DT‐678 inhibits platelet activation with lower tendency for bleeding compared to existing P2Y12 antagonists

Abstract The novel clopidogrel conjugate, DT‐678, is an effective inhibitor of platelets and thrombosis in preclinical studies. However, a comparison of the bleeding risk with DT‐678 and currently approved P2Y12 antagonists has yet to be determined. The objective of this study was to evaluate the bleeding tendency of animals treated with clopidogrel, ticagrelor, and DT‐678. Ninety‐one New Zealand white rabbits were randomized to one of 13 treatment groups (n = 7). Platelet activation was assessed by flow cytometry and light transmission aggregometry before and after the administration of various doses of DT‐678, clopidogrel, and ticagrelor. Tongue template bleeding times were also measured before and after drug treatment. Treatment with P2Y12 receptor antagonists caused a dose‐dependent reduction in markers of platelet activation (P‐selectin and integrin αIIbβ3) and aggregation in response to adenosine diphosphate stimulation. At the same doses required for platelet inhibition, clopidogrel and ticagrelor significantly prolonged bleeding times, while DT‐678 did not. DT‐678 and the FDA‐approved P2Y12 antagonists clopidogrel and ticagrelor are effective inhibitors of platelet activation and aggregation. However, unlike clopidogrel and ticagrelor, DT‐678 did not prolong bleeding times at equally effective antiplatelet doses. The results suggest a more favorable benefit/risk ratio for DT‐678 and potential utility as part of a dual antiplatelet therapy regimen.


| INTRODUC TI ON
Together with aspirin, purinergic P2Y 12 receptor antagonists, like clopidogrel and ticagrelor, are widely used in dual antiplatelet therapy (DAPT) for the prevention of thrombosis in patients with acute coronary syndrome (ACS). [1][2][3][4][5] Approximately, one million patients receive DAPT for ACS in the United States every year. Recent clinical trials have demonstrated the benefits of DAPT beyond 1 year and it is anticipated that long-term use of DAPT will steadily increase. 6,7 The most concerning adverse event associated with any antithrombotic therapy is bleeding. Head-to-head comparison of bleeding tendency between P2Y 12 antagonists is difficult since the classification of severity and clinical relevance of bleeding events differ in many of the large clinical trials. Additionally, even minor bleeding events, while not life-threatening in and of themselves, are significant since they are one of the most important reasons for antiplatelet therapy nonadherence which can leave patients at increased risk for thrombotic events. 8 Despite the approval of newer, more efficacious agents, clopidogrel continues to be broadly used in clinical cardiology. The comparative bleeding safety of clopidogrel compared to the newer agents like prasugrel and ticagrelor has been demonstrated in multiple large-scale clinical trials. 9,10 In the Platelet Inhibition and Patient Outcomes (PLATO) trial, ticagrelor significantly increased spontaneous bleeds, major bleeds, major plus minor bleeds, and major plus minor plus minimal bleeds compared to clopidogrel. Therefore, clopidogrel is the preferred agent for long-term management of patients. Clopidogrel, however, is subject to several limitations which include interpatient variability, delayed onset of action, and frequent drug-drug interactions. 11,12 In addition, approximately 30% of Caucasians and 60%-70% of Asians fail to respond to clopidogrel therapy due to polymorphisms in CYP2C19. 11,13,14 As a result, these patients have increased risk of major adverse cardiovascular events. 14,15 Our group has previously reported the development of DT-678 (née ClopNPT), a disulfide conjugate of the clopidogrel active metabolite (AM) with 3-nitropyridine-2-thiol. [16][17][18] In the presence of glutathione, DT-678 is readily converted to the AM through a disulfide exchange reaction as illustrated in Figure 1. 17 Our earlier studies have demonstrated significant inhibition of ex vivo platelet aggregation and thrombosis in mice and rabbits. 16,18 Furthermore, we have established that DT-678 releases the AM with a T max of <5 minutes in C57BL/6 mice via both oral and intravenous routes, and the plasma concentrations of the AM reached C max values of >1000 ng/mL after a 5 mg/kg intravenous dose or a 10 mg/kg oral dose. 18 These results suggest that DT-678 has favorable pharmacokinetic/pharmacodynamic properties that may potentially overcome the attenuated pharmacokinetic properties of clopidogrel and thus significantly improve the efficacy of antiplatelet therapy.
In this study, we sought to further evaluate the compound by comparing antiplatelet activity and bleeding tendency in animals treated with the approved P2Y 12 antagonists clopidogrel and ticagrelor, or DT-678. Our results demonstrate dose-dependent inhibition of platelet aggregation and activation by all agents. However, bleeding times were significantly prolonged by clopidogrel and ticagrelor, but not DT-678. These findings suggest that DT-678 may be safer in the clinical setting while maintaining similar antiplatelet efficacy.

| Animal care and use
The procedures used in this study were in accordance with the

| Surgical preparation of rabbits and administration of drugs
Ninety-one male New Zealand white rabbits (1.9-2.4 kg) were obtained from Charles River Laboratories, Inc (Wilmington, MA). All animals were acclimated for a minimum of 5 days and had free access to standard chow and fresh water prior to the study. Animals were maintained on an automated 12/12-hour light/dark cycle with 7:00 am as the start of the light phase. On the day of the study, rabbits were sedated and anesthetized to surgical unconsciousness F I G U R E 1 In the presence of glutathione (GSH), DT-678 releases the clopidogrel active metabolite (clop-AM) via a thiol exchange reaction without the requirement of CYP2C19 with ketamine (40 mg/kg, intramuscular [IM]), xylazine (5 mg/kg, IM), and isoflurane (1%-3%, inhaled). The surgical site was shaved, and the rabbits were placed on a 37°C heating pad. Isoflurane was administered through a mask that was placed over the mouth and nose. The right jugular vein was surgically isolated and instrumented with a polyethylene cannula for drug administration and blood collection. Respiratory rate, the lead II electrocardiogram, heart rate, and body temperature were monitored throughout the procedure.

| Collection of whole blood
Blood samples were collected from the jugular cannula into a syringe containing 3.2% sodium citrate as an anticoagulant (1:10 citrate to blood ratio) before (baseline) and 10 minutes, 1 hour, and 2 hours after drug treatment. The blood samples were divided into two parts: 1.5 mL was used to perform flow cytometry (baseline and 1 hour posttreatment time points only) while the remainder was used for platelet aggregometry (see below).

| Determination of platelet aggregation by light transmission aggregometry
Platelet reactivity was determined in platelet-rich plasma (PRP) obtained from whole blood samples using light transmission aggregometry. Whole blood samples (see above) were centrifuged at 150 g for 10 minutes at room temperature and the supernatant was collected.
The pellet was then centrifuged at 1500 g at room temperature for 10 minutes to obtain the platelet-poor plasma (PPP). Ex vivo platelet aggregation was assessed using a 4 channel aggregometer (Chronolog Corporation Model 700; Chrono-log Corporation). PRP was continually stirred and maintained at 37°C during the assay. The change in light transmission relative to PPP after stimulation with platelet agonists (ADP [20 µmol/L], AA [500 µmol/L], and collagen [2 µg/ mL]) was recorded.

| Determination of bleeding time in New Zealand white rabbits
To evaluate the bleeding risk of the P2Y 12 antagonists, bleeding times were measured using a Surgicutt ® device (Accriva Diagnostics), which creates a uniform 5-mm long and 1-mm deep incision on the upper surface of the tongue. The margins of the lesion were blotted every 10 seconds with filter paper until blood was no longer transferred from the tongue to the filter paper. The interval from the time the incision was created to the time that blood was no longer apparent on the filter paper is considered the tongue bleeding time.
Bleeding times were assessed before treatment and 2 hours after treatment.

| Statistical analysis
Data were analyzed using GraphPad Prism 7 software (GraphPad Software) and are presented as mean ± SEM. Statistical differences between drug treatment groups and vehicle were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test.

| P2Y 12 antagonists decrease α-granule secretion and the formation of integrin α IIb β 3
The effects of P2Y 12 antagonist treatment on α-granule secretion and the formation of integrin α IIb β 3 in rabbit platelets were meas-

| D ISCUSS I ON
Antagonists of purinergic P2Y 12 receptors are an important component in the pharmacological management of patients at risk for thrombotic events. Prescribed together with low-dose aspirin, these agents have been proven effective at reducing the risk of heart attack and stroke. Despite the availability of multiple P2Y 12 antagonists recommended for DAPT, interpatient variability persists in a significant number of ACS patients, which leads to increased risk of ischemic complications and reduced survival rate. Although clopidogrel is generally effective and well tolerated, it has well-documented clinical limitations such as interpatient variability, delayed onset of action, and drug-drug interactions. Approximately, 30% of Caucasians and up to 70% of Asians are resistant to clopidogrel. 14,19,20 Genetic polymorphisms in CYP2C19 are a main contributor to this lack of responsiveness. Patients who carry CYP2C19 loss-of-function mutations fail to effectively metabolize the clopidogrel prodrug to the pharmacologically AM. More recently a reversible P2Y 12 antagonist, ticagrelor, has been developed. This agent differs from previous thienopyridine agents in that it is not a prodrug and therefore does not require bioactivation. Ticagrelor, however, is subject to CYP3A4-mediated metabolism and its primary metabolite is also a potent P2Y 12 inhibitor. 21 Ticagrelor is prone to numerous adverse drug interactions due to the induction and inhibition of CYP3A4 by many clinically used drugs including ketoconazole, atazanavir, ritonavir, rifampin, dexamethasone, statins, and more. 22 Although the F I G U R E 2 Flow cytometric assessment of platelets activated with ADP. Representative scatter plots of platelets from animals treated with (A) vehicle, (B) 3.0 mg/kg DT-678, (C) 10.0 mg/kg clopidogrel, and (D) 3.0 mg/kg ticagrelor. (E) Pretreatment with antiplatelet agents caused a dose-dependent reduction in α-granule secretion (as measured by CD62P expression) and the formation of integrin α IIb β 3 (indicated by FITC-fibrinogen binding) in response to ADP activation. Double-positive (CD62P + fibrinogen + ) events were quantified in the upper right quadrant of individual animal flow cytometric dots plots. The data are presented as the mean ± SEM of seven separate experiments. **P < .01, ***P < .001, ****P < .0001 when compared with vehicle-treated group by one-way ANOVA followed by Dunnett's post hoc test. ADP, adenosine diphosphate newer agents like ticagrelor have improved clinical outcomes, they also increase the risks of bleeding. 23,24 The primary safety concern with ticagrelor is bleeding as indicated in the PLATO trial supporting the approval of ticagrelor by the FDA. 23 Patients taking ticagrelor are nine times more likely to discontinue the use of drug than those on clopidogrel.
Due to the numerous limitations with P2Y 12 antagonists, our research team has developed a novel conjugate of clopidogrel that spontaneously and nonenzymatically releases the AM after oral and intravenous administration. [16][17][18] Our earlier studies have demonstrated the rapid release of the AM within minutes of administration. 18 In this study, we report our findings comparing the antiplatelet and bleeding effects of DT-678 to clopidogrel and ticagrelor. The inhibitory effects of these agents on platelet activation were evident in the reduced surface expression of P-selectin and decreased binding of fluorescently labeled fibrinogen in response to ADP activation. In addition, ADP-induced platelet aggregation was dose-dependently inhibited by treatment with DT-678, clopidogrel, and ticagrelor. Importantly, however, tongue template bleeding times were only significantly prolonged by treatment of clopidogrel and ticagrelor and not DT-678 suggesting that the latter has a more favorable safety profile.
Preclinical assessment of bleeding risk is limited by the availability of standardized animal models. A great deal of effort has been devoted to characterizing the murine tail cut assay in assessing the bleeding tendency of antithrombotic drugs and genetic hemostatic disorders. The severity of the tail amputation, however, does not accurately replicate the clinical state. Furthermore, there is no common protocol for testing and as a result bleeding times vary considerably between laboratories. This inconsistency makes direct comparison of the adverse bleeding effects of drugs difficult. 25,26 Template bleeding tests in humans were first described by Milian in 1901 and were later improved by several others. [27][28][29] The tests involve making a small incision on the skin (often the  With respect to ticagrelor, the compound has been reported to have potential off-target effects on purinergic receptors in the vasculature leading to vasodilation. 33,34 Ticagrelor is structurally distinct from the thienopyridine class of antiplatelet agents and as such may possess differential actions at purinergic receptors in distinct tissues. In addition, increased circulating adenosine concentrations have also been reported in patients taking ticagrelor which might also explain some of the vascular effects associated with the drug. 35 It is possible that these vascular properties of ticagrelor may result in an increased bleeding tendency in the presence of simultaneous inhibition of platelet P2Y 12 . The importance of these effects is uncertain, however, as more recent reports in humans suggest no difference in the vascular effects of thienopyridines and ticagrelor. 36 An interesting observation from our results is that clopidogrel required an approximately 10-fold higher dosage than DT-678 to elicit similar antiplatelet effects. A likely explanation for this finding is that the clopidogrel prodrug undergoes a complicated metabolism pathway in which only 1%-5% of administered dose are converted to the AM. 37,38 DT-678, on the other hand is nonenzymatically converted to the AM and therefore all the administered dose is available for inhibition of P2Y 12 . This finding is potentially important in the context of Type II diabetic patients treated with DAPT. This subset of patients has an impaired ability to form the AM of thienopyridine P2Y 12 antagonists. 39,40 The underlying effect is hypothesized to result from dysregulation of cytochrome P450 (CYP450) enzymes. 41,42 CYP450-independent activation of the AM as with DT-678 may find utility in this unique population. It is important to note, however, that in the present study, rabbits were treated with a single intravenous injection of each drug. Further investigation is required to determine whether the observed effects persist with chronic oral administration.
We conclude that in an experimental bleeding model in rabbits, DT-678 did not significantly prolong bleeding time at doses that were capable of inhibiting platelet activation and aggregation.
Conversely, administration of clopidogrel or ticagrelor significantly prolonged bleeding time at equally effective antiplatelet doses.
Given its simplified activation pathway and favorable pharmacokinetics, our results suggest that DT-678 is a potentially useful alternative to existing P2Y 12 antagonists with improved predictability and safety.