Unique Features of the Molecule

image01Unique collagen mediated platelet activation Inhibitor.

image01 Inhibits collagen induced platelet activation.

image01 Novel mechanism of action as a specific inhibitor of collagen induced platelet aggregation and adhesion.

image01 Good pharmacokinetic properties and bioavailability.

Executive Summary

Platelet collagen interaction through its receptors (glycoprotein VI [GPVI] and α2β1 or GIa/IIa) is crucial for the initiation of intravascular thrombosis. Recent data suggest collagen receptors as promising target for a novel anti-platelet therapy. In the present study, a novel synthetic compound S002-333, conferred significant and better protection in mice model of collagen-epinephrine induced thrombosis with comparable prolongation in bleeding tendency in mice to that of standard drug Aspirin. Compound S002-333 specifically inhibited platelet aggregation induced by collagen, but not by other agents including ADP, arachidonic acid and TRAP. Furthermore, the compound also inhibited platelet aggregation induced by collagen-related peptide and convulxin, the specific agonists of the major collagen signaling receptor GP VI. Moreover, the compound exhibited a specific inhibitory activity against GP VI mediated platelet adhesion over collagen in a concentration dependent manner in both in vitro (human) and ex vivo (mice) assays. It also inhibited collagen induced dense granule secretion, thromboxane A2 generation, intra-platelet calcium mobilization as well as tyrosine phosphorylation of various proteins. S002-333 significantly prolonged the time of occlusion in mouse carotid artery following endothelial injury by ferric chloride. Therefore these results suggest that the compound S002-333 seems to target GPVI receptor and inhibit platelet activation by collagen. S002-333 is a safe and well-tolerated molecule that prevents platelet adhesion and consecutive thrombus formation at the site of vascular injury. The strategy is to block the very early source of acute vascular complications, thereby excluding the disadvantages of existing anti-platelet drugs that primarily rely upon targeting the consequences of platelet activation.

Background and Opportunity

Acute coronary artery disease is a major cause of death worldwide. The first response to vascular injury is adhesion of circulating platelets to exposed sub-endothelial matrix proteins, which triggers subsequent platelet aggregation. Among the macromolecular components of the subendothelial layer, fibrillar collagen is considered the most thrombogenic constituent, as it acts as a strong activator of platelets and supports platelet adhesion both in vitro and in vivo. GPVI is a 60-65 kDa type-I trans-membrane glycoprotein, which belongs to the immunoglobulin superfamily. Platelets deficient in GPVI show loss of collagen induced adhesion and aggregation in vitro (Sugiyama et al, 1987). Likewise, anti-GPVI monoclonal antibodies attenuate ex vivo platelet aggregation in response to collagen and collagen-related peptide (CRP), which mimics collagen triple helix. In different mouse models of endothelial denudation both the inhibition or absence of GPVI virtually abolished platelet-vessel wall interactions and platelet aggregation, thereby indicating GPVI as the major determinant of arterial thrombus formation.

There is compelling evidence for a crucial role of GPVI and G Ia/IIa in arterial thrombosis from studies in mice. It was demonstrated that thrombus formation in the injured carotid artery in mice is virtually abolished in the absence of functional GPVI (Massberg et al, 2003). This agrees with a recent study with markedly reduced platelet attachment and subsequent neointimal hyperplasia at sites of vascular injury in FcR γ-chain–deficient mice that lack GPVI. These developments implicate GPVI as a potential pharmacologic target for the treatment of ischemic cardiovascular disease.

Such a strategy might have a number of advantages. First, GPVI is exclusively expressed on platelets and megakaryocytes, which prevents the risk of side effects of anti-GPVI agents on other cell types. Second, GPVI deficiency is not associated with a major bleeding risk in humans and mice, suggesting that anti-GPVI therapy might be well tolerated. Thus, there is considerable interest in developing novel tools, such as receptor antagonists, or depleting antibodies to evaluate GPVI function in humans. The second of these procedures is a unique way to abolish collagen responses for the lifetime of the platelet. This has been demonstrated in mice in which the injection of the anti-GPVI antibody JAQ1 leads to internalization and degradation of GPVI in circulating platelets resulting in a prolonged “GPVI knock-out”–like phenotype. A similar mechanism of GPVI down-regulation appears to exist in humans, as one GPVI-deficient patient had developed antibodies against the apparently absent receptor, suggesting that she suffered from an acquired GPVI deficiency based on antibody-induced clearing of the receptor from her platelets. Understanding of these receptor recognition motifs within collagen has revealed new antithrombotic targets, which might lead to the development of new anti-platelet drugs to inhibit intravascular thrombus formation. Both commercial and academic groups have identified platelet collagen (GPVI and GpIa/IIa) receptors as attractive targets for antithrombotic therapy.

Highlights

The compound S002-333 inhibits collagen induced platelet activation:

image01 It has a novel mechanism of action as a specific inhibitor of collagen induced platelet aggregation and adhesion possibly mediated by the inhibition of platelet collagen receptors.

image01 It has no effect on the coagulation cascade (TT, PT, aPTT) and no adverse effect on vasoreactivity.

image01 It is orally active, and offered significant protection against collagen and epinephrine induced thrombosis, and prevented thrombus formation in AV-shunt and ferric chloride induced thrombosis.

image01 It has moderate effect on mice tail bleeding time with better efficacy, in contrast to existing anti-platelet drugs Aspirin and Clopidogrel.

image01 Preclinical safety pharmacological studies have been completed, exhibiting no adverse effect on cardiovascular, respiratory and neurological parameters. It has been tested in vitro for binding with 451 kinases, important GPCRs and hERG channel, which predict its safety.

image01Good pharmacokinetic properties and bioavailability.

Intellectual Property Right Protection (IPR)/ Patent Status

Patent filed: 2-alkyl/aryl sulphonyl-1,2,3,4-tetrahydro-9H-pyrido (3,4-b) indole-3-carboxylic acid esters /amides as antithrombotic agents

image01 Indian Application No. 01258DELNP2005, Dt. 31-Mar-05.

image01 United States Application No. 11/842674, Dt. 21-Aug-07.

image01 PCT Application No. PCT/IN04/00417, Dt. 27-Dec-04.