Madhu Chintala

Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs

ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.

Discovery of a novel, orally active himbacine-based thrombin receptor antagonist (SCH 530348) with potent antiplatelet activity.

The discovery of an exceptionally potent series of thrombin receptor (PAR-1) antagonists based on the natural product himbacine is described. Optimization of this series has led to the discovery of 4 (SCH 530348), a potent, oral antiplatelet agent that is currently undergoing Phase-III clinical trials for acute coronary syndrome (unstable angina/non-ST segment elevation myocardial infarction) and secondary prevention of cardiovascular events in high-risk patients.

Evidence for the Presence of a Proteinase-Activated Receptor Distinct From the Thrombin Receptor in Vascular Endothelial Cells

The thrombin receptor was the first cloned G protein-coupled receptor reported to be activated by proteolytic cleavage of its extracellular amino terminus. A second proteinase-activated receptor (PAR-2) was cloned recently and expressed in Xenopus laevis oocytes. PAR-2 was activated by trypsin and by a peptide (SLIGRL) derived from the new amino terminus. Since PAR-2 mRNA was detected in highly vascularized organs, we compared the physiological functions of the thrombin receptor and PAR-2 in vascular endothelium. Thrombin and trypsin both elicited endothelium-dependent relaxations in prostaglandin F2alpha (PGF2alpha)-contracted strips of porcine coronary artery. Whereas high doses of both thrombin or trypsin (10 U/mL) caused homologous desensitization, trypsin caused further relaxation of thrombin-desensitized tissues. Thrombin and PAR-2-derived peptides (SFLLRN and SLIGRL) both induced endothelium-dependent relaxations in PGF2alpha-contracted porcine coronary arteries. SFLLRN or SLIGRL (30 micronmol/L) also showed homologous desensitization but not cross desensitization. In the presence of the NO synthase inhibitor NG-monomethyl-L-arginine (1 mmol/L), both SFLLRN- and SLIGRL-induced relaxations were partially inhibited. SFLLRN elicited weak contraction in coronary arteries without endothelium, whereas SLIGRL had no effect. Intravenous injection of SFLLRN (1 mg/kg, bolus) into anesthetized rats elicited a transient depressor response followed by pronounced pressor response. In contrast, intravenous administration of SLIGRL (1 mg/kg, bolus) produced only a marked depressor response. Consistent with the in vivo data, SFLLRN contracted the endothelium-rubbed rat aortic rings and aggregated human platelets in vitro, whereas SLIGRL had no effect. The finding that both trypsin and SLIGRL induced endothelium-dependent relaxations indicates the presence of PAR-2 on endothelial cells. In addition, both trypsin and SLIGRL elicited relaxations in thrombin- or SFLLRN-desensitized tissue, suggesting that PAR-2 is distinct from thrombin receptor in vascular endothelium. The lack of PAR-2-mediated platelet aggregation or smooth muscle contraction suggested it might not share the pathogenic properties associated with the thrombin receptor in the vasculature.

Inhibition of endothelial derived relaxing factor (EDRF) aggravates ischemic acute renal failure in anesthetized rats

SummaryThe relative importance of endothelial derived relaxing factor (EDRF)/nitric oxide (NO) in maintaining kidney function in normal condition and in acute renal failure (ARF) were evaluated in inactin anesthetized rats. ARF was induced by unilateral occlusion of the left renal artery (40 min) followed by reperfusion, with the contralateral kidney serving as normal control. This protocol resulted in marked reductions in renal plasma flow (RPF), glomerular filtration rate (GFR) and increases in fractional sodium excretion (FENa) and urinary protein excretion in the post-ischemic kidney in comparison to the contralateral normal kidney. Administration of the nitric oxide (NO) synthase inhibitor NG — monomethyl-L-arginine (0.25 mg/kg per min, L-NMMA) exacerbated the ischemia-induced changes in renal functions as reflected by further reductions in urine flow (V), GFR, marked sodium wasting and renal edema. Pretreatment of the animals with NO precursor L-arginine (2.5 mg/kg per min, L-Arg) abolished the detrimental effects of L-NMMA in ARE In contrast, D-Arginine (2.5 mg/kg per min, DArg) failed to reverse the detrimental effects of L-NMMA. Infusion of L-Arg alone also resulted in improvements in RPF and GFR in the ischemic kidney. The results of the present study suggest that the function of the ischemic kidney is sustained by EDRF/NO and is thus more sensitive to NO synthase inhibition.

Antiplatelet and antiproliferative effects of SCH 51866, a novel type 1 and type 5 phosphodiesterase inhibitor.

SCH 51866 is a potent and selective PDE1 and PDE5 inhibitor. The antiplatelet, antiproliferative, and hemodynamic effects of SCH 51866 were compared with those of E4021, a highly selective PDE5 inhibitor. SCH 51866 inhibited PDE1 and PDE5 isozymes with a 50% inhibitory concentration (IC50) of 70 and 60 nM, respectively. SCH 51866 and E4021 inhibited washed human platelet aggregation induced by collagen with an IC50 of 10 and 4 microM, respectively, and attenuated (p < 0.05) the adhesion of 111indium-labeled platelets to the nylon filament-injured rat aorta. The doses of SCH 51866 and E4021 that inhibited platelet adhesion caused significant increases in platelet cyclic guanosine monophosphate (cGMP; p < 0.05). SCH 51866 (1-10 mg/kg, p.o. twice daily) but not E4021 (3-30 mg/kg, p.o twice daily) inhibited neointima formation in the carotid arteries of spontaneously hypertensive rats (SHRs) subjected to balloon angioplasty. Moreover, SCH 51866 (0.3-10 mg/kg, p.o.) elicited dose-dependent reduction in blood pressure in SHRs, whereas E4021 (3-30 mg/kg, p.o.) did not affect blood pressure in SHRs. In conclusion, the data suggest that inhibition of PDE1 and PDE5 isozymes by SCH 51866 exerts antiplatelet and vascular protective effects. In comparison, inhibition of PDE5 alone by E4021 exhibited antiplatelet effects without affecting neointima formation.

SCH 602539, a Protease-Activated Receptor-1 Antagonist, Inhibits Thrombosis Alone and in Combination With Cangrelor in a Folts Model of Arterial Thrombosis in Cynomolgus Monkeys

Objective—To determine the antithrombotic effects of SCH 602539, an analog of the selective protease-activated receptor (PAR)-1 antagonist vorapaxar (formerly SCH 530348) currently in advanced clinical development, and the P2Y12 ADP receptor antagonist cangrelor, alone and in combination. Methods and Results—Multiple platelet activation pathways contribute to thrombosis. The effects of SCH 602539 and cangrelor alone and in combination on cyclic flow reductions were evaluated in a Folts model of thrombosis in cynomolgus monkeys. The effects of these treatments on ex vivo platelet aggregation and coagulation parameters were also monitored. Dose-dependent inhibition of cyclic flow reductions was observed after treatment with SCH 602539 alone and cangrelor alone (P<0.05 versus vehicle for the 2 highest concentrations of each agent). The combination of SCH 602539 and cangrelor was associated with synergistic antithrombotic effects (P<0.05 versus vehicle for all combinations tested). The 2 highest doses of SCH 602539 inhibited platelet aggregation in response to PAR-1–selective high-affinity thrombin receptor agonist peptide by greater than 80% but did not affect platelet aggregation induced by other agonists; also, they did not affect any coagulation parameters. Conclusion—The combined inhibition of the PAR-1 and the P2Y12 ADP platelet activation pathways had synergistic antithrombotic and antiplatelet effects. The addition of a PAR-1 antagonist to a P2Y12 ADP receptor antagonist may provide incremental clinical benefits in patients with atherothrombotic disease, both in short- and long-term settings. These hypotheses need to be tested clinically.

Attenuation of Ischemic Acute Renal Failure by Phosphoramidon in Rats

The protective effects of phosphoramidon, a dual inhibitor of endothelin-converting enzyme and neutral endopeptidase (E.C. 24.11), on renal function in ischemic acute renal failure were investigated in anesthetized rats. Intravenous infusion of phosphoramidon (0.03 and 0.1 mg/kg per min) significantly suppressed tubular sodium wasting (measured by fractional excretion of sodium) and proteinuria in the postischemic kidney without modifying functional parameters in the contralateral normal kidney. Phosphoramidon (0.1 mg/kg/min) was associated with increased glomerular filtration in the ischemic kidney. In comparison, SCH 42354, a highly selective inhibitor of neutral endopeptidase at 0.3 mg/kg/min, did not inhibit endothelin-converting enzyme or afford renal protection. The data suggest that the protective action of phosphoramidon against ischemic acute renal failure is most likely mediated by inhibition of endothelin formation.

Design and synthesis of xanthine analogues as potent and selective PDE5 inhibitors.

We have discovered potent and selective xanthine PDE5 inhibitors. Compound 25 (PDE5 IC(50)=0.6 nM, PDE6/PDE5=101) demonstrated similar functional efficacy and PK profile to Sildenafil (PDE5 IC(50)=3.5 nM, PDE6/PDE5=7).

Discovery of SCH 900271, a Potent Nicotinic Acid Receptor Agonist for the Treatment of Dyslipidemia.

Structure-guided optimization of a series of C-5 alkyl substituents led to the discovery of a potent nicotinic acid receptor agonist SCH 900271 (33) with an EC50 of 2 nM in the hu-GPR109a assay. Compound 33 demonstrated good oral bioavailability in all species. Compound 33 exhibited dose-dependent inhibition of plasma free fatty acid (FFA) with 50% FFA reduction at 1.0 mg/kg in fasted male beagle dogs. Compound 33 had no overt signs of flushing at doses up to 10 mg/kg with an improved therapeutic window to flushing as compared to nicotinic acid. Compound 33 was evaluated in human clinical trials.

Inhibition of platelet adhesion and aggregation by E4021, a type V phosphodiesterase inhibitor, in guinea pigs

Abstract E4021 (sodium 1-[6-chloro-4-(3, 4-methylenedioxybenzyl)-aminoquinazolin-2-yl]piperidine-4-carboxylate sesquihydrate) is a highly selective and potent inhibitor of type V phosphodiesterase(PDE5). The in vitro and in vivo effect of E4021 on platelet function was evaluated, using echistatin, a potent disintegrin, as a positive reference agent. E4021 inhibits aggregatory response to collagen in washed human platelets (IC50 =5μM, vs. 0.14μM with echistatin). In the ex vivoplatelet aggregation assay using whole blood from treated guinea pigs, E4021 (9mg/kg i.v.) showed a moderate inhibition (43%) against collagen (0.125μg/ml), whereas echistatin (250μg/kg i.v.) exerted a 88% inhibition. The absence of endothelium-derived factors (NO) may account for the moderate in vitro and ex vivo antiplatelet activity of E4021. In an in vivo model of reversible platelet aggregation elicited by collagen (100μg/kg i.v.), both E4021 and echistatin attenuated the intrapulmonary platelet accumulation in guinea pigs (-36% and -44%, respectively). In addition, E4021 (9mg/kg i.v.) and echistatin (250μg/kg i.v.) caused a similar inhibition of platelet adhesion at sites of microfilament-induced vascular injury in guinea pigs (52% and 65%, respectively). The two agents in combination did not show additive effect, suggesting that E4021 inhibits platelet activation and impairs interactions of adhesion receptors with matrix proteins. E4021 caused a selective increase in cGMP concentrations in the platelets isolated from treated guinea pigs; cAMP was not affected. It is concluded that the antiplatelet activity of E4021 is mediated through cGMP mechanism by virtue of selective inhibition of PDE5 in the platelets.