DT-678
DT-678 (previously known as ClopNPT) is a novel antiplatelet agent being developed to prevent recurrence of heart attacks and strokes in patients with previous events, and to inhibit blood clot formation after percutaneous transluminal coronary angioplasty (PTCA) with or without the implantation of an arterial stent.
DT-678 was developed to overcome the need for metabolic activation of clopidogrel and has been described in the literature:
https://pubmed.ncbi.nlm.nih.gov/22584220/
https://pubmed.ncbi.nlm.nih.gov/27511819/
https://pubmed.ncbi.nlm.nih.gov/25230737/
https://pubmed.ncbi.nlm.nih.gov/23348501/
https://pubmed.ncbi.nlm.nih.gov/32268786/
https://pubmed.ncbi.nlm.nih.gov/31372229/
Novel Synthesis of DT-678
The creation, synthesis, and discovery of DT-678 is novel. For a long time, the pharmaceutical industry has desired to create a P2Y12 inhibitor to prevent platelet induced aggregation, that is specific, rapidly activated, lacking requirement for hepatic activation. Synthesis of compounds like DT-678 were found to be technically challenging as they could not be made by traditional medicinal chemistry methods. Diapin's patented innovations include the creation of novel biological enzymes to catalyze the formation of DT-678 from precursors. DT-678 when administered orally is rapidly absorbed and nearly completely reduced to its active form by plasma glutathione.
Figure 1 compares the metabolism of DT-678 to that of clopidogrel (also known as Plavix®). DT-678 is metabolically activated in plasma by glutathione to near complete (~99%) conversion to M5, the active metabolite of clopidogrel. In contrast, clopidogrel needs to undergo extensive metabolism in the liver including a CYP2C19 enzymatic step, to generate the M5 active metabolite. This conversion is inefficient yielding only 1-2% M5, plus results in generation in over 15 inactive metabolites. One of these metabolites, M4, has recently been described to be the cause of the increased bleeding and thus safety risk of clopidogrel.
The creation, synthesis, and discovery of DT-678 is novel. For a long time, the pharmaceutical industry has desired to create a P2Y12 inhibitor to prevent platelet induced aggregation, that is specific, rapidly activated, lacking requirement for hepatic activation. Synthesis of compounds like DT-678 were found to be technically challenging as they could not be made by traditional medicinal chemistry methods. Diapin's patented innovations include the creation of novel biological enzymes to catalyze the formation of DT-678 from precursors. DT-678 when administered orally is rapidly absorbed and nearly completely reduced to its active form by plasma glutathione.
Figure 1. Clopidogrel is Extensively Metabolized into Over 15 Metabolites
Figure 1 shows the formation of the AM (labelled M4) from clopidogrel (labelled M0) or DT-678. M4 represents only a small fraction of the metabolites produced by clopidogrel in the liver while it is the major metabolite of DT-678 formed in the plasma. Unlike DT-678, the major metabolites of clopidogrel includes M1 produced by a carboxyesterase and is 85% of all metabolites. Also, unlike DT-678, clopidogrel’s conversion to AM is dependent upon the liver CYP2C19 activity. CYP2C19 mutations that reduce or inactivate clopidogrel conversion activity are fairly common. As many as 60% of Asian and 30% of the Non-Asian populations are either hetero- or homozygous for these mutations. To try to overcome low clopidogrel conversion activity, these patients are sometimes prescribed clopidogrel at higher than recommended dose, which can increase bleeding events with little additional effect on antiplatelet activity.
The usefulness of DT-678 relates to known variants of CYP2C19, that are unable to metabolically activate clopidogrel. Sixty percent of Asians and 30% of Caucasians have reduced or absence of clopidogrel activation given a heterozygous or homozygous CYP2C19 variant, respectively. Furthermore, many patients that are type II diabetic have reduced hepatic levels of CYP2C19, making it difficult to obtain the benefit of clopidogrel anti-platelet effects at a recommended therapeutic dose. These patients are often administered higher levels of clopidogrel which may increase bleeding risk by an increased formation of undesirable metabolites. The value of DT-678 is that it can be converted to the active metabolite of clopidogrel (M5) independent of the genetic variation or expressed hepatic level of CYP2C19. Therefore, all patients may benefit from the anti-platelet properties of DT-678 independent of CYP2C19 activity.
Fig. 2. Platelet activation in response to ADP stimulation (10µM) was assessed by surface receptor flow cytometry in rabbits 1 h after drug treatment.
Dose (mg/kg)
Expression of CD62P and integrin αIIbβ3 are positively associated with platelet activation.
n = 5/group; * p <0.05 vs. vehicle, ** p <0.01 vs. vehicle, *** p<0.001 vs. vehicle when compared with vehicle treated group by one-way ANOVA followed by Dunnett’s post hoc test.
Fig. 3. Tongue bleeding time was assessed using a Surgicutt® device before and 1 h after drug treatment in rabbits.
Data are expressed as relative bleeding time vs. baseline. n = 5/group; * p <0.05 vs. vehicle, ** p <0.01 vs. vehicle when compared with vehicle treated group by one-way ANOVA followed by Dunnett’s post hoc test.
Since both compounds deliver the same active metabolite, it was hypothesized that one or more of the “inactive metabolites” of clopidogrel were, in fact, active and contributing to bleeding. A literature report was found that suggested that one of the “in active” metabolites (M5 above) might have activity (https://pubmed.ncbi.nlm.nih.gov/30262863/). Dr. Zhang was able to obtain a sample of this metabolite in sufficient quantities to evaluate its activity in mice.
Figure 4. Activity of metabolite in platelet aggregation assays from blood taken from M5 treated animals, and blood collected from a tail vein bleeding study in the same mice.
The structure of this metabolite, M5, is shown on top. The panel on the right shows that M5 did not act to inhibit platelet aggregation that was induced with either ADP or collagen. However, in the bleeding study, nearly 10X as much bleeding was observed. This result supports our hypothesis that the “inactive” metabolites do contribute to the bleeding seen with clopidogrel and that this bleeding is caused by a mechanism other than inhibition of platelet aggregation. Chemical analysis of clopidogrel shows the presence of 2 chiral centers, which means there are 4 stereoisomers of the AM. Dr. Zhang and Beijing SL Pharma were able to identify and isolate these 4 different stereoisomers of the AM in sufficient quantities to evaluate their activity.
Almost all of the activity of the Active Metabolite (AM) is present in 2 of the 4 stereoisomers. If the 2 less active isomers were eliminated, then the AM of DT-678 should have more activity than the AM from clopidogrel. A proprietary process was developed to manufacture DT-678 such that it contains only the 2 active stereoisomers. This process is currently a trade secret.
Figure 5. Platelet aggregation assays
DT-678 was then compared to clopidogrel in a platelet aggregation assay in rats by Tianjin Tiancheng New Drug Evaluation Co., Ltd. The results are shown in Table 1.
The respective ED50s demonstrate that DT-678 is approximately 30X more potent than clopidogrel in rat platelet aggregation assays. This should translate into an ability to use less DT-678 to get the same effect as clopidogrel, and therefore lower the chance of bleeding and off-target adverse events.
Table 1.
Table 2. Comparison of the PK of DT-678 and clopidogrel in DIO mice
The IV and PO doses were 5 and 10 mg/kg respectively and the plasma concentrations of the AM were determined by LC-MS/MS. DT-678 delivered higher plasma concentrations, 20-40X, of the AM in a much shorter time, 5 min vs. 30 min and 10 min vs 1 hr, via either the intravenous (IV) or oral (PO) routes of administration, respectively.
DT-678 improved upon clopidogrel’s slow onset of action. The slow onset limits the use of clopidogrel in emergency and surgical situations. To evaluate if DT-678 has a faster onset of action, pharmacokinetic analysis was performed in the PK core at the University of Michigan.
Taken together, the results of the discovery studies demonstrate that the AM of DT-678 (a subset of the AM of clopidogrel that has been proven to be active in patients) is more potent, has a faster onset of action, and causes less bleeding than clopidogrel (which is the gold standard for antithrombotics). This is a product profile that should allow DT-678 to compete in the marketplace with all of the generics in this space.