Bruce P. Damiano

Altered Vascular Injury Responses in Mice Deficient in Protease-Activated Receptor-1

Expression of protease-activated receptor-1 (PAR-1), a cell-surface receptor for thrombin, is increased in balloon-injured rat carotid artery and human atherosclerotic tissue. To examine the role of PAR-1 in vascular injury, we compared vascular injury responses in wild-type (WT) and PAR-1-deficient (PAR-1(-/-)) mice. Arterial injury was induced by inserting a flexible guidewire into the common carotid artery and withdrawing it 6 times with rotation. Bromodeoxyuridine, delivered subcutaneously by osmotic minipump, was used to measure cellular proliferation. Mice were perfusion-fixed at 1, 2, 5, 10, and 14 days after injury. Extensive endothelial damage, mural thrombosis, platelet adherence, and medial smooth muscle cell loss and necrosis were apparent at day 1 in both WT and PAR-1(-/-) mice. The incidence of thrombosis or platelet deposition in WT and PAR-1(-/-) mice declined from 100% at day 1 to 25% and 21%, respectively, at 14 days. Endothelial disruption, as assessed by Evans blue uptake, was maximum at day 1 and declined by day 14. This apparent endothelial regrowth was similar in WT and PAR-1(-/-) mice. Significant medial thickening at 14 days after injury was similar in WT (from 22.8+/-1.7 to 30.7+/-1.9 microm) and PAR-1(-/-) (from 23.2+/-2.1 to 30.5+/-2.2 microm) mice. Medial area also increased in response to injury but to a lesser extent in PAR-1(-/-) mice (from 0.0250+/-0.0044 to 0.0312+/-0.0047 mm(2)) than in WT mice (from 0.0266+/-0.0040 to 0.0398+/-0.0050 mm(2)). Neointima was variable and occurred in 6 of 13 WT and 5 of 12 PAR-1(-/-) mice. However, intimal area tended to be less in PAR-1(-/-) mice (0. 0016+/-0.0007 mm(2)) compared with WT mice (0.0082+/-0.0032 mm(2)), although this difference did not achieve statistical significance (P=0.06). Cell density was significantly greater in normal carotids from PAR-1(-/-) (6.4+/-0.5 x 10(3)/mm(2)) compared with WT (4.3+/-0. 8 x 10(3)/mm(2)) mice and remained elevated after injury. Vessel and lumen diameters tended to increase in WT mice after injury, whereas vessel diameter was unchanged and lumen diameter actually decreased in PAR-1(-/-) mice. Cell proliferation in injured carotid arteries was similar in PAR-1(-/-) and WT mice. These data suggest that PAR-1(-/-) may play a role in vascular injury responses in this mouse model via possible effects on extracellular matrix regulation.

Mer Receptor Tyrosine Kinase Signaling Participates in Platelet Function

Objective—Recently, mice made deficient in growth arrest–specific gene 6 product (Gas6) or in which Gas6 gene expression was inhibited were shown to have platelet dysfunction and to be less susceptible to thrombosis. The aim of this study was to define and characterize the relevant Gas6 receptor or receptors involved in platelet function. Methods and Results—Using RT-PCR and Western blot analysis we found that mer was the predominantly expressed subtype in mouse and human platelets, whereas axl and rse were not detected. We generated mer-deficient mice by targeted disruption of the mer receptor gene. Platelets derived from mer-deficient mice had decreased platelet aggregation in responses to low concentrations of collagen, U46619, and PAR4 thrombin receptor agonist peptide in vitro. However, the response to ADP was not different from wild-type platelets. Knockout of the mer gene protected mice from collagen/epinephrine–induced pulmonary thromoembolism and inhibited ferric chloride–induced thrombosis in vivo. Tail bleeding times, coagulation parameters, and peripheral blood cell counts in mer-deficient mice were similar to wild-type mice. Conclusion—Our data provide the first evidence that mer, presumably through activation by its ligand Gas6, participates in regulation of platelet function in vitro and platelet-dependent thrombosis in vivo.

Receptor-activating peptides distinguish thrombin receptor (PAR-1) and protease activated receptor 2 (PAR-2) mediated hemodynamic responses in vivo

Vascular expression and cellular functions of the thrombin receptor (PAR-1) and protease activated receptor 2 (PAR-2) suggest similar but distinct vascular regulatory roles. The vascular actions of PAR-1 and PAR-2 in vivo were differentiated by monitoring mean arterial pressure (MAP) and heart rate (HR) of anesthetized mice in response to intravenous SFLLRN (0.1, 0.3, and 1 mumol/kg) and SLIGRL (0.1, 0.3, and 1 mumol/kg), the respective receptor-activating sequences for PAR-1 and PAR-2, and TFLLRNPNDK (0.3, 1, and 3 mumol/kg), a synthetic peptide selective for PAR-1. All peptides dose dependently decreased MAP (order of potency: SLIGRL > SFLLRN > TFLLRNPNDK). SLIGRL induced a more prolonged hypotension with a slow return to baseline, whereas SFLLRN- and TFLLRNPNDK-induced hypotension was followed by a rapid return towards baseline and a sustained moderate hypotension. SFLLRN and TFLLRNPNDK, but not SLIGRL, decreased HR. N omega-Nitro-L-arginine methyl ester HCl (L-NAME), an inhibitor of nitric oxide synthesis, attenuated the cumulative hypotensive response to SLIGRL but had no effect on the SFLLRN and TFLLRNPNDK hypotension. However, L-NAME revealed a rebound hypertension in response to SFLLRN and TFLLRNPNDK but not SLIGRL. In conclusion, activation of either PAR-1 or PAR-2 in vivo results in hypotension. In addition, only PAR-1 activation induced hypertension following L-NAME, reflecting concurrent PAR-1-mediated vasoconstriction. Thus, these different hemodynamic responses in vivo suggest distinct physiological or pathophysiological roles for PAR-1 and PAR-2 in local vascular regulation.

Nonpeptide Urotensin-II Receptor Antagonists: A New Ligand Class Based on Piperazino-Phthalimide and Piperazino-Isoindolinone Subunits

We have discovered two related chemical series of nonpeptide urotensin-II (U-II) receptor antagonists based on piperazino-phthalimide (5 and 6) and piperazino-isoindolinone (7) scaffolds. These structure types are distinctive from those of U-II receptor antagonist series reported in the literature. Antagonist 7a exhibited single-digit nanomolar potency in rat and human cell-based functional assays, as well as strong binding to the human U-II receptor. In advanced pharmacological testing, 7a blocked the effects of U-II in vitro in a rat aortic ring assay and in vivo in a rat ear-flush model. A discussion of U-II receptor antagonist pharmacophores is presented, and a specifically defined model is suggested from tricycle 13, which has a high degree of conformational constraint.

Characterization of functional urotensin II receptors in human skeletal muscle myoblasts: comparison with angiotensin II receptors

The properties of urotensin II (U-II) receptor (UT receptor) and angiotensin II (ANG II) receptor (AT receptor) in primary human skeletal myoblasts (HSMM) and differentiated skeletal myotubes (HSMMT) were characterized. Radiolabeled U-II and ANG II bound specifically to HSMM with Kds of 0.31 nM (2311 receptors/cell) and 0.61 nM (18,257 receptors/cell), respectively. The cyclic segment of U-II peptide, CFWKYC, was the minimal sequence required for binding, with the WKY residues essential. Inhibitor studies suggested AT1 is the predominant ANG II receptor. After radioligand binding, under conditions designed to minimize receptor internalization, half the bound U-II was resistant to acid washing suggesting that U-II binds tightly to its receptor in a quasi-irreversible fashion. The AT1 receptor-bound radioligand was completely removed under the same conditions. RT-PCR detected the expression of mRNAs for UT and AT1 receptors. Western blotting showed that U-II and ANG II signaled via ERK1/2 kinase. UT receptor was not lost upon differentiation into myotubes since both mRNA for UT receptor and U-II binding were still present. ANG II receptors were also present as shown by ANG II-induced calcium mobilization.

1,2,4-Triazolo[3,4-a]pyridine as a novel, constrained template for fibrinogen receptor (GPIIb/IIIa) antagonists

Conformationally constrained analogues of the GPIIb/IIIa antagonist elarofiban (RWJ-53308) have been synthesized and biologically evaluated. The 1,2,4-triazolo[3,4-a]pyridine scaffold provided potent antagonists with favorable pharmacodynamic and pharmacokinetic attributes in dogs. Compounds 12a and 13a exhibited enhancements in oral bioavailability, t(1/2), and ex vivo duration of action (inhibition of ADP-induced platelet aggregation) relative to elarofiban.

Design, Synthesis, and Biological Evaluation of (2R,αS)-3,4-Dihydro-2-[3-(1,1,2,2-tetrafluoroethoxy)phenyl]-5-[3-(trifluoromethoxy)-phenyl]-α-(trifluoromethyl)-1(2H)-quinolineethanol as Potent and Orally Active Cholesteryl Ester Transfer Protein Inhibitor

With the goal of identifying a CETP inhibitor with high in vitro potency and optimal in vivo efficacy, a conformationally constrained molecule was designed based on the highly potent and flexible 13. The synthetic chemistry efforts led to the discovery of the potent and selective 12. In high-fat fed hamsters, human CETP transgenic mice, and cynomolgus monkeys, the in vivo efficacy of 12 for raising HDL-C was demonstrated to be comparable to torcetrapib.

Solid-phase parallel synthesis applied to lead optimization: Discovery of potent analogues of the GPIIb/IIIa antagonist RWJ-50042

Abstract A study of β-turn peptide mimetics, related to the C-terminal γ-chain of fibrinogen and containing a nipecotic acid scaffold, led to RWJ-50042 (1), an interesting fibrinogen receptor (GPIIb/IIIa) antagonist. To enhance potency, we employed solid-phase parallel synthesis for the preparation of over 200 analogues in a protocol of optimization cycles. This strategy produced several promising nipecotamide analogues, such as 25, which is 35 times more potent than 1 in vitro.

Discovery and clinical evaluation of 1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-[2,2-difluoro-2-phenylethylamino]pyrazinone (RWJ-671818), a thrombin inhibitor with an oxyguanidine P1 motif.

We have identified RWJ-671818 (8) as a novel, low molecular weight, orally active inhibitor of human alpha-thrombin (K(i) = 1.3 nM) that is potentially useful for the acute and chronic treatment of venous and arterial thrombosis. In a rat deep venous thrombosis model used to assess antithrombotic efficacy, oral administration of 8 at 30 and 50 mg/kg reduced thrombus weight by 87 and 94%, respectively. In an anesthetized rat antithrombotic model, where electrical stimulation of the carotid artery created a thrombus, 8 prolonged occlusion time 2- and 3-fold at 0.1 and 1.0 mg/kg, i.v., respectively, and more than doubled activated clotting time and activated partial thromboplastin time at the higher dose. This compound had excellent oral bioavailability of 100% in dogs with an estimated half-life of approximately 3 h. On the basis of its noteworthy preclinical data, 8 was advanced into human clinical trials and successfully progressed through phase 1 studies.

Orally efficacious thrombin inhibitors with cyanofluorophenylacetamide as the P2 motif

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K(i)=1.2nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.