitive target gene for each NR.

Activity data from in vitro induction studies for a NME should be:

– normalized to a negative control – compared to an ‘appropriate’ positive control– considered significant when they are 40% of the positive

control complemented with protein or mRNA data