1. Clopidogrel (Plavix) By Oksana Ekkert

2. Objectives
At the end of this presentation, participants should be able to:
Describe CYP2C19 enzyme function and its variant alleles.
Describe how CYP2C19 polymorphism affects the metabolism of the drugs.
Understand the mechanism of action and metabolism of clopidogrel.
Identify multiple factors involved in clopidogrel response variability.
Understand the importance and the nature of clopidogrel-PPI drug interaction.

3. The routes of elimination for the 200 drugs sold by prescription in the United States according to the RxList data listed in April

4. CYP2C19
CYP2C19 is primarily present in hepatic tissue, but a significant amount is also found in the gut wall, particularly the duodenum.
Protein of 490 amino acids.
Located in a densely packed region on chromosome 10 along with CYP2C8, 2C9, and 2C18 genes.
This CYP2C isozyme, the least expressed, was the first to be discovered due to its marked genetic polymorphism, resulting in (S)-mephenytoin-poor (PM) and extensive metabolizer (EM) phenotypes. Although not initially of any particular clinical interest, the CYP2C19 isozyme was later found to show a marked preference for proton-pump inhibitors (PPI) like omeprazole and pantoprazole, and numerous additional important drugs have been identified as major CYP2C19 substrates in recent years. CYP2C19 plays a prominent role in the metabolism of several first- and second-generation antidepressants, cyclophosphamide, proguanil, voriconazole, propranolol.

5. Variant alleles in CYP2C19 genotype.
Nucleotide
change
Effect
Europeans
Blacks
Asians *1
Wild type
85%
82%
65% *2
681G→A
Truncated protein
13-19%
10-25%
20-30% *3
636G→A
<1%
0-2%
5-13%
*17
-806C→T -3402C→T
Increased translation
18% 4%
One of the most common, with an allelic frequency of 15% to 30%, is designated CYP2C19*2. This polymorphism causes a splicing defect and a complete loss in enzyme activity, presenting phenotypically as a poor metabolizer.
The CYP2C19*17 variant is associated with ultrarapid metabolizers and seems relatively common in Swedes (18 percent), Ethiopians (18 percent), and Chinese (4 percent).

6. Variant Alleles Extensive metabolism: CYP2C19*1/*1
Intermediate metabolism:
CYP2C19*1/*2 or *1/*3
Poor metabolism:
CYP2C19*2/*2, *2/*3 or *3/*3 (also *4,*5) 
Ultrarapid: CYP2C19*17 /*17
The PM phenotype results from two null alleles, leading to the absence of functional CYP2C19 protein, whereas extensive metabolizers carry at least one functional allele.
Poor metabolizers have markedly reduced or absent enzyme activity.
Intermediate metabolizers have reduced enzyme activity.
Ultrarapid metabolizers have high enzyme activity.
The investigators found that the presence of the *17 polymorphism was indeed associated with a higher level of platelet inhibition, as measured with multipleelectrode platelet aggregometry, in a gene-dose– dependent manner (ie, homozygotes had greater platelet inhibition than heterozygotes, who had greater inhibition than wild types).

7. Comparison of pro-drugs and active drugs and clinical consequences
Drug type
Metabolizer
Phenotype
Effect
on drug
metabolism
Potential consequence
Prodrug
Needs metabolism to work
(clopidogrel)
Poor to intermediate
Slow
Poor drug efficacy, patient at risk of therapeutic failure due ↓ levels of active drug.
Accumulation of prodrug, patient at increased risk of drug-induced side effects.
Ultrarapid
Fast
Good drug efficacy, rapid effect. Possible accumulation of active drug → potential of adverse effects.
Active drug
Metabolized to inactive drug (omeprazole)
Accumulation of active drug, patient is at increased risk of drug-induced side effects. Patient requires lower dosage .
Poor drug efficacy, patient is at risk of therapeutic failure. Patient likely will require higher dosage.

8. Clopidogrel Anti-platelet agent
In 2005, world’s 2nd highest selling drug--U.S. sales $5.9 billion
Effective (with aspirin) for secondary prevention of MI and stroke, and thrombosis prevention after percutaneous coronary interventions (e.g., stent placement, angioplasty)
• Despite a short half-life ~2hrs, the irreversible binding of clopidogrel ’ s active metabolite to the platelet receptor leads to a prolonged pharmacodynamic effect.

9. ADP Clopidogrel ATP + P2X1 P2Y1 P2Y12 AC
15% active metabolite
HOOC
* HS N O Cl
OCH3 N S O Cl
CH3 C
P2X1
P2Y1
Gastro-intestinal absorption Gq
G12
“Rho”
Ca2+ flux
PIP2
Shape change
P2Y12
PLCβ
Shape change + Gi
IP3
DAG
Hepatic CYP Biotransformation
PKC αi βγ
Ca2+
mobilization
MLCK-P AC
PI3K
85% inactive metabolites
(Esterases in blood)
GP IIb/IIIa
receptor activation
Granule secretion
PKB/Akt
Rap1b
The remaining 15% of the absorbed clopidogrel is rapidly and extensively metabolized by the liver and the plasma concentration of the parent compound is below the detection limit beyond 2 h post dosing.
. Purinergic receptors and mechanism of action of clopidogrel. Clopidogrel is a pro-drug of which approximately 85% is hydrolyzed by esterases in the blood to inactive metabolites and only 15% is metabolized by the cytochrome P450 (CYP) system in the liver into an active metabolite. The active metabolite irreversibly inhibits the adenosine diphosphate (ADP) P2Y12 receptor. The P2X1 receptor, which uses adenosine triphosphate (ATP) as an agonist, is involved in platelet shape change through extracellular calcium influx and helps to amplify platelet responses mediated by other agonists. Activation of the P2Y1 receptor leads to alteration in shape and initiates a weak and transient phase of platelet aggregation. The binding of ADP to the Gi-coupledP2Y12 receptor liberates the Gi protein subunits ai and bg, resulting in stabilization of platelet aggregation. The ai subunit inhibits adenylyl cyclase (AC) and, thus, reduces cyclic adenosine monophosphate (cAMP) levels, which diminishes cAMP-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP-P). The status of VASP-P modulates glycoprotein (GP) IIb/IIIa receptor activation. The subunit bg activates the phosphatidylinositol 3-kinase (PI3K), which leads to GP IIb/IIIa receptor activation through activation of a serine-threonine protein kinase B (PKB/Akt) and of Rap1b GTP binding proteins. Prostaglandin E1 (PGE1) activates AC, which increases cAMP levels and status of VASP-P. Solid arrows indicate activation; dotted arrows indicate inhibition.
Acts by binding to ADP receptors on platelets, preventing platelet aggregation and thrombosis
• Great variability in response to clopidogrel
– % of individuals are resistant
GP IIb/IIIa receptor activation
Initiation of Platelet Aggregation
cAMP
Stabilization of Platelet Aggregation
VASP
VASP-P AC Gs
PGE1
cAMP
GP IIb/IIIa receptor activation
Angiolillo DJ et al JACC 2007

10. Given that only an estimated 2% of ingested clopidogrel ends up bound to platelets, it is easy to appreciate that small changes in its metabolism may substantially affect platelet p2y12 inhibition.
Hepatic metabolism is essential to the generation of the active metabolite of clopidogrel, specifically, 2 sequential oxidative steps through the cytochrome P450 (CYP) system.2 This superfamily of proteins, which encompasses 57 genes and 18 families, is involved in 80% of oxidative drug metabolism, as well as the metabolism of many other exogenous and endogenous chemicals, including arachidonic acid and eicosanoids, steroids, lipids, bile acids, and vitamins.3 We now know that a variety of P450 enzymes contribute to clopidogrel metabolism. The first metabolic step, which leads to 2-oxo-clopidogrel, is catalyzed by 3 enzymes (CYP1A2, CYP2B6, and CYP2C19), whereas the second step, which culminates in the active metabolite, involves 4 enzymes (CYP2B6, CYP2C9, CYP2C19, and CYP3A4).
Estimates of the contribution ratio (fm P450) of CYP1A2, CYP2B6, and CYP2C19 to 2-oxo-clopidogrel formation were 35.8, 19.4, and 44.9%, respectively, suggesting that the contribution of CYP2C19 to this oxidation process was higher compared with the other two P450s.
Estimates of the fm P450 of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 for the active metabolite formation were 32.9, 6.76, 20.6, and 39.8%, respectively, suggesting that the contribution of CYP3A4 to the active metabolite formation was greater compared with the other three P450s.
T.E. Klein, J.T. Chang, M.K. Cho, K.L. Easton, R. Fergerson, M. Hewett, Z. Lin, Y. Liu, S. Liu, D.E. Oliver, D.L. Rubin, F. Shafa, J.M. Stuart and R.B. Altman, "Integrating Genotype and Phenotype Information: An Overview of the PharmGKB Project" (220k PDF), The Pharmacogenomics Journal (2001) 1,

11. First oxidative step: conversion of clopidogrel to 2- oxo-clopidogrel
CYP1A2 (responsible for 36% of conversion): genetic polymorphisms: 16 identified SNPs
CYP2B6 (responsible for 19% of conversion): genetic polymorphisms: 29 identified SNPs
CYP2C19 (responsible for 45% of conversion): genetic polymorphisms: 25 identified SNPs
Second oxidative step: conversion of 2-oxo- clopidogrel to the active metabolite
CYP2B6: responsible for 33% of conversion
CYP2C9 (responsible for 7% of conversion): genetic polymorphisms: 34 identified SNPs
CYP2C19: responsible for 20% of conversion
CYP3A4 (responsible for 40% of conversion): genetic polymorphisms: 20 identified SNPs
With the CYP2C19 enzyme involved in both steps, contributing to an estimated 45% of 2-oxo-clopidogrel generated and 21% of its conversion to active metabolite, it is understandable why so much recent research has focused on the potential impact of drugs and genetic polymorphisms that influence the activity of this enzyme.

12. Not only CYP2C19 genetics, but--
Genetics of CYP2C9*3 and ABCB1 have been shown to be important

13. Clopidogrel Response Variability 20% do not have adequate response
Intestinal Absorption
Poor compliance
Inadequate administration
Variable absorption
Genetic polymorphisms
CYP2C19PMs, CYP2C9*3, ABCB1
Drug-drug interactions
Genetic polymorphisms P2Y12 receptor
Alternate pathways of platelet activation
Hepatic Metabolism
Cytochrome P450 pathway
Active Metabolite
P2Y12 Receptor
(irreversible inhibition)
GP IIb/IIIa receptor expression
O’Donoghue M, Wiviott SD. Circulation. 2006;114:e600-e606
Simon T et al.NEJM. 2009;363-75
Feher G et al. Clin Genetics. 2009; 75:1-18.

14. Mechanisms of Clopidogrel Response Variability
Limited absorption capacity with ceiling effect at 600mg loading dose7
Esterases 85%
Clopidogrel Bisulfate
Inactive Carboxylic Acid Metabolite
Intestinal Absorption ?
P-glycoprotein
(MDR1 3435T genotype)2
15%
CYP3A4
CYP3A5
CYP2C19
CYP2C9
CYP3A4 inducers: rifampin
CYP3A4 inhibitors: erythromycin
Hepatic P450
Cytochromes
2C19 Genetic polymorphisms 2C19 inhibitors
2C9*3 Genetic polymorphisms
CYP1A2
CYP2B6, 2C19
Smoking (induction)
Multistep Conversion
The delayed onset of action of clopidogrel is one of its limitations, thus, it needs a loading dose to shorten it when rapid inhibition is required, such as in the context of ACS or PCI. 600mg loading dose is widely used given the positive result evidence.
There are about 5%-40% low responders or resistant patients identefied.
Variability factors in brief: genetic, cellular, and clinical factors.
Genetic factors:
1. Gene ABCB1 codes for MDR1 Pgp involved in clopidogrel absorption. Patient with variant alleles may have reduced active metabolite generation after administration of a loading dose of clopidogrel. Conflicting evidence.
2. CYP isoenzymes involved in metabolism of clopidogrel: CYP3A4, 3A5, 2C9 and 2C19 polymorphisms.
Clopidogrel-induced antiplatelet effects may be affected by several cellular factors.
For example, an accelerated platelet turnover has been suggested to diminish clopidogrel responsiveness.
Upregulation of P2Y1 and P2Y12 receptors to be implicated in clopidogrel variability in response.
Active Thiol Metabolite
P2Y12 Receptor
Inhibition of Platelet Aggregation (Wide Response Variability)1
1. Gurbel PA et al. Thromb Res. 2007;120: Taubert et al. Clin Pharmacol. 2006;80: von Beckerth et al. Eur Heart J.2007;28:

15. Why do we need PPI’s with clopidogrel?
Deepak LB, et al. Circulation 2008; 118:

16. Ki (μM) values of PPI’s for CYP2C19 enzyme
Brand
Generic
Ki (μM)
Model
Inhibitor
HLMa
rCYP2C19b
Ticlopidine
HLM 0.31±0.05
rCYP2C ±0.04
Prilosec
omeprazole
6.2±0.8
2.4±0.05
Aciphex
rabeprazole
21.3±2.8
(2.4±0.1)c
18.8±1.3
(2.8± 0.1)d
Nexium
esomeprazole
8.6±1.0
7.9±0.5
Prevacid
lansoprazole
0.45±0.07
0.74±0.09
Protonix
pantoprazole
69.4±9.2
15.3±1.1
a,b marker reaction used was S-Mephenytoin 4-Hydroxylation
c,d rabeprazole thioether; HLM=human liver microsomes
Lower Ki = greater inhibition.
Different PPIs available are metabolized by CYP isoforms mainly CYP2C19 and Cyp3A4.
Omeprazole is primarily metabolized by CYP2C19 as well as inhibit CYP2C19.
Li X, Andersson TB, Ahlstrom M, Weidolf L. Comparison of inhibitory effects of the proton pump-inhibiting drugs omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole on human cytochrome p450 activities. Drug Metab Dispos August 1;32(8):821-7.

17. Risk of All-Cause Mortality and Recurrent ACS in Patients Taking Clopidogrel and PPI
Neither clopidogrel nor PPI
PPI without clopidogrel
Clopidogrel + PPI
Clopidogrel without PPI
0.70
0.60
0.50
0.40
0.30
0.20
0.10
Deaths or Recurrent ACS
Proportion of
In this retrospective cohort study by Ho and colleagues, concomitant use of clopidogrel and a proton pump inhibitor (PPI) after hospital discharge for ACS was associated with an increased risk of adverse outcomes than use of clopidogrel without a PPI, suggesting that use of PPI may be associated with attenuation of benefits of clopidogrel after ACS.
Of 8205 patients with ACS taking clopidogrel after hospital discharge, 63.9% (n=5244) were prescribed PPI at discharge, during follow-up, or both and 36.1% (n=2961) were not prescribed PPI. Median follow-up after hospital discharge was 521 days (interquartile range, days). Death or rehospitalization for ACS occurred in 20.8% (n=615) of patients prescribed clopidogrel without PPI and 29.8% (n=1561) of patients prescribed clopidogrel plus PPI.
In multivariable analysis, use of clopidogrel plus PPI at any point in time was associated with an increased risk of death or rehospitalization for ACS compared with use of clopidogrel without PPI (adjusted odds ratio, 1.25; 95% CI, ).
In multivariable analyses with medication use as a time-varying covariate, periods of use of clopidogrel without PPI were associated with a significantly lower risk of adverse events compared with periods without the use of either clopidogrel or PPI (P<.001). However, this association appeared to be attenuated when comparing periods of use of clopidogrel plus PPI use with periods without use of either clopidogrel or PPI (shown in figure on slide). 90
180
270
360
450
540
630
720
810
900
990
1080
Days Since Discharge
Ho PM, Maddox TM, Wang L, et al. JAMA. 2009;301(9):
Ho PM, Maddox TM, Wang L, et al. Risk of adverse outcomes associated with concomitant use of clopidogrel and proton pump inhibitors following acute coronary syndrome. JAMA. 2009;301(9):

18. Considerations for Healthcare Providers
Patients receiving clopidogrel for MI or stroke may not receive the expected antiplatelet activity if omeprazole is used concurrently.
Separating the time of administration of clopidogrel and omeprazole does not reduce the chance of the interaction.
The FDA does not have sufficient drug interaction information to provide recommendations for concurrent use of other PPIs.
There is no evidence that H2 antagonists (other than cimetidine) interfere with antiplatelet activity of clopidogrel. Both cimetidine and omeprazole are available in nonprescription (OTC) forms and patients should be educated to avoid these drugs if receiving clopidogrel.
Concurrent use of cimetidine, esomeprazole, etravirine, erythromycin, felbamate, fluconazole, fluvoxamine, fluoxetine, ketoconazole, voriconazole and ticlopidine should also be avoided because they may also reduce clopidogrel’s antiplatelet activity.
Rifampin has been shown to increase the concentrations of active metabolite through CYP3A4 induction.
At high concentrations in vitro, clopidogrel inhibits P450 (2C9). Accordingly, clopidogrel may interfere with the metabolism of phenytoin, tamoxifen, tolbutamide, warfarin, torsemide, fluvastatin, and many non-steroidal anti-inflammatory agents, but there are no data with which to predict the magnitude of these interactions. Caution should be used when any of these drugs is coadministered with clopidogrel.
In conclusion, the findings of this meta-analysis suggest that there is conflicting and inconsistent evidence on the impact of the clopidogrel PPI interaction on cardiovascular outcomes, and certainly no evidence of any effect on overall mortality. Clinicians should carefully weigh up the real dangers of an increase in gastrointestinal haemorrhage events [13] before routinely avoiding the use of PPIs in patients taking clopidogrel.
List all the clopidogrel interactions found in the program, cyp3a4 inducers and cyp2c19 inducers

19. In Conclusion
The totality of all of the CYP2C19 polymorphism data suggests that it would be appropriate to begin genotyping all potential patients and thus identify those patients who would be at increased risk for thrombosis or bleeding if treated with clopidogrel.
Question 2c9 involvment

20. References
Wienkers LC, Heath TG (2005) Nat Rev Drug Discov 4:825–833 (top 200)
S. R. Steinhubl. Genotyping, Clopidogrel Metabolism, and the Search for the Therapeutic Window of Thienopyridines Circulation February 2, :
Plavix prescribing information. Accessed February 20, 2010.
Kazui M, Nishiya Y, Ishizuka T, Hagihara K, Farid NA, Okazaki O, Ikeda T, Kurihara A. Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. Drug Metab Dispos. 2010;38:92–99.
Sibbing D, Koch W, Gebhardt D, Schuster T, Braun S, Stegherr J, Morath T, Schomig A, Kastrati A. Cytochrome 2C19*17 allelic variant, platelet aggregation, bleeding events, and stent thrombosis in clopidogrel-treated patients with coronary stent placement. Circulation ;121:512–518.
Klotz U, Schwab M, Treiber G. CYP2C19 polymorphism and proton pump inhibitors. Basic Clin Pharmacol Toxicol 2004; 95: 2–8.
Beckerath N, Taubert D, Pogatsa-Murray G, et al. Absorption, metabolization, and antiplatelet effects of 300-, 600-, and 900-mg loading doses of clopidogrel: results of the ISAR-CHOICE Trial. Circulation 2005;112:
Li X, Andersson TB, Ahlstrom M, Weidolf L. Comparison of inhibitory effects of the proton pump-inhibiting drugs omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole on human cytochrome p450 activities. Drug Metab Dispos August 1;32(8):821-7.