Comparison of clopidogrel antiplatelet activity in diabetic versus nondiabetic patients with acute coronary syndromes and patients undergoing elective stent placement Thesis Submitted for fulfillment of master degree in Clinical Pharmacy, Faculty of pharmacy, Cairo University Prepared by Mina Wageh Mohareb Bachelor of Pharmaceutical Science Assiut University Under Supervision of Prof. Dr. Samar Farid Professor of Clinical Pharmacy Department of Clinical Pharmacy, Cairo University Ass.Prof.Dr. Nermeen Ahmed Sabry Dr. of Clinical Pharmacy Department of Clinical Pharmacy, Cairo University Dr. Mohamed Abd Elghany Karim Dr. of Cardiology Faculty of Medicine , Assiut University 2016 Abstract Introduction and aim of the study In clinical practice, oral antiplatelet therapy with adenosine diphoshate (ADP) p2y12 inhibiting activity such as thienopyridines (clopidogrel) represent a corner stone in the prevention of major cardiovascular events in those subjects at high risk (primary prevention) and in those who have already experienced a cardiovascular event (secondary prevention). Although antiplatelet therapy reduce ischemic events in patients with high risk for stent thrombosis, the individual inhibitory effects of clopidogrel shows large variability. Prospective studies implementing different methods of platelet function testing have reported that a certain proportion of patients shows attenuated response to clopidgrel, which is associated with increased risk of ischemic events. Diabetes mellitus presents with a high incidence of atherosclerotic vascular disease and patients with type 2 diabetes mellitus who already suffered an ischemic event are at higher risk of recurrence than non– diabetic patients. The rationale for aggressive antiplatelet therapy in diabetics is strengthened by the observation that circulating platelets from patients affected by diabetes mellitus show multiple abnormalities. Some studies which evaluated functional platelet parameters during combined aspirin and clopidogrel treatment showed that diabetic patients exhibit lower degree of platelet inhibition after clopidogrel administration in comparison with non-diabetic patients. The aim of this study was to determine the effect of diabetes mellitus on clopidogrel activity(measured by ADP- induced aggregation after clopidogrel administration) and concentration of clopidogrel active metabolite in cardiac patients undergoing PCI. Patients and methods This is an observational study made on IHD patients undergoing elective PCI. Recruited patients were categorized according to DM status into diabetic group (N= 30) and non-diabetic group (N=33). All patients received clopidogrel-loading dose of 600 mg before PCI. Platelet function was assessed using light transmittance aggregometry (LTA) technique at baseline (before clopidogrel loading administration), 24 hour after clopidogrel loading dose administration and 7-10 days after PCI. Clopidogrel active metabolite concentrations were measured using liquid chromatography/ mass spectrometry technique during one day of loading dose clopidogrel administration. All patients administered a maintenance dose of 75 mg clopidogrel for at least one year after percutaneous coronary intervention (PCI). All patients were followed up for two years year after PCI (by clinic visits once monthly and phone calls twice monthly) for recurrence of acute cardiac events. Results There was no statistically significant difference between diabetic and non-diabetic groups with respect to 10 µm adenosine di-phosphate (ADP) induced platelet aggregation - measured at base line (P = 0.64), 24 hour after PCI (P = 0.874) and 7-10 days after PCI (P = 0.643). Diabetics were non-significantly different from non-diabetics regarding post-PCI acute stent thrombosis (P = 0.945), sub-acute stent thrombosis (P=0.945), unstable angina(P = 0.29) and cardiac death (P = 0.64). Both diabetic and non-diabetic groups were comparable regarding active clopidogrel metabolite concentration (0.987). There was significant strong negative correlation between 24 hour ADP aggregation and active clopidogrel concentration in both diabetics (R = -0.8479 and P = 0.00001) and non- diabetics (R = -0.8895 and P = 0.00001). Also, strong negative association could be detected between 7-10 days ADP aggregation and active clopidogrel concentration in both diabetic group (R = -0.7233, P = 0.00001) and non-diabetic groups (R = -0.893, P = 0.00001). Using multiple linear regression test, Diabetes as a co-morbidity could not be found as a significant predictor for 24-hour ADP aggregation (p = 0.469), 7-10 days ADP aggregation (P = 0.769), or post PCI acute cardiac events (P = 0.865). Conclusion Diabetic patients may have comparable post-PCI platelet aggregation, post-PCI acute cardiac events and active clopidogrel concentration with non-diabetic patients when high loading dose 600 mg clopidogrel is administered before percutaneous coronary intervention (PCI). Introduction It has been anticipated that by year 2030, 8.6 million adults in Egypt can have diabetes making it the country with the tenth largest population of diabetes in the world . Patients with diabetes mellitus (DM) suffer from high risk of cardiovascular diseases . DM patients undergoing percutaneous coronary intervention (PCI) are mainly at high hazard for recurrent cardiovascular events. Dual antiplatelet therapy with aspirin and clopidogrel is the standard therapy for acute coronary syndromes (ACS) and for patients undergoing PCI with stenting, inclusive of DM patients . However, diabetic patients undergoing PCI are susceptible to more recurrent ischemic events than non-diabetics despite being maintained on usual dual antiplatelet therapy . High platelet reactivity (HPR) and/or suboptimal reaction to antiplatelet agents might justify partly the high recurrent rates of ischemic events in DM patients . For instance, diabetic patients show less responsiveness to daily 81 mg of aspirin compared to non-diabetic patients, overcome in part by higher dose of aspirin . The ADP P2Y12 receptor blockade with clopidogrel was superior in decreasing recurrent ischemic events over aspirin alone in diabetic patients with coronary artery disease . Angiolillo et al, validated that, DM patients have a lower level of platelet inhibition versus non-diabetic patients each in the acute phase, following administration of 300 mg loading dose (LD) of clopidogrel, and in the chronic phase, whereas on 75 mg/day maintenance dose (MD). It has been reported that, 600 mg clopidogrel LD is associated with higher degree of platelet inhibition and lower probability of clopidogrel non-responsiveness in comparison to 300 mg clopidogrel LD . However, platelet inhibitory activity of 600 mg clopidogrel LD during PCI has not been well-established in diabetic patients compared to non-diabetics . Aim of the Study Aims The main purpose of the present observational study was to examine the differences in the platelet function and post-PCI acute cardiac events between diabetic and non-diabetic coronary artery disease (CAD) patients after administration of 600 mg loading dose of clopidogrel. Methodology This study was a prospective observational single center study conducted to observe the effect of diabetes mellitus on patients’ response of already prescribed loading dose of clopidogrel administered before elective PCI. Recruitment methods Study subjects and study site Study subjects were recruited from those patients who were admitted to the catheterization unit in Assuit University Teaching Hospital for PCI during the period between April 2011 and March 2012. Patients were screened for the following inclusion and exclusion criteria before their approvals were sought. Inclusion criteria  Adult patients > 18 years old,  patients with documented history of coronary artery disease (CAD),  patients had diagnostic catheterization within 2 weeks before stenting, and  diabetic patients(insulin and non-insulin dependent) for the diabetics arm group (diabetes defined as serum glucose level ≥ 126 mg/dl or administration of anti-hyperglycemic drug (141). Exclusion criteria  Patients who received any glycoprotein IIb/IIIa inhibitors before the procedure,  patients who had a family or personal history of bleeding, serious bleeding tendency, history of intracranial hemorrhage, or any sign of active bleeding,  patients with platelet count < 100 x 103/µl, Hemoglobin level < 10 g/dl, and Haematocrit level < 30%, (142)  patients undergone major surgery within one week of enrollment,  patient with any known hypersensitivity to clopidogrel,  patients with uncontrolled hypertension who were not responsive to hypotensive drugs,  patients with severe decompensated liver diseases,  patients with creatinine clearance less than 25 ml/min,  patients with current gastrointestinal disorders such as severe diarrhea, severe constipation, inflammatory bowel disease, paralytic illus or any other disorder that may dramatically affect gastrointestinal absorption,  patients taking medications that may increase likelihood of life threatening hemorrhage by serious pharmacodynamic synergistic interaction (such as antithrombinalfa, antithrombin iii, apixban, argatroban and bivalirudin) or by stimulation of hepatic metabolizing enzymes responsible for clopidogrel bioactivation ( such as carmabazepine rifampin, rifabutin and St.John Wart) (143), or  patients taking medications that may seriously decrease serum levels and antiplatelet effect of clopidogrel by inhibition of CYP 450 enzymes responsible for clopidogrel bioactivation (such as variconazole, esmoprazole, fluconzole, itraconazole, erythromycin, clarithromycin and etravirine) (143). Study design This was a prospective, randomized, and observational parallel study using the simple restricted randomization technique. The study was approved by the research and ethics committee of Faculty of Pharmacy, Cairo University (Appendix I), which followed the tenets of the Declaration of Helsinki (162). Informed consent was obtained from all subjects or their surrogate after explaining the nature, purpose, and potential risks of the study (Appendix II). The recruited patients were divided according to the presence of diabetes mellitus (DM) as a co-morbidity into two groups; diabetic and non-diabetic groups. Diabetes was defined as a fasting blood glucose concentration ≥ 126 mg/dL or administration of anti-hyperglycemic medications (141).While the patients in the other (non-diabetic group) were selected based on normal fasting blood glucose(less than 126 mg/dl) and no previous administration of hypoglycemic drugs. Baseline screening All recruited patients in both groups were screened at baseline (before clopidogrel initiation and conducting PCI) for the following data: A. Biochemical laboratory data This included measuring serum glucose level (mg/dL), serum creatinine level (g/dL), total cholesterol(mg/dL), serum triglycerides(mg/dL), serum sodium level (meq/L), serum potassium level (meq/dL), prothrombin concentration(%), and platelet count. B. Medical history In this section, current smoking status (any amount of daily cigarette smoking (Smoking is a practice in which a substance-mainly tobacco is burned and the resulting smoke breathed in to be tasted and absorbed into the bloodstream) (163), previous history of hypertension, previous history of myocardial infarction, previous history of unstable angina, previous PCI with one or more stents, previous coronary artery bypass graft were recorded. C. Medication history Previous chronic administration of beta-blockers, angiotensin converting enzyme inhibitors or statins, calcium channel blockers and long acting nitrates were recorded (most widely used classes in patients with coronary artery disease) (85-88), in order to exclude the effect of medication interaction effect (other than clopidogrel) on ADP aggregation results and post-PCI cardiac events in both study groups. Patients in both groups received clopidogrel with a loading dose of 600 mg immediately before undergoing PCI, followed by 75 mg maintenance dose for at least one-year post PCI. Study outcomes and Follow-up evaluation A. Platelets aggregation The primary outcome of this study was measuring ADP induced platelet aggregation using light transmittance analysis technique at baseline before clopidogrel loading, 24 hours and 7-10 days after initiating clopidogrel therapy. Platelet aggregation was quantitatively measured by recording the transmission of a light beam across a suspension of constantly agitated platelets in an APACT-4004 aggregom- eter (LABiTec, Ahrensburg, Germany) (140). The blood is collected in 3.8% sodium dihydrate citrate (one part per nine parts of blood) and centrifuged at 190 g for 15 min at ambient temperature. The cPRP is stored at ambient temperature in stoppered tubes under CO to avoid pH 2