Introduction  

Coronary artery disease (CAD) is one of the most detrimental cardiac disorders associated with significant morbidity and mortality worldwide [1, 2]. It implies cardiac ischemia, myocardial infarction, and sudden cardiac death and is usually caused by coronary atherosclerosis [3].

Recent guidelines consider percutaneous coronary intervention (PCI, formerly known as angioplasty with stent) one of the most common medical procedures performed for the treatment of CAD. It is a safe procedure in patients with stable coronary artery disease (SCAD) and it is indicated whenever medical treatment fails to improve the prognosis [4].

Anti-platelet therapy as adjunctive therapy for PCI is recommended before, during, and after elective PCI to reduce the risk of ischemic events. Aspirin has been the cornerstone anti-platelet drug in patients undergoing PCI. In addition to aspirin, recent guidelines have shown improved outcomes with newer antiplatelet drugs to reduce the incidence of ischemia [5].

There are two types of adenosine diphosphate (ADP) purinergic receptors P2Y1 and P2Y12. The P2Y12 is the predominant receptor involved in the process of platelet aggregation [6].

Different oral P2Y12-inhibitors are used frequently in clinical practice. Differences in the pharmacokinetic and pharmacodynamic properties of these agents such as the mechanism of their binding to the receptor site, their half-lives, the onset and offset of action and adverse effects are important factors in the determination of the most suitable regimen for loading and maintenance therapy [7].

Clopidogrel is a thienopyridine ADP receptor antagonist that produces an effective inhibition of platelet aggregation. Being a prodrug, hepatic biotransformation of clopidogrel via CYP450 pathway is essential for its anti-platelet activity. It causes irreversible receptor inhibition. Clopidogrel has a slow offset which may be problematic in some patients. In addition, several genetic and non-genetic factors influence the drug pharmacokinetics, producing inter-individual variability in response to clopidogrel [8].

Differently from thienopyridines, which block the receptor site directly, ticagrelor acts non-competitively, moreover, it has a faster onset of action with no need for previous metabolic activation contrary to clopidogrel [9].

This work was designed to compare the clinical safety and efficacy of clopidogrel versus ticagrelor, in patients undergoing elective PCI with the determination of actual plasma concentrations and the platelet inhibitory effect of both drugs with the help of ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and light transmission aggregometry (LTA) respectively.

Materials and Methods

Study Population

This study was self-funded and was conducted at the critical care department, Faculty of Medicine. The study protocol was approved by the Research Ethics Committee (REC), approval number (N-11-2019), and all patients gave written informed consent for participation. A total of 60 patients diagnosed with SCAD, aged between 18-80 years were enrolled in the study and scheduled for elective PCI with a single drug-eluting stent. Half of the patients received clopidogrel, 600 mg oral loading dose, and 75mg daily after the PCI. Oppositely, the oral loading dose of ticagrelor recommended for the other thirty patients was 180 mg and the maintenance dose was 90 mg twice daily thereafter. Aspirin 325 mg was the preferred loading dose.

The detailed history of the patients was recorded at the time of admission, before the PCI.

Patients were followed clinically during and after elective PCI for the occurrence of death, major adverse cardiac events (MACE), and major bleeding. During the follow-up period, dyspnea or any adverse side effect was also reported. Follow-up visits were for about 6 months. Patients with a history of acute myocardial infarction, bleeding disorder, anemia, thrombocytopenia, administration of thrombolytic drugs or anticoagulants within the 7 days before PCI, known intracranial vascular malformation, allergy to clopidogrel or ticagrelor, and those with creatinine clearance < 45 mL/min, elevated liver enzymes or other indication of clinically significant hepatic dysfunction were excluded from the study.

Sample collection

2-3 hours after a high loading dose with clopidogrel 600 mg or ticagrelor 180 mg, about 5 ml aliquot of blood was drawn from all patients in the two studied groups and collected in test tubes containing ethylene diamine tetra acetic potassium salt (EDTA-K) to evaluate the maximum plasma concentration of both drugs (Cmax). Blood centrifugation at 4000 round per minute (rpm) for about 10 minutes was performed to obtain plasma samples which were separated and stored at −70 °C until analysis by UPLC-MS/MS. Another 5 ml aliquot of blood was drawn in citrated tubes  and centrifugated at 800 rpm for 15 minutes  to obtain platelet rich plasma for assessment of alterations in light transmission of  the stirred platelets exposed to ADP using LTA, samples were kept at room temperature and not refrigerated and the test was performed immediately.

Chemicals

Clopidogrel bisulphate, ticagrelor, rasagiline, methanol, formic acid, and acetonitrile were purchased from Sigma -Aldrich, Egypt. ADP was obtained from Biodata -USA. Drug-free human plasma was obtained from Vacsera, Egypt.

UPLC MS/ MS study

• Chromatographic conditions

The analytes were separated in Poroshell 120EC- C18 (4.6x50) mm, 2.7 μm column (Agilent Technologies, USA) in case of ticagrelor and Luna® Omega 1.6 μm polar- C18 (150x2.1mm) column (Agilent Technologies, USA) in case of clopidogrel. The mobile phase was a mixture of acetonitrile and 0.2% formic acid. The flow rate was 0.8 mL/min.in case of ticagrelor and 0.3 ml/min in case of clopidogrel. Nitrogen was used as collision gas. The released eluent entered the mass spectrometry (MS) interface, using electrospray ionization in the positive ion mode. The multiple reaction monitoring (MRM) mode was used for the detection of specific transitions of the analytes.

• Stock solutions

Each clopidogrel and ticagrelor and rasagiline (used as internal standard) were weighed at 0.01 gm and dissolved in methanol to a concentration of 100 μg/ml. Dilution was performed to achieve different drug concentrations.

These concentrations were (20, 50, 100, 500, 1000, 1500, 2000, 4000) ng / ml in case of ticagrelor and (0.5, 1, 5, 10, 15, 20, 50, 100) ng/ml in case of clopidogrel and a rasagiline sample at a concentration of 1500 ng/ml was also prepared. Blank samples (plasma without drug or internal standard) and zero samples (plasma with internal standard) were prepared.

The plasma calibration standards were produced by the addition of a volume of 450 μL plasma with 50 μl of the different measured drug concentrations then vortexed for 20 seconds after that about 50 μl of rasagiline (1500 ng/ml) was added then vortexed for 20 seconds, extraction was done by protein precipitation by spiking 1ml of acetonitrile to each sample and vortex for 1 min followed by centrifugation at 6000  rpm for 30 minutes. Lastly, 10 μL of the supernatant was then introduced to UPLC- MS/MS. The most sensitive mass transition, mass to charge ratio (m/z) for the precursor/ product ions was from 322 to 212, from 523.2 to 153, and from 172 to 117 for clopidogrel, ticagrelor, and rasagiline respectively.

• Calibration curve

The peak area obtained was recorded and then the calibration curve was constructed by plotting the measured peak area ratios versus different concentrations of standard samples. Linearity was determined by a linear regression equation with the calculation of the correlation coefficient.

• Sample preparation

Briefly, 500 μL Plasma containing the analyte was spiked with 50 μl of rasagiline (1500ng/ml) then Vortexed for 20 seconds. Precipitation of proteins was done by incorporating 1ml of acetonitrile into each sample. The mixture was vortexed for 1 minute followed by centrifugation for about 30 minutes at 6000 rpm. The resultant supernatant (10 μL) was then injected onto the UPLC- MS/MS.

Pharmacodynamic study

Determination of maximal ADP induced platelet aggregation was done using

Light transmission aggregometer Chrono log: (Havertown, USA).

As the platelets aggregate, changes in the light transmission were registered and calculated as the percentage of maximal ADP- induced aggregation. The aggregometer was calibrated by placing a cuvette containing platelet-poor plasma in the test well (100% light transmission)

Statistical methods

The statistical package for the Social Sciences (SPSS) version 26 (IBM Corp, Armonk, NY, USA) was used for coding data. Data were organized using mean and standard deviation (SD). An unpaired t-test was used to compare between groups. A Chi-square test was performed for comparing categorical data.

P-values less than 0.05 indicate statistically significant findings.

Results and Discussion

Baseline data

The detailed history of the patients which includes age, sex, body mass index, cardiovascular risk factors, and medications on admission was taken with no statistically significant difference between both groups as presented in Table 1.

Values were expressed as mean +SD or number of patients and percentage (n.and (%))

Clinical data

As shown in Table 2, the results revealed that ticagrelor was effective in reducing MACE but this reduction was not significant compared to clopidogrel (P-value = 0.472). No mortality was detected in the study. In the ticagrelor group, the occurrence of major bleeding was significantly higher contrary to the clopidogrel group (P-value =0.038). In addition, there was a significant increase in dyspnea in ticagrelor-treated patients compared to clopidogrel (P-value =0.007).

Values were expressed using several patients and percentage (n and (%))

Pharmacokinetic study for clopidogrel

The calibration curve for clopidogrel was linear based on the previously mentioned concentrations plus blank and zero samples. as shown in Figure 1.

The Cmax  (Mean ± SD, ng/ml) calculated 2-3 h after administration of clopidogrel 600mg was 54.33 ± 28.3, with a range: 15-98, Figure 2.

Pharmacokinetic study for ticagrelor

The calibration curve for ticagrelor was linear based on the previously mentioned concentrations plus blank and zero samples Figure 3.

and The Cmax  (Mean ± SD, ng/ml) calculated 2-3 h after administration of ticagrelor 180 mg was 972.80 ± 189.7 as presented in Figure 4.