CYP2C8, 2C9 and 2C19 Induction by Model P450 Inducers in Human Hepatocytes: A Correlative Study with CYP1A2,

2B6 and 3A4 Induction in Hepatocytes from Multiple Donors

Albert P. Li, Ph. D. In Vitro ADMET Laboratories Inc.

Columbia, MD and Malden, MA

lialbert@invitroadmet.com

U. S. FDA requires CYP3A4 inducers to be evaluated for CYP2C induction

If CYP3A induction results are positive, then induction of CYP2C should be studied either in vitro or in vivo.

http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm292362.pdf

CYP2C Induction in Human Hepatocytes

• Role of CYP in drug toxicity and DDI

• Current understanding of the mechanism of CYP2C induction

• Human hepatocyte-derived individual differences in P450 induction

• Correlation of CYP2C with CYP1A, 2B, and CYP3A induction with human hepatocytes from multiple donors

• Conclusion and future directions

CYP2C9 and Drug Toxicity

• Sulfonylureas – Association between CYP2C9 slow metabolizer genotypes and

severe hypoglycaemia on medication with sulphonylurea hypoglycaemic agents (Holstein A , Plaschke A , Ptak M et al. Br J Clin Pharmacol 2005 ; 60 : 103-106)

– CYP2C9*2 Allele Increases Risk for Hypoglycemia in POR*1/*1 Type 2 Diabetic Patients Treated with Sulfonylureas (Ragia G et al. Exp Clin Endocrinol Diabetes 2014; 122: 60–63)

• Bosentan – Association of CYP2C9*2 With Bosentan-Induced Liver Injury

(Markova et al, Clinical Pharmacology & Therapeutics (2013); 94 6, 678–686. doi:10.1038/clpt.2013.143)

CYP2C and Drug-Drug Interactions

• Human CYP2C8, CYP2C9, CYP2C18, and CYP2C19 represent approx. 20% of hepatic P450 and metabolize more than 20% of all therapeutic drugs as well as a number of endogenous compounds.

Review: Chen and Golstein, Current Drug Metab (2016)

Clinically significant CYP2C9 DDI

• Victims: – Sulfonylureas (glibenclamide, glimepiride, and

glipizide) – Nonsteroidal antiinflammatory drugs (NSAIDs):

ibuprofen, diclofenac, naproxen, piroxicam, and tenoxicam,

– Hypoglycemic Agents: tolbutamide, glipizide – Angiotensin II Blockers: losartan, irbesartan

• Perpetrators: – Inducers: rifampin, phenobarbital, hyperforin – Inhibitors: fluconazole, amiodarone

Mechanism of CYP2C Induction

• Xenobiotic-mediated induction: Nuclear receptors CAR, PXR, VDR, and GR

• Constitutive expression: HNF4α, HNF3γ, C/EBPα and RORs

• Maximal transcriptional induction of CYP2C genes: coordinative cross-talk between drug responsive nuclear receptors, hepatic factors, and coactivators.

Nuclear Receptors and CYP2C9 Induction

• Ferguson, Stephen S., et al. "Human CYP2C8 is transcriptionally regulated by the nuclear receptors constitutive androstane receptor, pregnane X receptor, glucocorticoid receptor, and hepatic nuclear factor 4α." Molecular pharmacology 68.3 (2005): 747-757.

• Chen, Yuping, et al. "The nuclear receptors constitutive androstane receptor and pregnane X receptor cross-talk with hepatic nuclear factor 4α to synergistically activate the human CYP2C9 promoter." Journal of Pharmacology and Experimental Therapeutics 314.3 (2005): 1125-1133.

• Chen, Yuping, Stephen S. Ferguson, Masahiko Negishi, and Joyce A. Goldstein. "Identification of constitutive androstane receptor and glucocorticoid receptor binding sites in the CYP2C19 promoter." Molecular pharmacology 64, no. 2 (2003): 316-324.

CYP2C9 Induction

• Regulation of Human CYP2C9 Expression by Electrophilic Stress Involves Activator Protein 1 Activation and DNA Looping (Makia, Goldstein et al; Molecular Pharmacology August 2014 vol. 86 no. 2 125-137)

– oxidative stress generated by exposure to electrophilic xenobiotics and metabolites induces the expression of CYP2C9 and CYP2C19 in human hepatocytes

Protein-Protein Interaction

• Altered CYP2C9 Activity Following Modulation of CYP3A4 Levels in Human Hepatocytes (Ramsden, Tweedy et al, DMD November 2014 vol. 42 no. 11 1940-1946)

– siRNA silencing and recovery evaluation in long-term cultured human hepatocyes show inverse correlation of CYP2C9 with CYP3A4 activity

CYP2C8, 2C9 and 2C19 Induction by Model P450 Inducers in Human Hepatocytes: A Correlative

Study with CYP1A2, 2B6 and 3A4 Induction

Cryopreserved Plateable Human Hepatocytes: Gold Standard for

P450 Induction Studies

IVAL Plateable Cryopreserved Hepatocytes

Human Cynomolgus Monkey Beagle Dog

CD-1 Mouse SD Rat Wistar Rat

OnDemand™ Human Hepatocytes

14

Objectives

• Overall evaluation of inducibility of CYP2C8, CYP2C9 and CYP2C19 in human hepatocytes from 9 donors – Inducers: Omeprazole (AHR); phenobarbital (CAR);

Rifampin (PXR)

• Identify donors with highest sensitivity to CYP2C induction

• Correlate CYP2C induction with CYP1A, CYP2B and CYP3A induction to provide insight towards relative roles of AHR, CAR, and PXR in CYP2C induction

Procedures

• Plateable human hepatocytes from 9 donors • 96-well matrigel-collagen sandwich culture • Day 1: Thaw, plate and matrigel overlay • Day 2: Initiation of treatment with omeprazole,

phenobarbital and rifampin • Days 3, 4: repeat treatment (72 h treatment) • Day 5: RNA extraction • RT-PCR quantification of CYP1A2, 2B6, 2C8, 2C9,

2C19 and 3A4 gene expression • Delta-delta Ct calculation of fold induction

Donor Demorgraphics Lot Ethnicity Sex Age BMI

Plateable/ Suspension

HH1032 C F 68 26.17 Plateable HH1051 C M 23 25.30 Plateable HH1052 C M 44 24.60 Plateable HH1053 C F 33 21.10 Plateable HH1083 C F 45 36.30 Plateable HH1089 C F 57 28.40 Plateable HH1085 H F 77 30.3 Plateable HH1103 C/H F 44 29.39 Plateable

Results

CYP1A2, 2B6, and 3A4 Induction in Human Hepatocytes (9 Donors)

CYP1A2 CYP2B6 CYP3A4

OMP PB RIF OMP PB RIF OMP PB RIF

Donor mean sd mean sd mean sd mean sd mean sd mean sd mean sd mean sd mean sd

HH1032 36.81 5.78 2.25 0.18 0.85 0.22 1.85 0.33 5.76 0.60 4.02 0.70 4.45 0.67 19.41 2.85 13.89 1.15

HH1051 17.59 2.85 1.33 0.28 0.21 0.03 3.11 0.34 10.98 1.90 9.65 1.81 3.76 0.38 13.52 2.37 12.84 1.93

HH1052 31.89 0.86 2.20 0.25 0.68 0.06 5.99 0.81 9.59 0.66 7.53 0.60 11.36 2.13 38.00 5.54 44.13 7.03

HH1053 23.37 3.15 28.54 3.91 1.90 0.32 3.81 1.13 1.31 0.55 10.76 0.61 4.34 0.55 0.58 0.17 19.66 2.74

HH1072 88.93 12.01 4.36 0.52 1.24 0.29 4.46 0.83 13.45 0.97 10.07 2.06 4.53 0.84 22.40 6.27 18.47 5.15

HH1083 66.06 6.10 3.88 1.12 0.88 0.11 4.11 0.48 6.74 0.88 6.03 0.47 3.58 0.34 10.20 0.58 14.51 1.06

HH1085 43.32 4.18 1.83 0.19 0.80 0.08 4.93 0.84 8.23 0.77 11.41 2.34 5.07 0.86 13.63 0.96 27.56 5.78

HH1089 64.14 10.66 1.15 0.30 0.56 0.12 5.10 1.62 3.56 1.24 4.76 0.33 4.47 1.52 7.50 2.32 14.08 1.45

HH1103 59.29 4.42 2.87 0.19 1.14 0.11 5.20 0.24 11.14 2.80 8.90 1.77 2.36 0.38 25.20 4.63 18.98 6.09

OMP: Omeprazole (50 uM); PB: Phenobarbital (1000 uM); RIF: Rifampin (20 uM)

CYP2C8, CYP2C9 and CYP2C19 Induction in Human Hepatocytes (9 Donors) CYP2C8 CYP2C9 CYP2C19

OMP PB RIF OMP PB RIF OMP PB RIF

Donor mean sd mean sd mean sd mean sd mean sd mean sd mean sd mean sd mean sd

HH1032 0.92 0.14 2.04 0.29 1.58 0.14 0.90 0.10 2.10 0.32 1.40 0.24 0.65 0.05 0.89 0.05 0.72 0.11

HH1051 2.24 0.34 6.13 0.59 1.84 1.18 2.16 0.50 4.94 1.44 2.13 1.40 1.22 0.33 3.48 1.13 1.53 1.08

HH1052 0.92 0.08 2.89 0.33 1.67 0.18 1.20 0.11 2.41 0.31 2.22 0.25 1.26 0.08 1.77 0.07 1.26 0.15

HH1053 2.31 0.56 1.88 0.77 8.98 1.21 1.25 0.26 1.63 0.73 5.60 0.97 2.31 0.38 2.04 0.59 4.33 0.76

HH1072 1.72 0.60 4.63 0.79 1.95 0.91 1.88 0.48 4.63 0.89 2.07 0.91 0.93 0.23 2.20 0.50 1.13 0.41

HH1083 1.20 0.39 3.49 0.35 1.96 0.54 1.14 0.08 3.18 0.20 2.01 0.26 0.63 0.05 1.08 0.09 0.67 0.10

HH1085 0.69 0.12 3.53 0.30 5.49 1.90 1.21 0.12 2.58 0.41 2.99 0.41 1.12 0.10 1.72 0.16 1.96 0.09

HH1089 1.88 0.48 4.63 0.89 2.07 0.91 1.06 0.35 1.23 0.28 1.83 0.34 1.30 0.48 1.31 0.20 1.36 0.45

HH1103 0.82 0.12 4.85 1.24 3.02 0.79 0.99 0.21 2.27 0.26 1.79 0.32 0.39 0.09 0.84 0.10 0.55 0.11

OMP: Omeprazole (50 uM); PB: Phenobarbital (1000 uM); RIF: Rifampin (20 uM)

Individual Difference

Individual Difference in P450 Induction: CYP1A2, CYP2B6, CYP3A4

RIF (20 uM); PB (1000 uM); OMP (50 uM)

-20.00

0.00

20.00

40.00

60.00

80.00

100.00

120.00

Fold

Ind

uct

ion

Donors

CYP1A2

0.00

2.00

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6.00

8.00

10.00

12.00

14.00

16.00

Fold

Ind

uct

ion

Donors

CYP2B6

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

HH1032 HH1051 HH1052 HH1053 HH1072 HH1083 HH1085 HH1089 HH1103 Average

Fold

Ind

uct

ion

Donors

CYP3A4

Rifampin

Phenobarbital

Omeprazole

Individual Difference in P450 Induction: CYP2C8, CYP2C9, CYP2C19

RIF (20 uM); PB (1000 uM); OMP (50 uM)

0.00

2.00

4.00

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Fold

Ind

uct

ion

Donors

CYP2C8

-1.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

HH1032 HH1051 HH1052 HH1053 HH1072 HH1083 HH1085 HH1089 HH1103 Average

Fold

Ind

uct

ion

Donors

CYP2C9

Rifampin

Phenobarbital

Omeprazole

0.00

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6.00

Fold

Ind

uct

ion

Donors

CYP2C19

HH1053: Dose-Response HH1053

Inducer CYP1A2 CYP2B6 CYP3A4 CYP2C8 CYP2C9 CYP2C19

Omeprazole (μM) Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

0.08 1.42 0.23 1.36 0.26 1.2 0.15 1.53 0.28 1.38 0.17 1.21 0.09

0.4 2.03 0.26 1.23 0.06 1.38 0.06 1.54 0.26 1.44 0.2 1.2 0.23

2 5.29 0.7 1.51 0.32 1.86 0.27 1.9 0.47 1.53 0.3 1.29 0.27

10 14.69 2.59 3.91 0.58 5.45 0.32 3.12 0.43 1.55 0.08 1.87 0.21

25 21.14 0.3 3.84 0.16 5.31 0.35 2.7 0.3 1.53 0.12 1.9 0.12

50 23.37 3.15 3.81 1.13 4.34 0.55 2.31 0.56 1.25 0.26 2.31 0.38

100 27.32 1.77 7.36 1.01 6.23 0.57 1.74 0.54 1.4 0.05 2.8 0.27

Phenobarbital (μM)

4 1.16 0.28 1.48 0.13 1.25 0.13 1.37 0.27 1.4 0.19 1.19 0.06 20 1.93 1.31 2.81 0.76 1.73 0.06 1.55 0.44 1.32 0.26 1.03 0.12 50 1.6 0.23 5.83 1.14 2.91 0.72 2.96 0.69 2.02 0.38 1.49 0.22

100 1.5 0.19 7.7 1.13 5.79 0.76 3.09 0.43 2.09 0.29 1.49 0.04 250 1.67 0.23 12.1 1.37 10.19 3.13 4.99 1.05 3.4 0.92 2.8 0.84 500 1.81 0.05 10.62 2.23 10.33 1 6.31 0.49 3.36 0.28 3.08 0.21

1000 28.54 3.91 1.31 0.55 0.58 0.17 1.88 0.77 1.63 0.73 2.04 0.59 Rifampin (μM)

0.016 1.59 0.09 1.35 0.21 2.13 0.22 1.03 0.34 1.23 0.21 1.08 0.12 0.08 1.72 0.4 1.65 0.39 4.18 1.29 1.55 0.78 1.46 0.46 1.19 0.29 0.4 1.33 0.43 3.8 2.53 4.04 4.1 3.17 2.48 2.29 1.39 1.6 0.63 2 1.48 0.25 6.17 1.92 10.74 3.22 2.64 2.03 2.27 0.92 1.32 0.88

10 1.71 0.14 9.92 1.9 16.6 1.13 6.42 0.8 4.46 0.14 3.04 0.39 20 1.9 0.32 10.76 0.61 19.66 2.74 8.98 1.21 5.6 0.97 4.33 0.76 40 1.58 0.12 8.02 5.42 19.24 3.48 11.14 2.08 6.65 1.24 5.5 0.86

HH1051: Dose Response HH1053

Inducer CYP1A2 CYP2B6 CYP3A4 CYP2C8 CYP2C9 CYP2C19

Omeprazole (μM) Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

0.08 0.98 0.07 0.85 0.06 1.07 0.19 1.95 0.96 1.72 0.69 1.94 0.94

0.4 1.18 0.24 0.83 0.2 0.88 0.26 1.25 0.2 1 0.27 1.03 0.36

2 1.99 0.37 1.19 0.16 1.08 0.38 1.49 0.59 1.04 0.35 0.86 0.38

10 4.6 0.73 2.41 0.47 2.66 0.34 1.88 0.05 1.79 0.42 1.23 0.17

25 11.5 1.15 3.28 0.58 4.11 0.73 2.48 0.27 2.09 0.35 1.76 0.46

50 17.59 2.85 3.11 0.34 3.76 0.38 2.24 0.34 2.16 0.5 1.22 0.33

100 22.79 3.68 2.25 0.54 3.55 1.01 2.86 0.8 2.24 0.73 1.23 0.34

Phenobarbital (μM)

4 0.92 0.06 0.79 0.1 0.76 0.14 1.28 0.43 1.42 0.41 1.5 0.35 20 0.89 0.12 0.86 0.05 0.85 0.13 1.65 0.21 1.62 0.44 1.75 0.78 50 0.71 0.05 1.07 0.08 0.84 0.2 1.13 0.21 1.23 0.47 0.96 0.33

100 0.8 0.1 1.57 0.23 1.89 0.49 1.93 0.29 1.9 0.47 1.42 0.42 250 0.91 0.1 3.67 0.53 4.84 0.82 3.53 0.92 3.01 0.77 2.01 0.25 500 1.07 0.14 8.08 0.34 10.56 1.53 4.15 0.86 2.89 0.7 1.86 0.1

1000 1.33 0.28 10.98 1.9 13.52 2.37 6.13 0.59 4.94 1.44 3.48 1.13

Rifampin (μM)

0.016 0.82 0.11 1.02 0.34 1.08 0.36 1.11 0.2 1.19 0.18 1.37 0.28 0.08 0.67 0.12 1.34 0.09 1.62 0.13 1.13 0.79 1.3 1.04 1.39 1.06 0.4 0.44 0.06 3.81 0.61 3.67 0.83 1.09 0.48 0.96 0.31 0.81 0.24 2 0.3 0.08 8.21 1.03 6.8 1.2 1.82 0.85 1.57 0.54 1.04 0.47

10 0.25 0.04 10.74 1.64 11.06 0.81 1.83 1.01 2.38 1.37 1.27 0.68 20 0.21 0.03 9.65 1.81 12.84 1.93 1.84 1.18 2.13 1.4 1.53 1.08 40 0.17 0.02 11.57 2.16 21.51 2.7 2.55 1.78 3.13 1.55 2.11 1.04

Correlation Analysis

• Pool results with 9 donors with the three model inducers: omeprazole (50 uM), phenobarbital (1000 uM) and rifampin (50 uM)

• Plot fold induction of one CYP vs another to identify isoforms with best correlations, thereby revealing similar mechanisms of induction

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Fold CYP2B Induction

CYP3A4 vs CYP2B6

Phenobarbital

Rifampin

Omeprazole

CYP3A4 (PXR) vs CYP2B6 (CAR) Fold Induction

Correlation of CYP1A2, CYP2B6 and CYP3A4 Induction

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n

Fold CYP3A4 Induction

CYP1A2 vs CYP3A4

y = 0.3585x + 2.3629 R² = 0.6827

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CYP2B6 vs CYP3A4

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Fold CYP2B6 Induction

CYP1A2 vs CYP2B6

Correlation of CYP2C and CYP3A4 Induction

y = 0.1505x + 1.0477 R² = 0.3369

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CY

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C8

In

du

ctio

n

Fold CYP3A4 Induction

CYP2C8 vs CYP3A4

y = 0.0903x + 1.0953 R² = 0.3071

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du

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Fold CYP3A4 Induction

CYP2C9 vs CYP3A4

y = 0.0268x + 1.1071 R² = 0.0498

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Fold CYP3A4 Induction

CYP2C19 vs CYP3A4

Correlation of CYP2C and CYP2B6 Induction

y = 0.3889x + 0.26 R² = 0.4236

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In

du

ctio

n

Fold CYP2B6 Induction

CYP2C8 vs CYP2B6

y = 0.2744x + 0.3513 R² = 0.5337

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C9

In

du

ctio

n

Fold CYP2B6 Induction

CYP2C9 vs CYP2B6

y = 0.1175x + 0.6471 R² = 0.1809

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nd

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ion

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CYP2C19 vs CYP2B6

Correlation of CYP2C8, CYP2C9 and CYP2C19 Induction

y = 1.345x - 0.0927 R² = 0.7164

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CY

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C8

In

du

ctio

n

Fold CYP2C9 Induction

CYP2C8 vs CYP2C9

y = 0.3555x + 0.4302 R² = 0.5875

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CYP2C19 vs CYP2C8

y = 0.5755x + 0.1856 R² = 0.6096

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CYP2C19 vs CYP2C9

Summary and Conclusions

Summary

• CYP2C induction was evaluated in human hepatocytes from 9 donors and compared to CYP1A2, CYP2B6 and CYP3A4 – Isoform dependent fold induction:

CYP1A2>CYP3A4>CYP2B6>CYP2C8=CYP2C9>CYP2C19

• Individual variations was observed – While all lots exhibited robust response to CYP1A2, CYP2B6, and

CYP3A4 induction, only 2 of the 9 lots were also inducible (>2 fold induction) for CYP2C8, CYP2C9, and CYP2C19

• Phenobarbital was the best positive control for CYP2C induction

• Correlation demonstrated that CYP2C induction correlates best among CYP2C isoforms followed by CYP3A4 and CYP2B6, with no correlation with CYP1A2 induction

Conclusion

• Careful selection of human hepatocyte lots is important for the evaluation of CYP2C induction

• Mechanism of CYP2C9 induction is different from CYP1A induction, confirming that AHR is not involved

• Correlation of CYP2C induction with CYP2B6 and CYP3A4 induction is consistent with the roles of CAR and PXR

• Best correlations are among CYP2C8, CYP2C9 and CYP2C19, suggesting that similar mechanism for the three isoforms which is different from CYP2B6 and CYP3A4 induction

U. S. FDA requires CYP3A4 inducers to be evaluated for CYP2C induction

If CYP3A induction results are positive, then induction of CYP2C should be studied either in vitro or in vivo.

http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm292362.pdf

• Careful selection of human hepatocyte lots is important for the evaluation of CYP2C induction