1. Chief Scientific Officer, CellzDirect, Inc.
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

2. 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.

3. Enzyme Induction in Humans
Induced Plasma
Drug CYPs Conc (µm) NR Drugs affected in vivo
Carbamazepine 3A, 2B CAR Praziquantel, Itraconazole,
Cyclosporin A
SJW 3A, 2C AhR, PXR Indinavir, Digoxin,
Cyclosporin, Theophylline
Phenytoin 2C, 3A, 2B >10 CAR Warfarin, Praziquantel, Quinidine, Cyclosporin A
Phenobarbital 2C, 3A, 2B CAR, PXR Warfarin, Cyclosporin A
Rifampin 2C, 3A, 2B PXR Tolbutamide, Warfarin
Cyclosporin, Oral contraceptives
Troglitazone 3A, 2B PXR Cyclosporin A, Terfenadine
Avasimibe 2C, 3A, 2B PXR Midazolam, Warfarin, Digoxin

4. Inducible P450 Enzymes in Human Liver
P450 Enzyme Inducers
CYP1A2 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-inducible
CYP4A11 Non-inducible (?)

5. CAR
RXR
XREM / PBREM
AhR
ARNT
XRE / DRE
PXR
XREM / PXRE
CYP1A, UGT1A1, SULT1A1
CYP2B, CYP3A, CYP1A,
CYP2A, CYP2Cs, OATP2
UGT1A1, MRP2, SULT1A1
CYP3A, CYP2B, CYP2Cs,
CYP7A, MDR1, MRP2,
OATP2, GSTA-2, UGT1A1,
AldHs, Carboxyesterase 2, 3

6. Faucette S et al. Drug Metab Dispos 2004
Strong Mod Weak
CLZ CMZ DTBA
RIF SPZ
RIT
PHN PB
Strong Mod Weak
CLZ PB SDM
RIF SPZ DEX
RIT* CMZ PHN
DTBA
Faucette S et al. Drug Metab Dispos 2004

7. Co-regulation of CYP2C9 and CYP3A4 by Avasimibe

9. 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.

10. Treat with NME at 3-4 conc. for 1-3 days
In Vitro Protocol for Enzyme Induction
Treat with NME at 3-4 conc. for 1-3 days
mRNA Content
RT/PCR (TaqMan)
Include Positive Controls:
3-MC/BNF
Phenobarbital/Phenytoin
Rifampin
Cultured Hepatocytes
Enzyme Activity
LC-MS/MS assay
HPLC assay
Radiometric assay
Protein Content
Western Immunoblotting
Major CYP target gene for each nuclear receptor:
CYP1A2 (AhR), 2B6 (CAR), 3A4 (PXR)

11. CYP3A4 Induction in Human Hepatocytes

12. Effects of Inducers on CYP3A4
mRNA Expression

13. 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

14. 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