Fuentes QE et al . Platelets, 2013;24:255-62.

1. Pharmacogenomics of cardiovascular drugs – The Example of ThienopyridinesProf. GerardSIESTIFCC Worldlab 2014 Istanbul, June 23, 2014Biomarkers and Personalized Pharmacotherapy Session

2. CVD remains the leading cause of death in the developed countries and the second common cause worldwide. Fuentes QE et al. Platelets, 2013;24:255-62.

3. Antianginals Antihypertensives Antiarythmics Cardiotonics Vasodilatators Anticoagulants Antiaggregating agents Diuretics Antidiabetics Hypolipemic drugs Anti-inflammatory drugs Thyroidtherapy Anti smoking drugs Hormonal replacementtherapy Contraceptives Cardiovascular drugs classes

5. Clinical Indications • Prevention of ischaemic events in atherosclerotic patients • Acute coronary syndrome without ST-segment elevation (NSTEMI) • ST elevation MI (STEMI) • Prevention of thrombosis after stenting (Clopidogrel + ASA) • Alternative for aspirin intolerancy

6. CARDIOVASCULAR DRUGS METABOLIZED THROUGH CYP 2C SUB-FAMILIES : • (S)Warfarin • Aceclofenac • Acenocoumarol • Bupropion • Candesartan • Carvedilol • Celecoxib • Clopidogrel • Diclofenac • Flurbiprofen • Fluvastatin • Glibenclamide • Glimepiride • Glipizide • Glyburide • Ibuprofen • Indapamide • Indometacin • Irbesartan • Lornoxicam • Losartan • Mefenamicacid • Meloxicam • Naproxen • Nateglinide • Phenylbutazone • Piroxicam • Rosiglitazone • Suprofen • Tenoxicam • Tienilicacid • Tolbutamide • Torasemide • Valsartan It seemsthat CYP 2J2 is not heavilyinvolved in drugmetabolism.

7. Schematic representation of the metabolism of Clopidogrel and Prasugrel Holmes et al. J Am Coll Cardiol 2010; 56:321-341

8. Antiplatelet Drug ClopidogrelPathway (PK)  Website: http://www.pharmgkb.org - Posted by Sangkuhl K. on February 20, 2008.

9. Mechanism of Action • On platelet - adenosine diphosphate (ADP) receptor (P2RY12) blocker • Blocking activation of the glycoprotein IIb/IIIapathway • Inhibiting platelet aggregation

10. Inflammation

11. Inflammation is a complex and highly orchestrated process that has a domino-like effect on the initiation and development of a wide range of systemic illnesses including CVD Gabay C, Kushner I. N Engl J Med, 1999;340:448-54.

12. Inflammation participates in all stages of atherosclerosis; from the initial lesion to the thrombotic complications. Atherosclerosis is recognized currently as a chronic inflammatory disease of large arteries. Hansson GK. N Engl J Med, 2005;352:1685-95. Fuentes QE et al. Platelets, 2013;24:255-62.

13. Procedure-evoked inflammatory response is the main causative mechanism of Post-PCI in-stent restenosis (ISR) Jukema JW, et al. Nat Rev Cardiol, 2011;9:53-62. Kim MS, et al. Cardiovasc Ther, 2011;29:190-8.

14. CYP enzymes play an indispensable role in the regulation of inflammation thorough: • Contribution to the metabolism of drugs with proven or potential anti-inflammatory properties; ex. Thienopyridines; Clopidogrel and Prasugrel • Biosynthesis of endogenous bioactive lipid mediators that modulate inflammation; ex. AA derivatives; epoxyeicosatrienoic acids (EETs)

15. Clopidogrel and arachidonicacid are metabolized by the sameCYPs 2C

16. Arachidonic acid metabolism Arachidonicacid (esterified) cPLA2 Arachidonicacid (free) COX CYP2C8 CYP4A11 LOX CYP2J2 CYP2C9 CYP4F2 Prostaglandins Leukotrienes 12/15-HETE CYP2E1 CYP2C19 20-HETE • Vasodilatation • Anti-inflammation • Fibrinolysis • VSMC antimigratory • Cardioprotection 5,6-ETT 8,9EET 11,12-EET 14,15-EET Leukotrienes sEH X inhibition GSH-T 5,6-DHET 8,9-DHET 11,12-DHET 14,15-DHET GSH-EET • Less biological activity ABCB1 Excretion Siest G, 2009 (adaptedfromTheken KN & Lee CR. Pharmacogenomics 2007, 8(10):1369-1383)

17. AA: a 20-c polyunsaturated omega-6 fatty acid that resides in the cell membrane phospholipids ShahabiP, Siest G, Visvikis-Siest S. Influence of Inflammation on Cardiovascular Protective effects of Cytochrome P450 Epoxynase-derived Epoxyeicosatrienoic Acids. Drug Metab Rev. 2014 Feb;46(1):33-56

18. CLOPIDOGREL TREATMENT The Problem • Inter-individual variability in response to Clopidogrel • Up to 30% of patients do not display adequate antiplatelet response Simon et al. N Engl J Med 2009;360:363-75 Gurbel et al. Circulation 2003;107:2908–13

19. Inter-individual variability in response to Clopidogrel - Prasugrel Up to 30% of patients do not display adequate antiplatelet response Gurbel et al. Circulation 2003;107:2908–13

20. Clinical Consequence Hyporesponsiveness is associated with poorer clinical outcomes after an acute coronary syndrome, particularly after percutaneous coronary intervention (PCI) Bonello et al. JACC 2008;51:1404-11

21. Causes of Response Variability Account for only a small fractionofvariation in response • Genetic Factors (Polymorphisms) • Cellular Factors Accelerated platelet turnover Increased ADP exposure Up-regulation of P2Y12 pathway Up-regulation of P2Y-independent pathway (TXA2, thrombin) • Clinical Factors Under-dosing/poor compliance Poor absorption Drugs interaction (Ca-blockers, PPIs) Smoking DM/insulin resistance Obesity Angiolillo et al. JACC 2007;49:1505–16

22. Polymorphisms of 2C19

23. Polymorphisms of 2C19Simon et al. N Engl J Med 2009;360:363-75 Outcome according to the CYP2C19 loss-of-function alleles

24. CYP2C19 loss-of-function (CYP2C19*2) and gain-of-function (CYP2C19*17) polymorphisms have been shown to be associated, respectively, with an attenuated and strengthened response to both clopidogrel and prasugrel

25. Otherdrugmetabolizing enzymes influences on Clopidogrelmetabolism • CYP 3A4 One polymorphism (12 G > A) ? – Discrepancy • ABCB1 C3435 T influence (-) French people • (+) Amish • CYP 3A5 IncreasedatherothrombicriskwithItraconazole • CYP 3A5 inhibition • CYP 2B6 Strong inhibition by Clopidogrel • (Bupropion, Cyclophosphamide = substrates)

26. Ethnicdifferences CYP 2C19*2 - Chinese 50% - African American 34% - White 25% - Mexican American 19% CYP 2C19, 17* - Swedish 17% - Ethiopian 17% - Chinese 4%

27. Clopidogrel – Drug interactions • Clopidogrelusuallygivenwith aspirine risk of MI  but not  death • risk of gastrointestinalbleeding • Proton pumpinhibiters • ≠ betweenOmeprazol and others • Caution • Verapamil – no problem • Atorvastatin ? – inhibition of CYP 3A5 • Calcium antagonists • Ketoconazole – CYP 3A4/A5 inhibitions • Drugs or pathologyincreasingIL6 → inhibition of carboxylesterases and DME

29. INTEREST TO FOLLOW THE INFLAMMATORY MARKERS

30. CLOPIDOGREL: EFFECTS ON INFLAMMATORY MARKERS

31. Baseline inflammatory marker levels in function of CYP epoxygenase polymorphisms No significant association between plasma biomarker levels and CYP2C19*2, CYP2J2*7, CYP2C8*3,CYP2C9*2 and CYP2C9*3 polymorphisms in healthy population ShahabiP, Siest G. et al. Int J MolSci 2013;18:16402-13.

32. Causes of Response Variability Account for only a small fractionofvariation in response • Genetic Factors (Polymorphisms) • Cellular Factors Accelerated platelet turnover Increased ADP exposure Up-regulation of P2Y12 pathway Up-regulation of P2Y-independent pathway (TXA2, thrombin) • Clinical Factors Under-dosing/poor compliance Poor absorption Drugs interaction (Ca-blockers, PPIs) Smoking DM/insulin resistance Obesity Angiolillo et al. JACC 2007;49:1505–16

33. Smoking Interactions CYP2C19 polymorphisms and/or smoking and/or a CYP2C19 polymorphisms-smoking interaction may contribute to the inconsistency in the results of different studies regarding anti-inflammatory effects of thienopyridines

34. Smoker’s Paradox (Controversial) Clopidogrel-treated smokers appears to have a greater platelet inhibition and lower aggregation when compared to non-smoker patients

35. Thienopyridines may exert pleiotropic anti-inflammatory effects Inflammation-reducing effects of these drugs have not been a persistent finding and the results of different studies in this field are sometimes contradictory

36. Genetic-determined Low Response to Thienopyridines is Associated with Higher Systemic Inflammation in Smokers PaymanShahabi, MD,1* Thomas Cuisset, MD, PhD,2,3,4* Bernard Herbeth, PhD,1 Pierre Emmanuel Morange, MD, PhD,3,4,5 Charlotte Grosdidier, MD,3,4,5 Maria G. Stathopoulou, PhD,1 Gérard Siest, PharmD, PhD,1¥ Marie-Christine Alessi, MD, PhD,3,4,5† Sophie Visvikis-Siest, PhD,1† 1UMR INSERM U1122, IGE-PCV, Université de Lorraine, Faculté de Pharmacie, Nancy, France 2 Département de Cardiologie, CHU Timone, Marseille, France 3 INSERM, UMR1062, Nutrition, Obesity and Risk of Thrombosis, Marseille, France 4 Faculté de Médecine, Aix-Marseille Université, Marseille, France 5 Laboratoire d'Hematologie, CHU Timone, Marseille, France *Equal first authors † Equal last authors Under review in the International Journal of Cardiology

37. Patient with ACS Marseille Study Enrollment (N = 1128) PCI Discharge Clo. 75 mg/d; (n = 118) Clo. 150 mg/d; (n = 631) Pra. 10 mg/d; (n = 309) Clinical assessment 1-month visit Blood sample* Clinical assessment 6-month visit * Genotyping for CYP2C19*2, *17 *Measurement of CRP, Clinical assessment 12-month visit

38. Smoking and CYP2C19*2 polymorphism

39. Interaction between CYP2P19 loss-of-functionallele and smoking

40. Gawron-Skarbek & al. Nutr. Metab. Cardiovasc. Dis. 2014 Jun;24(6):e21-3

41. The findings could be explained by the following mechanisms • Nuclear factor-kappa B (NF-kB) • Reactive oxygen species (ROS) • Endothelial nitric oxide (NO) • Tissue factor (TF) The oxidative antioxidative status in plasma and cells is also important

42. stimulatory effect Inhibitory effect • ShahabiP., • Siest G., • Visvikis- • SiestS. • Drug Metab • Rev. 2014 • Feb;46(1):33-56

43. Genotyping CYP, CRP and Inflammation Interaction of CYP2C19 polymorphisms and smoking could make a significant contribution to identifying the patients who are at high risk of post-PCI clinical and procedural complications. CRP could give additional clinical information. At this time, there is no convincing data to support that routine CYP2C19 genotyping, as a tailored thienopyridine treatment, would be an effective strategy in clinical practice. The results of the current study may provide additional incentive for the clinicians to incorporate pharmacogenetics testing (including point-of-care genetic testing)into their daily clinical practice.

48. GENOTYPING CLINICAL DECISION SUPPORT

49. Josh Denny – 20th International Symposium on Microsomes and Drug Oxidations 5/19/2014

50. Josh Denny – 20th International Symposium on Microsomes and Drug Oxidations 5/19/2014