Page 1
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
Page 2
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 2
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
Page 3
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 3
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
Page 4
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 4
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
Page 5
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 5
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
Page 6
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 6
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
Page 7
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 7
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
Page 8
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 8
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
Page 9
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 9
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
Page 10
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 10
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
Page 11
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 11
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
Page 12
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 12
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
Page 13
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 13
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
Page 14
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 14
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
Page 15
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 15
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
Page 16
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 16
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
Page 17
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 17
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
Page 18
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 18
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
Page 19
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 19
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
Page 20
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 20
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
Page 21
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 21
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
Page 22
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 22
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
Page 23
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 23
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
Page 24
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 24
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
Page 25
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 25
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
Page 26
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 26
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
Page 27
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 27
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
Page 28
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 28
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
Page 29
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 29
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
Page 30
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 30
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
Page 31
Introduction
Why study the Cytochrome P4503A
(CYP3A) Family?
Dr. Erin Schuetz
The screen versions of these slides have full details of copyright and acknowledgements 31
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