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Introduction

Why study the Cytochrome P4503A

(CYP3A) Family?

Dr. Erin Schuetz

The screen versions of these slides have full details of copyright and acknowledgements 1

Introduction

Why study the

Cytochrome P4503A

(CYP3A) Family?

1

CYP3A is the major CYP family

in Human Liver(Shimada et al, JPET 270:414-423, 1994)

U�K�OW�

CYP2E1

7%

CYP3A

29%

CYP2C

(2C8, 9, & 19)

18%

CYP2D6

1.5%

CYP1A2

13%

CYP2A6

4%

CYP2B6

0.2%

27%

2

Proportion of Drugs Metabolized by Individual Cytochrome P450’s

CYP3A4/5

CYP2C9/10CYP1A2

CYP2A1

CYP2C1

CYP2E1

CYP2D6

CYP3A is involved in the oxidative metabolismOf ~50% of all drugs

3

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


CYP3A5

CYP3AP1

CYP3A7CYP3AP2CYP3A4

CYP3A43

Adapted from: Finta & Zaphiropoul os; Gene 260:13- 23, 2000

Human CYP3AGene Locus on Chromosome 7q21

• CYP3A4 > CYP3A5 > CYP3A7 are the

most important for drug metabolism in the adult

4

CYP3A4

• First isolated as HL5, then HLp and P450NF

• Two original cDNA sequences, 3A3 and 3A4, with only 14 nucleotide differences

• PCR studies with specific primers suggest 3A3 is not expressed

• Publications with HL5, HLp, P450NF, 3A3 and 3A4 are examining the same CYP

5

Heterogeneous Hepatic CYP3A Distribution

• CYP3A distribution is predominantly pericentral.

• inter-individual differences reflect, in part, a

variable number of CYP3A (+) hepatocytes.

pericentral →periportal

Central Venous

Portal Venous

•Major CYP in human liver,

•about 30 % on average and up to 60% in an induced liver

•40-fold range of expression6

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


CYP3A Activity

• Broad substrate selectivity, MW of

substrates ranges from acetaminophen (151) to cyclosporin (1201)

7

CYP3A in Drug MetabolismDrug Substrates

Calcium channel blockers: diltiazem, felodipine, nemodipine, nifedipine, nisoldipine, nitrendipine, verapamil

Immunosuppresive agents: cyclosporine A, tacrolimus

Steroids: budesonide, cortisol, 17ß-estradiol, progesterone, testostrone

Macrolide antibiotics: clarithromycin, erythromycin, troleandomycin

Chemotherapeutic agents: cyclophosphamide, tamoxifen

Nonsedating antihistamines: astemizole, loratadine, terfenadine

Sedative hypnotics: alprazolam, midazolam, triazolam

Opioids: alfentanyl, fentanyl, sufentanil

methadone

Lipid Lowering agents: Lovastatin, simvastatin, atorvastatin, cerivastatin

HIV protease inhibitors: indinavir, nelfinavir, ritonavir, saquinavir

Others: cisapride, quinidine

8

CYP3A4• Variations in the expression of CYP3A

Influences the metabolic clearance of numerous exogenous and endogenous compounds

• Major drug-drug interactions result from CYP3A4

inhibition,

induction, andactivation (cooperativity)

9

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


CYP3A Inhibition

•Clarithromycin*, erythromycin*

(not azithromycin)

•Diltiazem* (verapamil and nifedipine less likely to inhibit)

•Itraconazole*, ketoconazole*, fluconazole

•Ritonavir*, indinavir

•Amiodarone

•grapefruit juice

•Fluoxetine,

* potent10

Screening for CYP3A Inhibition

using minimal best practice

studies•In Vitro Test Systems

•Human liver microsomesRecombinant cDNA expressed CYP enzymes

•CYP3A4 substrates to use:

strongly recommend that threeStructurally unrelated substrates be used(midazolam; erythromycin; nifedipine)

TD Bjornsson et al., Drug Metab Dispo 31:815-32,; 200311

Wrighton et al Developed a noveltestosterone 6 beta-hydroxylase activity assay to

study CYP3A-mediated metabolism and inhibition in vitro

Fayer JL et al., J Pharmacol Toxicol Methods 46:117-23, 2001

•Assay is superior to existing methods

Ease of sample preparationshort run times

low detection limits

12

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


No inhibitor control

Ketoconazole 100nM

Ketoconazole 20nM

Nifedipine 50µM

Nifedipine 20µM

Naringenin 100µM

Naringenin 20µM

Optimization a robust testosterone assay methodology for

high throughput Ki determination

Mic

ros

om

es

10

µM

tes

tos

tero

ne

Mic

ros

om

es

25

µM

tes

tos

tero

ne

Mic

ros

om

es

50

µM

tes

tos

tero

ne

Mic

ros

om

es

10

0µ

M te

sto

ste

ron

e

Mic

ros

om

es

17

5µ

M te

sto

ste

ron

e

Mic

ros

om

es

25

0µ

M te

sto

ste

ron

e

0.2mg/ml microsomes100µl volume10 minute incubation

LC-MS analysis - 6β-OHtestosterone

Fayer JL et al., J Pharmacol Toxicol Methods 46:117-23, 2001 13

3 day reproducibility of Kinetic parameters and Ki v alues

Ketoconazole Ki

1 2 50

5

10

15

20

25

day of experiment

keto

con

azo

le K

i (n

M)

Naringe nin K i

1 2 50

5

10

15

20

25

day of experiment

na

ring

en

in K

i (µ

M)

N if edipine Ki

1 2 50

5

10

15

20

25

day of experiment

nif

edip

ine

Ki

(µM

)

T estosterone K m

1 2 50

10

20

30

40

50

60

day of experiment

test

ost

ero

ne

Km

(µ

M)

T estostero ne Vm ax

1 2 50 .0 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

day of experiment

tes

tost

ero

ne

ac

tivit

y

Testosterone Vmax = 0.65 ± 0.03 nmol/min/mg (sd) 5.2 % (%sd)

Testosterone Km = 36.7 ± 3.0 µM (sd) 8.1 %

Ki ketoconazole = 14.7 ± 1.5 nM (sd) 10 %

Ki nifedipine = 16.8 ± 1.3 µM (sd) 7.4 %

Ki naringenin = 15.8 ± 2.0 µM (sd) 12 %

Error bars indicate standard error of the estimate 14

CYP3A Catalytic Activity

can also be activated

• Activation is an increase in the activity of the CYP

without increasing CYP levels

• Metabolism may be stimulated (activated) by the

substrate (homotropic) or by another agent

(heterotropic)

• The substrate itself or an additional agent may

activate a CYP (most often with CYP3A)

15

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


CYP3A Activation

• Example - alpha Nathoflavone activates CYP3A activity

• The Activation mechanism not understood, potential explanations include:

• Two drug molecules in active site simultaneously

• CYP3A4 has an allosteric binding site

• Multiple conformers of CYP3A4; identification of substrate groupings

16

17

Intestinal CYP3A and First-Pass

Drug Metabolism

CYP3A has a major rolein limiting the oral bioavailability

of many drugs

18

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Intestinal CYP3A4

Major CYP in epithelialbarrier of small intestine,

70% of CYPS present

19

Crypts

Intima

EnterocytesLumen

Serosal

CYP3A4 (+) expression is confined to the

“absorptive” enterocytes of the

mucosal villi.

Greater expression

proximal vs distal intestine

Courtesy of P.B. Watkins, MD20

21

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Heterogeneous Distribution of CYP3A4 Protein

in Small Intestine

• Although there is significant inter-individual variability, CYP3A4 protein declines by 50%, on average D→I.

0.0

5.0

10.0

15.0

20.0

25.0

30.0

0 4 8 12 16 20

Section Small Intestine (~30 cm each)

CYP3A4Villin

HI-32

Paine et al., 1997

22

23

Intestinal CYP3A First-Pass

Metabolism and Drug-Drug Interactions

24

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Oral Bioavailability of Select CYP3A Substrates

Drug Indication F (%)

Buspirone anxiety 4 ± 4

Terfenadine antihistamine < 2

Lovastatin hypercholesterolemia < 5

Saquinavir HIV 4 - 14

Felodipine hypertension 15 ± 8

Verapamil angina, arrhythmia 22 ± 8

Tacrolimus immunosuppression 25 ± 10

Erythromycin bacterial infections 35 ± 2

Diltiazem angina, hypertension 44 ± 10

Midazolam sedation 44 ± 17

Oral absorption

25

CYP3A F (%) KTZ Dose (mg) AUCi/AUC Author/Year

Terfenadine < 2 200, bid 16-76 Honig, 1993

Nisoldipine ~ 5 200, qd 25.3 Honig, 1999

Midazolam 44 ± 17 400, qd 15.9 Olkkola, 1994

Triazolam ~ 40 200, bid 13.7 Greenblatt, 1998

Alprazolam 88 ± 16 200, bid 1.8 Schmider, 1999

200, bid 4.0 Greenblatt, 1998

Quinine 76 ± 11 100, bid 1.4 Mirghani, 1999

Zolpidem 72 ± 7 200, bid 1.7 Greenblatt, 1998

Ketoconazole inhibits intestinal CYP3A increasing

Bioavailability of co-administered CYP3A substrates

26

First Pass Drug Interactions with Midazolam: A Standardized CYP3A Probe

MDZ Route % AUC Increase

ketoconazol e (400 mg, 4d) p.o. 1490

itraconazole (200 mg, 4d) p.o. 980

saquinavir (3600 mg, 5d) p.o. 418

erythromycin (1.5 g, 7d) p.o. 340

clarithromycin (1g, 6d) p.o. 330

diltiazem (180 mg, 2d) p.o. 275

verapamil (240 mg, 2d) p.o. 240

fluconazol e (400 mg, 1h) p.o. 123

roxithromycin (300 mg, 6d) p.o. 47

cimetidine (800 mg, 1d) p.o. 35

Data compiled from recent literature (1993-98)

27

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Summary

• Many drugs are metabolized by hepatic CYP3A4

• Intestinal CYP3A4 contributes to the incomplete oral bioavailabilityof many drugs (e.g., midazolam, verapamil, felodipine, lovastatin)

• Hepatic and intestinal CYP3A4 expression is highly variable between individuals ; there is also a heterogenous distribution of CYP3A4 along the length of the gastrointesti nal tract (highest in

proximal jejunum)

• Drugs with significant intestinal and hepatic CYP3A4 metabolic extraction are highly suscepti bl e to pronounced changes in AUC

with potent inhibitors and inducers of the enzyme

28

Genetic Contributions to

Variable Intestinal CYP3A-

Dependent Drug Metabolism

29

Ozdemir Paradox

•Determined there is a significant genetic Component to CYP3A expression.

•However, none of the CYP3A4 sequence

variations are frequent enough toexplain human variation in CYP3A4

expression.

30

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


CYP3A4 Gene Variants

• Flanking Region: multiple SNPs (CYP3A4*1B)

� High allele frequency AA (35-67%) vs C (2-10%)

� SNP in putative enhancer region (NFSE)

� Conflicting in vitro and in vivo data - causality questioned

• Coding SNPs: 17 unique amino acid changes to date (*2 -*19)

� Low allele frequency C, possibly higher in other racial groups

� Supporting in vitro data for some variants:

� *17, F189S, decreased Vmax (5-25% of control); allele, 2% (C)

� *18, L293P, increased Vmax (170% of control); allele, 2% (A)

Lamba et al., Adv Drug Deliv Rev 54:1271, 2002http://www.imm.ki.se/CYPalleles/cyp3a4.htm

31

CYP3A5• cDNA sequence 88% similar to CYP3A4

• Polymorphically expressed in liver

• Contributes significantly (>50%) to the total CYP3A content in 33% of Caucasians and 50% of African-Americans (Nature Genetics 27:282-291,2001)

• Detected through out GI, highest in stomach and large intestine

• Expressed in kidney

• Catalytic activity relatively poorly understood

32

Polymorphic Hepatic CYP3A5

3A5 standards (pmol)

Anti-CYP3A5

A B C D 1 0.5 0.25 0.125 E F G H

Analysis of microsomes from different human livers (A-H) indicates marked inter-individual

variability in specific enzyme content

Paine et al., 1997

33

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


MF Paine et al, Drug Metab Dispos 33:426-33, 2005

CYP3A5 is polymorphically expressed in

Human Intestine (duodenum)

34

Characterizing CYP3A5mRNA Splice Variants identified the

most common inactivating allele, CYP3A5*3

Kuehl et al, Nature Genetics, 2001

a.

b.

35

CYP3A5*3 --the Major Inactivating Variant Allele

Kuehl et al., 2001; Lin et al.,

2002

1 2 3 4 5 6 7 8 9 10 11 12 133B

CYP3A5*3 (G)CYP3A5*1 (A)

CYP3A5

protein

1 2 3 4 5 6 7 8 9 10 11 12 135’ UTR 3’ UTR

(wt-CYP3A5 mR�A) (SV1-CYP3A5 mR�A)

A→G

truncated, inactive protein

1 2 3 4 5 6 7 8 9 10 11 12 13

36

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Exon-by-exon sequencing would

have missed the CYP3A5*3

causative SNP

3 43B3B

1618 nt 377 nt

141 nt

[A/G]

52 nt 99 nt

Most exon-by-exon sequencing strategies, flank the exons

With intronic primers ~200 nt or less from the exon border

Sequencing the alternative mRNA transcripts in “CYP3A5

Non-expressors revealed the mechanism of polymorphic

CYP3A5 expression

*

37

CYP3A5 Inactivating alleles

CYP3A5*3: intron-3 SNP (6986A>G)

� Creates aberrant splice site; unstable mRNA and truncated protein

� Multiple haplotypes (*3(A-J)), all contain causal 6986A>G SNP)

• CYP3A5*6: exon-7 SNP (14690G> A)

� Splicing defect causes skipping of exon-7;

� higher allele freq in AA

• CYP3A5*7: exon-11 (27131- 32insT)

� Frameshift mutation, premature stop codon; higher allele freq in AA

http://www.imm.ki.se/CYPalleles/cyp3a5.htm38

All Whites with a low CYP3A5 content

had a CYP3A5*3/*3 genotype

39

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


African Americans with low

CYP3A5, had primarily a CYP3A5*3/*3 genotype;

some persons with 3A5*6 or 3A5*7 inactivating alleles

40

CYP3A5 =50% of hepatic CYP3A protein in CYP3A5 expressors

0

50

100

150

200

250

300

350

400CYP3A5CYP3A4CYP3A5

Genotype

Caucasian Livers

*3/*3

*3/*1

*1/*1

41

CYP3A5 =50% of intestinal CYP3A proteinin CYP3A5 expressors

0

5

10

15

20

25

30

35

CYP3A5CYP3A4CYP3A5

Genotype

Caucasian Intestines

*3/*3

*3/*1

*1/*1

42

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Midazolam Hydroxylation is 2.2-2.5-fold higher (P=0.03) in

Livers with at least one CYP3A5*1 allele

Non-expressor Non-expressor

ANOVA indicates that there is no statistically significant difference

between CYP3A4 content in CYP3A5 expressors and non-expressors,

All of the difference is due to CYP3A5.

43

% of Individuals Expressing

CYP3A5 Based on CYP3A5

GenotypeEthnic Group % Expressing CYP3A5

Caucasians 30Japanese 30

Mexicans 30

Chinese 40African Americans >50

Southeast Asians

(not Chinese or Japanese)

60

Pacific Islanders 57

Southwestern America n Indians 80

44

Relative Catalytic Activities of

CYP3A4, 3A5 and 3A7

45

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Summary and Conclusions

• 10 substrates and 15 biotransform ations examined with

CYP3A4, 3A5 and 3A7 (1 & 4 OH midazolam, alprazolam and triazolam, 2 & 4 OH estradiol, N-demethyl diltiazem, 6B OH testosterone, oxidized nifedipine, O-dealky benzyloxytrifluoromethylcoumarin, N-desmethyl & 14 OH clarithromycin, N-desmethyl tamoxifen)

• Except for clearance to 1'-OH MDZ where CYP3A4 = CYP3A5; rank order was CYP3A4> CYP3A5>> CYP3A7

• Regioselectivity of metabolism by CYP3A4 and CYP3A5 was similar; CYP3A7 often different, for example

4-OH MDZ & TZ >1-OH, 2α−OH Test = 6β−OH

• These results demonstr ate an equal or reduced metabolic

capability for CYP3A5 compared with CYP3A4, and a significantly lower capability for CYP3A7.

JA Williams et al., Drug Metab Dispo 30:883-91, 2002 46

CYP3A4 Matched Human Liver Microsomal Panelare a robust tool to compare the metabolic

contribution of CYP3A5 to CYP3A4

210 ± 91 *

117 ± 68 *

93 ± 39

10

Group 2

CYP3A4/5

10N =

Group 1

CYP3A4 only

96 ± 36Total CYP3A

3.6 ± 1.0CYP3A5 (pmol/mg)

93 ± 36CYP3A4 (pmol/mg)

* p < 0.01

Equivalent mean P450 reductase and cytochrome b5 activity for groups 1 and 2

47

Huang et al., 200448

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


CYP3A5 influences the

clearance of some CYP3A

substrates in vivo

49

CYP3A5Genotype is associated with

Tacrolimus clearance

Haufroid et al. 200450

In Vivo CsA (cyclosporin A) Disposition andCYP3A5Polymorphism

Haufroid et al. 2004

Kidney Tx Patients

51

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Conclusions

• CYP3A5*1 genotype contributes to a “higher than average” oral clearance of some drugs:

�Genotyping may help predict 1st-dose tacrolimus

requirements

�Effect appears (based on published data) to be substrate-dependent - why?

• First-pass metabolism by intestinal CYP3A4/5 is a major determinant of the low oral bioavailability of tacrolimus.

52

CYP3A5 genotype

affects the extent of some

inhibitor drug-drug

interactions

53

Azole Inhibition Kinetics: CYP3A4 is more potently inhibited by azoles than CYP3A5

Ketoconazole

CYP3A4 noncompetitive 5.57 3.13 26.7 ±1.7

CYP3A5 noncompetitive 4.62 4.64 109 ±20

Fluconazole

CYP3A4 noncompetitive 3.87 2.36 9.2 ± 0.5

CYP3A5 noncompetitive 4.76 4.13 84.6 ±12.9

Inhibition Type Vmax Km Ki ±A.S.E.

Vmax : nmol/mi n/nmol; Km : µM; Ki : nM(ketoconazole) , µM (fluconazol e)

Gibbs et al, DMD 27:180- 192,

199954

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Effect of Itraconazole on MDZ Metabolic Clearance

• subjects with *1/*1 genotype (CYP3A5 expressors) less susceptible to inhibition than *3/*3 homogygotes (CYP3A5 non-expressors)

Parameters Measured after ITZ Treatment

Yu et al., CPT, 2004

55

Conclusions – In Vivo Interaction Data

• CYP3A5 genotype has a modest (negative) effect on the potency of inhibition by azole

antifungals and contributes to inter-individual variability in the inhibition response

56

Substra te/In hibitor

/induc er

R eference

Sta tins Lipid l owe r

respon se

CY P3A5 exp ressors

smaller dec rease in

serum cho lestero l

Kiv isto KT et al .,

Pha rmacogene tics

14 :523 - 525, 2004

Afla tox in Afl atox in-a lbumin

addu c t form ation

Inc reased f orm ation of

mu tagen ic AFB1 - exo -

8 ,9-epox ide

Wo jnow ski et a l.,

Pha rmacogene tics

14 :691 - 700, 2004

3A5 pro tein CYP3A d rug

oxida tions/li ver

microso mes

(Japan e se)

CY P3A5 con tribut es to

d rug ox ida tions –

midazo lam &

tes tost erone 6b -

hyd r oxy lation

Ya m aori et al. ,

Dr ug Metab

Pha mracok ine t

19 :120 - 9, 2004

Inhi bitors Or al

Itraconazo le/M DZ

sy stemi c

Greater inh ib ito ry

po tency for 3A4>3A5

translates in vivo

(gre ater i nh ibition i n

3A5 non-exp r esso r s)

Yu et al. , C PT

76 :104 - 12, 2004

Ind ucers rifamp in Greater induc tion i n

CY P3A5 non -

exp ressor s

Fl oyd e t al.,

Pha rmacogene tics

13 :595 - 606, 2003

Functional consequence of Polymorphic

CYP3A5 expression

57

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Substrate/Inhibitor/inducer

Reference

Tacrolimus Organ anti-rejection CYP3A5 expressors

require high er dose;

less nephrotoxicity

4 i ndependent

studies

Antibiotic

ABT-773

Dose-dependent

effects

Ef fect of CYP3A5

genotype was dose-dependent

Katz et al., CPT

75:516-28 , 2004

Midazolam/cancer

patients

In vivo

Clearance of MDZ

faster in CYP3A5

expressors

Wong et al., CPT

75:529-38 , 2004

Oxycodone 3A5 intrinsic

clearance > 3A 4OCD N-

demethylation

Postoperative pain

may be influencedby polymorphic

CYP3A5

Lalovic et al.,

DMD 32:447-54,2004

Saquinavir Protease inhibitor CYP3A5 expressors

have lower systemic

concentrations

2 i ndependent

studies

Functional consequence of Polymorphic

CYP3A5 expression

58

CYP3A7

• First isolated as HFLa, then HLp2

• cDNA sequence 88% related to CYP3A4

• Detected in fetal liver, endometrium and placenta

• Catalytic properties rarely examined, except known to

catalyze the 16-OH of dehydroepiandrosterone 3-

sulfate

59

JS Leeder et al., J Pharm Exp Therap 31:626-635, 2005

CYP3A7 is the most abundantly expressed CYP3A

Isoform in fetal liver

CYP3A7

CYP3A5

CYP3A4

60

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


CYP3A7 is usually expressed only in fetal lifeExpression declines after birth. CYP3A4 is

Not significantly expressed in fetal life, butIncreases after birth

paradoxically,

Some adults express CYP3A7 - why?

61

-188 -129CYP3A4

CYP3A7*1C

CYP3A7

CYP3A7*1C --promoter contains a set of seven tightly

Linked variants that replaced 60 bp of the CYP3A7 (fetal)

promoter with the identical region from the CYP3A4 (adult) promoter

(-alters 3 transcription factor binding sites

hypothesized that these 60 nucleotides are important for the

Increased expression of hepatic CYP3A4, and loss of CYP3A7

Expression in most persons after birth.

ADULT expression

FETAL expression

???

62

GENOTYPE

CYP3A4

CYP3A7*1C

CYP3A7

-188 -129

Adults who express CYP3A7 after birth are more

Likely to carry the CYP3A7*1C allele

Nature Genetics 27:383-391, 2001

Replacement of part of fetal CYP3A7 promoter with part of the

“adult” CYP3A4 promoter leads to adult expression of CYP3A7

Express

Adult CYP3A7

YES

NO

63

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Mechanisms of and

Screening for

CYP3A4 Induction

CYP3A4

gene transcription appears to be under

the control of a complex mixture of signaling pathway

that respond to endogenous and exogenous receptor ligands.

64

CYP3A4 Inducers

contribute to CYP3A

drug-drug interactions

•Barbiturates carbamazepin e, phenytoin*

•Rifampin*

•Troglitazone

•St.John’s wort

* potent

Hepatic and Intestinal CYP3A4 can be induced by rifampin

and possibly other PXR ligands

65

Rifampin induces intestinal CYP3ADifferential effect on

Drugs with Different Gut First-Pass Extraction

• Both midazolam and zolpidem are selective CYP3A substrates.

Zolpidem: F = 72%, AUC(i)/AUC ratio = 0.29

Midazolam: F = 30%, AUC(i)/AUC ratio = 0.04

Clin Pharmacol Ther 62:629-34, 1997Clin Pharmacol Ther 59:7-13, 1996

rifampin

control

control

rifampin

66

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Gold Standard assay to screen for Induction of CYP3A activity

Is primary cultures of human hepatocytes

Schuetz EG et al, JBC 276:39411-18, 2001 67

PXR/SXR

Pregnane X ReceptorSteroid & Xenobiotic Receptor

Willson & Kliewer, Nat Rev Drug Discovery 1:259-266, 2002

Mechanism of “induced” drug-drug interactions

68

Tissue-Specific Induction of CYP3A4

Tracks with PXR Expression

Northern blot of PXR

mRNA content in

human tissues. Major

inducible organs are

those that express

PXR.

J Clin Invest 102:1016-23, 1998

69

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Screening for PXR activation using In vivo transactivation assays

CV1 cells Monkey kidney cells

Hek293

Human embryonic kidney cells

70

• Structurally diverse molecules can induce CYP3A via the same biochemical pathway; only rifampin is a biopsy proven inducer in the small intestine.

N

CNH2O

O

CH3

OH

OHO

H

CH3 H

HOCH3

F

NH

OCH

3

CH

NN

NCH3

OH

OH

O

O

CH3

CH3

O

H3CO

O

CH3

OH

HO

CH3

CH3

OH

CH3

O

H3C

rifampin

dexamethasone

carbamazepinephenobarbital

NH

NH

O

O

CH3CH2

O

Other In Vivo

CYP3A4 Inducers

Phenytoin

Prednisone

Hyperforin

Troglitazone

Nevirapine

Efavirenz

Clotrimazole

Ritonavir

Modafinil

71

Additional Ligands of PXR

• Endogenous ligands: glucocorticoi ds, pregnanes , some bile

acids,

vitamin E,oxysterol s

• Environmental contaminants: organochl ori ne pesticides and

polychlorinated biphenyls

72

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Glucocorticoids (nM)

excess

Glucocorticoidreceptor

Pharmacol ogical concentr ati ons of endogenous molecules activate PXR to

induce metaboli zing and transport genes to enhance steroid elimination

Target genes

73

Xie et al Nature. 2000 Jul 27;406(6794):435-9.

PXR MEDIATES DEX INDUCTION

Dex induces CYP3A and

induction is lost in PXR-KO mice

74

Constitutive Androstane Receptor

CAR, NR1I3

PXRE

CAR

CYP3A4 CYP3A4

Phenobarb

Phenytoin

carbamazepine

CAR also regulates CYP3A4 by binding to the “PXRE”

75

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


A master regulator of INTESTINAL CYP3A4

Vitamin D Receptor (VDR)

VDR1,25-dihydr oxy

Vitamin D3

Vitamin D3:

Constitutive intestinal CYP3A4 expression

may be controlled in part by a VDR-signaling pathway that

involves binding of 1,25-D3

–(+) effects in cell culture (intestinal) via VDR

Mol Pharmacol 51:741, 1997

76

Induction of CYP3A4 in LS180 Cells by 1,25-Vitamin D

mRNA

ProteinEt. Br.

CYP3A4CYP3A4

CT 1 10 100

1,25-D3 (nM)

HL14 CT 0.1 1 10 100

1,25-D3 (nM)

Pgp

Thummel et al., Mol Pharmacol 60:1399-1406, 2001

1,25-(OH)2-

D3

enhances

CYP3A4

and MDR1

transcription

in a human

intestinal

cell l ine

(LS180) that

expresses

both VDR

and PXR.

28S

77

Mutation of CYP3A4-ER6Abrogates Activation by 1,25-Din Transiently Transfected LS180 Cells

0 1 10 100

1,25 Vitamin D3

3.0

2.0

1.0

0.0

T G A A C T c a a a g g A G G T C A (wt)

A T A (mt)

CYP3A4-ER6

One of the PXR binding Sites in the CYP3A4Promoter recognized by VDR

WT

mt

78

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Activation of CYP3A4 PXRE by 1,25-D3 and hVDR/RXR

cells co-transfec ted

with hVDR or

hPXR expressi on

plasmids and

CYP3A4 reporter

plasmid.

1,25-D3 does

not activate CYP3A4

In the presence of

hPXR.

1,25-D3 activated gene

transcription only in

the presence of hVDR.

79

SUMMARY:

CYP3A is regulated by multiple nuclear hormone receptors that all bind to the

PXR response elements in the

CYP3A4 5’-flanking region

CARCAR PXRPXR

CYP3A4CYP3A4

PHENYTOINPHENYTOIN

DexamethasoneDexamethasone

RifampinRifampin

St. John’s wortSt. John’s wort

VDRVDR

1,25-dihydr oxy

Vitamin D3 (VD3)

CYP3A4

80

Regulation of Human CYP3A4 Expressionby Circulating Hormones

Growth Hormone:– (+) effects in hepatocytes via GHR; may influence

sexually

dimorphic CYP3A4 expression in liver (F>M)

J Clin Endocrinol 83:2411, 1998

HNF-4:– HNF-4 has a major role in regulating both basal and

PXR inducible expression of CYP3A4

Tirona et al., Nat Med. 2003 Feb;9(2):220- 4

81

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


Summary

Sources of CYP3A

Inter-individual Variation

• Genetics: polymorphic expression, gender and

ethnic differences.

• Induction: exposure to drug or xenobiotic increases

the activity of the drug metabolizing enzymes.

• Inhibition or inactivation: drugs compete for the same

enzyme or enzyme may be "killed" in the process of

metabolizing drug.

82

CYP3A expression and

activity

is also regulated

by the MDR1 gene that encodes

the efflux transporter

P-glycoprotein

83

Functional Interactions of

the product of the MDR1 gene/P-

glycoprotein (Pgp) and CYP3A

SUBSTRATES

INDUCERS

INHIBITORS

Endogenous

RegulatorsCYP3A

84

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


85

MDR1/Pgp Transporter regulates the intracellular

Concentration of some PXR ligands, and the

magnitude of CYP3A induction

Mdr -/- mice

High intracellular drug

HIGH CYP3A induction

Mdr +/+ mice

Low intracellular drug

Low CYP3A induction

CYP3APXRRXR CYP3A PXR RXR

86

MDR1/Pgp Transporter Effects the

Hepatic Concentration of CYP3A

substrate and CYP3A metabolism

Mdr -/- mice

High intracellular Erythromycin

Higher rate of CYP3A

Metabolism of erythromycin

Mdr +/+ mice

Low intracellular ERM

Lower rate of CYP3A

metabolism

CYP3A

Mol Pharm 58:8863- 8869, 2000

CYP3A

87

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


CYP3A4PXR

CYP3A activity is governed by a

dynamic interplay between multiple CYP3A Gene products,

Nuclear Hormone Receptors and Drug Efflux Proteins

HNF4

MDR1/P-Glycoprotein

CYP3A5

RXR

SUMMARY

88

Why study the

CYP3A

Family?

89

Recent US Market Withdrawal/Restricted

Use/�on-Approval - Some Examples

Terfenadine* (1998)

Fenfluramine/Phentermine (-1998)

Mibefradil* (1998)

Bromfenac (1998)

Astemizole* (1999)

Grepafloxacin (1999)

Drug X* - (non-approvable in 1999)

Trovafloxacin -restricted use; June 1999

Troglitazone (2000)

Cisapride* (2000)< http://www.fda.gov/medwatch/safety.htm > * Related to drug-drug interactions 90

Introduction


(CYP3A) Family?

Dr. Erin Schuetz


What do they have in common?

1. Terfenadine (1985- 1998)

2. Mibefradil (1997- 1998)

3. Astemizole (1988 - 1999)

4. Drug X (non approvable, 1999)

5. Cisapride (1993- 2000 )

6. Troglitazone (2000)

Unacceptable

risk/benefit ratio

QTc prolongation CYP3A

4: substrate

1: inhibitor

1: inducer

Drug-drug

interactions

Huang, EUFEPS, 11/200091

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