Evaluation of the antiplatelet effect of generic ticagrelor 90 mg (ticaspan^®) alone and in combination with aspirin 75 mg as compared to ticagrelor (innovator): An in vitro study

ABSTRACT

Objective:

To evaluate an in vitro antiplatelet effect of generic ticagrelor 90 mg (ticaspan) alone and in combination with aspirin 75 mg as compared to the innovator formulation of ticagrelor alone and in combination with aspirin among healthy Indian volunteers.

Methods:

18 volunteers were enrolled and platelet viability was tested using lactate dehydrogenase (LDH) assay in six of 18 volunteers. In 12 volunteers, maximum platelet aggregation (MPA) and percentage inhibition of platelet aggregation (PI) were assessed using a platelet aggregometer in six study groups.

Results:

There was no significant increase in LDH levels when platelets were incubated with an innovator or generic drug alone and in combination with aspirin as compared to the dimethyl sulfoxide [DMSO] group. All five study groups showed a significant reduction in the MPA values compared to the DMSO group (P < 0.01). The extent of decrease in MPA observed with the generic drug was not significantly different from the innovator drug (P = 0.325). Similarly, the MPA observed with the two combination groups did not differ from each other (P = 1.000), but it was significantly different from the MPA observed with aspirin (P = 0.039, each). The PI of platelet aggregation was significantly more in four study groups [generic drug alone; innovator alone; generic drug + aspirin; and innovator drug + aspirin] (P < 0.01) as compared to the aspirin group.

Conclusion:

The generic ticagrelor and its combination with aspirin demonstrated an antiplatelet effect equivalent to the innovator drug and its combination with aspirin.

Introduction

Ticagrelor, a selective, direct-acting, reversible P2Y12 platelet receptor antagonist,[1,2] has multiple benefits over clopidogrel, including a rapid onset of action, improved efficacy of inhibition of adenosine diphosphate (ADP)-induced platelet aggregation, and temporary platelet inhibition due to its short half-life (8–12 h) combined with the reversible receptor binding. In addition, it inhibits nonplatelet P2Y12 receptors on vascular smooth muscles, where they cause vasoconstriction and increase myocardial perfusion. There are lesser interindividual variations in the antiplatelet effect.[1,3,4]

The use of cost-saving generic medicines instead of innovator drugs majorly contributes to the reduced healthcare cost globally.[5] These generic drugs are marketed at substantially low cost (approximately 20–90% reduced cost) compared to their innovator drug[5] and are presumed equally efficacious. Due to limited knowledge and insufficient awareness about the safety and efficacy of these drug formulations, physicians are reluctant to prescribe generic drugs.[6] Generic medicines can be marketed once they achieve bioequivalence compared to an innovator.[5] It is argued that bioequivalence may not always correlate with therapeutic equivalence due to several factors, including interindividual variations.[7] Hence, to increase the acceptance of generic drugs in clinical practice, it is necessary to demonstrate comparable pharmacodynamic properties between the innovator and generic drug by conducting clinical studies.

The present study aimed to evaluate the in vitro antiplatelet effect of the generic ticagrelor 90 mg (ticaspan) alone and in combination with aspirin as compared to innovator ticagrelor 90 mg among healthy Indian volunteers. In patients who have experienced an acute coronary syndrome (ACS), combining aspirin with a P2Y12 receptor antagonist has been proven to provide an additional reduction in the risk of ischemic events during the initial year following the syndrome.[2,8,9] As per the various guidelines, dual antiplatelet therapy (DAPT) of aspirin (75–100 mg) once daily and ticagrelor (90 mg) twice daily is considered as a routine choice of regimen for the management of patients with ACS.[10,11] Therefore, a combination of ticagrelor with aspirin was also included in this study to compare between a combination of generic ticagrelor and innovator ticagrelor with aspirin.

Materials and Methods

The study protocol was approved by the Institutional Ethics Committee of Seth G.S. Medical College and K.E.M. Hospital, Mumbai (EC/PHARMA-18/2019).

Study participants

Male volunteers aged between 18 and 65 years were screened by clinical history, physical examination including weight and body mass index, and laboratory investigations including hematological findings [hemoglobin, complete blood count within 10% of the normal range] to confirm their health status. Biochemical tests including serum serum glutamic-pyruvic transaminase, serum glutamic-oxaloacetic transaminase and serum blood urea nitrogen and serum creatinine, fasting blood glucose, serum low density lipoprotein, serum cholesterol, and serum triglycerides were conducted at screening. Abnormilities within 1.5 times the upper limit of the normal range were detected. Patients with abnormal laboratory findings and abnormal electrocardiogram results were excluded from the study.

Recruitment of volunteers was done informally through advertisements in the hospital campus and by word of mouth. Written informed consent was obtained from all the volunteers before recruitment.

Volunteers with a history of intake of medications in the past 30 days (like aspirin, inhibitors of platelet receptors [abciximab, tirofiban, eptifibatide, ticlopidine, clopidogrel, prasugrel, cangrelor, ticagrelor, phosphodiesterase inhibitors, anticoagulants] known to affect platelet function), known case of diabetes mellitus, history of smoking, intake of alcohol or caffeine in the preceding 24 h, presence of bleeding/clotting disorders, history of intake of drugs from other systems of medicine like Homeopathy, Unani, etc., in the past 8 weeks, history of cardiovascular, musculoskeletal, hepatobiliary, genitourinary, neurological or any psychiatric condition, history of having participated in another investigational drug trial in the prior 30 days, history of positive screening result for hepatitis B and/or hepatitis C virus, and history of HIV or any immune deficient conditions were excluded.

The study was conducted on platelets harvested from eligible healthy volunteers.

Study drugs

• Generic drug: ticaspan (ticagrelor generic) manufactured by USV Pvt. Ltd Batch No. DT 1905101A, Manufacturing date – May 2019, Expiry date – April 2022.

• Innovator drug: brilinta - manufactured by AstraZeneca LOT No. 60033803. Manufacturing date – January 2019, Expiry date – December 2021.

The working solutions of concentrations of 20 μg/mL of both drug formulations were prepared freshly in 0.5% dimethyl sulfoxide (DMSO).

Vehicle: DMSO – EMPLURA, Cat No. 1.16743.0521; SF4S640258, Merck Sigma Ltd Mumbai. The (0.5%) DMSO in deionized water was used to dissolve generic and innovator drugs and served as vehicle control in this study.

Product used for combination with study drugs: Working solution of 1 mg/mL was prepared by dissolving aspirin (acetylsalicylic acid) procured from Sigma Chemical Co., St. Loius, Mi, USA in DMSO (0.5%).

Platelet aggregating agents

A 50 μM solution of ADP manufactured by Sigma Chemical Co., St. Loius, Mi, USA was used as an inducer of platelet aggregation. All the solutions and dilutions were prepared fresh daily.

Procedure for harvesting platelets [Figure 1]

Figure 1.

Study procedure. LDH, lactate dehydrogenase; MPA, maximum platelet aggregation

At visit 1, clinical history, physical examination, and laboratory investigations (by collecting 10 mL of blood from the antecubital vein for screening) were conducted. Eligible volunteers called for visit 2 within 7 days. At visit 2, after overnight 12 h of fasting, for platelet viability assay, 25 mL of venous blood was collected by venipuncture, while for the platelet aggregation assay, 50 mL of venous blood was collected by venipuncture. The blood samples were collected in citrated tubes and were centrifuged at 800 rpm for 8 min to obtain platelet-rich plasma (PRP). After removing PRP, the blood sample was centrifuged at 4000 rpm for 15 min to obtain platelet-poor plasma (PPP).

Platelets from PRP were counted using an automated cell counter (Diatron Abacus Hematology Analyzer). The platelet count was adjusted to 2.5 × 10⁸/mL using autologous PPP. The plasma with the adjusted platelet count was called working PRP (WPRP).

Study procedures: [Figure 1]

The study was conducted in two parts as follows:

Part 1: Platelet viability

Platelet viability was tested using lactate dehydrogenase (LDH) assay. Appropriate volumes of either 0.5% DMSO, study drugs, aspirin, or a combination of aspirin and study drugs were added to 450 μL of WPRP to make the total volume 500 μL. The tubes were incubated at 37ºC for 3 min, followed by centrifugation at 3000 rpm for 10 min. LDH levels in IU/L in each supernatant were measured using commercial LDH assay kits, manufactured by Transasia Biomedicals LTD, HP (code no 121020), on a semiautoanalyzer (ERBACHEM 5 plus) at 340 nm.[12,13,14,15]

Part 2: Effect of study drugs on ADP-induced platelet aggregation

Platelet aggregation study was carried out on a platelet aggregometer (Chrono-log Model 700 Whole blood/Optical Lumi aggregometer, Havertown, PA, USA). The WPRP (450 μL) was prewarmed at 37-C for 5 min. Appropriate volumes of either 0.5% DMSO, study drugs, aspirin or a combination of aspirin and study drugs were added to these WPRP to make 475 μL of volume and incubate at 37-C for 3 min. Then, the WPRP was transferred to the reading wells. The autologous PPP served to adjust the transmission to 100%. ADP was added to the WPRP cuvettes in a volume of 25 μL with continuous stirring to get a final concentration of 2.5 μM. The aggregation pattern reflected by the optical change was recorded as the curve of percent aggregation versus time for 10 min.[16,17,18,19,20] The maximum platelet aggregation (MPA) was calculated on the aggregometer (using AGGRO/LINK8 and Vw Cofactor Software packages). The percentage inhibition (PI) shown by test/reference drug was calculated from the values of MPA using the formula:

Statistical analysis

Statistical analysis was done using GraphPad InStat (version 3.06) GraphPad Software Inc. The results were expressed as mean± (standard deviation [SD]). Student’s paired t test was used to compare postincubation LDH release (IU/L) of platelets in the vehicle group with preincubation values. One-way ANOVA followed by a posthoc test of Dunnet was used to compare postincubation LDH release (IU/L) of platelets of the selected concentrations of the study drug with the vehicle group. For MPA and PI, different groups were compared using repeated measures ANOVA, followed by posthoc Dunnett Multiple Comparisons tests. The P-value of <0.05 was considered significant.

Results

Of the 24 volunteers screened, 18 were enrolled in this study. Platelet viability assay was tested in 6 volunteers and the platelet aggregation study was done in the remaining 12 volunteers. The details of recruitment are summarized in a flowchart [Figure 2].

Figure 2.

Healthy volunteers disposition

Part 1: Platelet viability

The findings of the effect of the generic drug alone, innovator drug alone, and the combination groups on LDH values of PRP from platelets are presented in Table 1. The LDH released by platelets treated with DMSO (vehicle) preincubation and postincubation were comparable (P = 0.898). There was no significant increase in LDH values postincubation with the generic drug alone, innovator drug alone, a generic drug with aspirin, and innovator drug with aspirin compared to the postincubation values of the DMSO group (P = 1.000).

Table 1.

Effect of various study drugs and their combination on LDH release in platelets (pre- and postincubation)

Study Groups (n=6/group)	LDH release (IU/L)
Preincubation
0.5% DMSO	260.83 (35.83)
Postincubation
0.5% DMSO	260.17 (40.41)
Aspirin	257.67 (40.11)
Generic drug	256.50 (35.93)
Innovator drug	257.33 (30.54)
Generic drug+ aspirin	259.33 (36.62)
Innovator drug + aspirin	260.83 (38.26)

All values represent as mean (SD). LDH, lactate dehydrogenase; DMSO, dimethyl sulfoxide; SD, standard deviation

Part 2: Effect of study drugs on ADP-induced platelet aggregation

All five study groups showed reduced MPA values compared to the DMSO (vehicle) group (P < 0.01). The generic and innovator drugs in combination with aspirin showed significantly less MPA than aspirin alone (P = 0.039, each). The MPA observed with the generic drug alone was not significantly different from the innovator drug (P = 0.325). Similarly, MPA observed with the combination of generic drug and aspirin was comparable with that of the innovator drug and aspirin (P = 1.000). The MPA observed with a combination of the generic drug with aspirin was not significantly different from the generic drug alone (P = 1.000). Likewise, the MPA observed with the combination of the innovator drug with aspirin was not significantly different from the innovator drug alone (P = 1.000). [Figure 3]

Figure 3.

Maximum platelet aggregation (MPA). *P < 0.01 versus DMSO, ^#P = 0.039 versus aspirin. Others, P = 0.325, generic drug versus innovator drug; P = 1.000, generic drug + aspirin versus innovator drug + aspirin, generic drug + aspirin versus generic drug, innovator drug + aspirin versus innovator drug. DMSO, dimethyl sulfoxide

Compared to the aspirin group, a significant increase in the PI of platelet aggregation was observed in all other study groups. However, the PI observed with these four groups did not differ significantly from each other [Figure 4].

Figure 4.

Percentage inhibition (PI) of platelet aggregation. *P = 0.005 versus aspirin. ^#P = 0.001 versus aspirin

Discussion

Antiplatelet therapy has shown consistent clinical benefits in the management of patients with atherosclerotic cardiovascular and cerebrovascular diseases, particularly for secondary prevention of cardiovascular events.[21] Current guidelines recommend DAPT of aspirin plus a P2Y12 inhibitor to prevent atherothrombotic events in patients with ACS.[22] Although clopidogrel is the standard choice of oral P2Y12 inhibitor for platelet inhibition, evidence suggests that prasugrel and ticagrelor provide more intensive platelet inhibition, leading to improve clinical outcomes.[11,21,22,23]

Based on the evidence from clinical trials, clinical efficacy and safety of ticagrelor are established in various patient populations, including patients with ACS, a history of myocardial infarction (MI), having a high risk of developing an atherothrombotic event, and those undergoing coronary intervention.[2,24] Long-term administration of ticagrelor in patients with myocardial ischemia is associated with higher platelet inhibition and improved peripheral endothelial function compared with prasugrel.[25] A combination of ticagrelor with low-dose aspirin (75–150 mg) has been indicated for the management of atherothrombotic events in patients with ACS who have been treated with either invasive or noninvasive modalities or have a history of MI at least one year earlier and a high risk of developing an atherothrombotic event, coronary artery disease, type 2 diabetes mellitus, and history of percutaneous coronary intervention, who are also at high risk of developing an atherothrombotic event.[26]

In the present in vitro study, the antiplatelet effect of the generic formulation of ticagrelor was compared with that of the innovator formulation to provide evidence supporting its in vitro equivalence with the innovator formulation. The platelet viability assay findings found no additional LDH release after the incubation of platelets with the generic drug and innovator drug when used alone and in combination with aspirin. This indicates that the study drugs did not affect platelet viability.

Inhibition of ADP-induced platelet aggregation is a standard method to evaluate pharmacodynamic parameters for P2Y12-receptor inhibitors. Evidence suggests that ticagrelor once a day (100–400 mg dose) effectively achieved fast and concentration-dependent inhibition of ADP-induced platelet aggregation in healthy volunteers.[27] Another pharmacodynamic study involving healthy volunteers receiving multiple doses of ticagrelor showed that post 12 h of drug induction, there was a gradual reduction in inhibition of platelet aggregation along with decreasing plasma concentration of the study drug, implying reversible action of ticagrelor.[28] A randomized controlled trial conducted in 44 healthy male study participants demonstrated that the inhibition of ADP-induced platelet aggregation (at 2 h and at 24 h) achieved by ticagrelor monotherapy and ticagrelor-based DAPT treatment was comparable, indicating a similar impact of ticagrelor monotherapy and ticagrelor-based DAPT treatment on platelet reactivity and coagulation activation. Therefore, ticagrelor was effective in ADP-induced platelet inhibition, irrespective of aspirin use.[29] Furthermore, pharmacodynamic assessment in the Ticagrelor Monotherapy and Platelet Reactivity trial demonstrated that the maximal amplitude of platelet activation at 4 weeks was comparable between ACS patients treated with ticagrelor alone versus those treated with a combination of ticagrelor and aspirin, highlighting the similar ADP-induced platelet inhibition with both treatment groups.[30]

Assessment of antiplatelet activity in our study revealed that both the innovator drug alone and its combination with aspirin, as well as the generic drug alone and its combination with aspirin, showed significant antiplatelet activity in vitro as compared to vehicle (DMSO). There was no significant difference between the antiplatelet efficacy as reflected by the results of MPA and PI of platelet aggregation between the generic and the innovator drug alone or in combination with aspirin. The two combination groups had significantly lower platelet aggregation compared to aspirin alone, highlighting that aspirin alone is not enough for achieving a higher level of platelet inhibition in patients with ACS.[8]

Thus, from the present study results, the generic formulation of ticagrelor 90 mg (ticaspan^®) shows potential to be used as an antiplatelet agent in clinical practice alone and in combination with aspirin with the added advantage of reduced cost. Lack of similar in vitro pharmacodynamic studies comparing generic ticagrelor with the innovator drug limits the interpretation of our results. Further, validation of the present study findings should be done in controlled clinical trials in patients requiring antiplatelet therapy.

Conclusion

The generic formulation of ticagrelor 90 mg (ticaspan^®) alone and in combination with aspirin inhibited ADP-induced platelet aggregation in an in vitro assay. The antiplatelet effects of the ticaspan alone and in combination with aspirin were found to be identical to the innovator drug and its combination of with aspirin.

Financial support and sponsorship

The study was sponsored by USV Pvt Ltd, Mumbai.

Conflicts of interest

Dr. Santosh Revankar is an employee of USV Pvt. Ltd, Mumbai. All other authors have no conflict of interest to declare.