Utilizing conformational constraints in conjunction with various structural considerations, we have synthesized a series of cyclic disulfide peptides that are highly potent and selective antagonists for the platelet integrin alpha IIb beta 3 (GPIIb/IIIa). The affinities of the peptides for alpha IIb beta 3 were determined by platelet aggregation assays and an alpha IIb beta 3 ELISA. Their affinities for alpha 5 beta 1 and alpha v beta 5 integrins were also determined in respective ELISA assays. Structure-activity relationship studies suggest that R-G-D-Ar-R (Ar = hydrophobic residue) is the essential pharmacophore that is responsible for their high alpha IIb beta 3 binding affinity, very high selectivity, and distinct biological properties. One of these analogues, TP9201, has been shown to inhibit platelet-mediated thrombus formation without associated prolongation of template bleeding time. The arginine residue adjacent the carboxy terminus of the R-G-D-Ar sequence could function as the biological effector element that determines this distinct and unexpected biological property.
Vascular calcification is a mortality risk factor for stage 5 chronic kidney disease patients. We investigated the role of phosphorus and vitamin D analogs in the pathogenesis of vascular calcification using in vivo, ex vivo, and in vitro models. Our results demonstrate that uremic rats receiving a hyperphosphatemia-inducing diet did not exhibit aortic calcification despite elevated levels of serum phosphorus and calciumphosphorus (CaxP) product. The vitamin D analog 1␣-hydroxyvitamin-D 2 [1␣(OH)D 2 ] at 0.17 g/kg raised serum calcium, phosphorus, CaxP product, and aortic calcification in the uremic rats, but 19-nor-1␣,25(OH) 2 D 2 (19-nor) at the same dose had no significant effect. At 0.67 g/kg, both 1␣(OH)D 2 and 19-nor had similar effects on serum calcium, phosphorus, and CaxP product, but only 1␣(OH)D 2 induced significant aortic calcification. Only aortic rings from 1␣(OH)D 2 -treated uremic rats exhibited a significant increase in 45 Ca uptake ex vivo. When aortic rings from normal rats or a primary culture of human coronary artery smooth muscle cells were treated with phosphorus or vitamin D analogs in vitro, high phosphorus induced calcium accumulation and/or 45 Ca uptake in a dose-or time-dependent manner, whereas vitamin D analogs including 1␣(OH)D 2 up to 100 nM had no significant effect despite the presence of a functional vitamin D receptor. However, serum from 1␣(OH)D 2 -treated uremic rats induced 45 Ca uptake into smooth muscle cells cultured in high phosphorus. These results suggest that the regulation of vascular calcification in vivo cannot be easily replicated in the ex vivo or in vitro models, and high phosphorus and some vitamin D analogs such as 1␣(OH)D 2 exert interactive effects on modulating vascular calcification.
A polyclonal antiserum to platelet membrane glycoprotein GPIIb/IIEa was used to detect antigenically related molecules on a diverse panel of human cells. Umbilical vein eadothelial cells, erythroleukemic HEL cells, and diploid fetal lung GM1380 fibroblasts expressed GPIIb/HIa-related molecules, as judged by immunofluorescence and immunoprecipitation of surface-labeled proteins. The GPIIb and GPMa subunits were both present and were of similar molec. ular weight in these cell types. These molecules were synthetic products of the cells, as shown by immunoprecipitation of intrinsically labeled proteins. Promyeloid U937 cells could be induced by 4p-phorbol 12-myristate 13-acetate to synthesize and express GP1Ib/IIIa-related molecules on their cell surface. The GPIIb/Hlla-related molecules were not precisely identical in the various cell types, based on slight differences in electrophoretic mobility and their failure to react with monoclonal antibodies specific for each subunit of platelet GPIIb/ma. These results suggest the existence of a widely distributed family of GPTIb/Illa-related molecules. This family of "cytoadhesins" may share a common function in cellular adhesive reactions.
We have discovered a novel class of endothelin (ET) receptor antagonists through pharmacophore analysis of the existing non-peptide ET antagonists. On the basis of this analysis, we determined that a pyrrolidine ring might replace the indian ring in SB 209670. The resultant compounds were readily prepared and amenable to extensive SAR studies. Thus a series of N-substituted trans,trans-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)pyrroli din e-3- carboxylic acids (8) have been synthesized and evaluated for binding at ET(A) and ET(B) receptors. Compounds with N-acyl and simple N-alkyl substituents had weak activity. Compounds with N-alkyl substituents containing ethers, sulfoxides, or sulfones showed increased activity. Much improved activity resulted from compounds where the N-substituents were acetamides. Compound 17u (A-127722) with the N,N-dibutylacetamide substituent is the best of the series. It has an IC(50)=0.36 nM for inhibition of ET-1 radioligand binding at the ET(A) receptor, with a 1000-fold selectivity for the ET(A) vs the ET(B) receptor. It is also a potent inhibitor (IC(50)=0.16 nM) of phosphoinositol hydrolysis stimulated by ET-1, and it antagonized the ET-1-induced contraction of the rabbit aorta with a pA(2)=9.20. The compound has 70% oral bioavailability in rats.
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