GSK1562590, a slowly dissociating urotensin-II receptor antagonist, exhibits prolonged pharmacodynamic activity ex vivo.

BACKGROUND AND PURPOSE: Recently identified antagonists of the urotensin-II (U-II) receptor (UT) are of limited utility for investigating the (patho)physiological role of U-II due to poor potency and limited selectivity and/or intrinsic activity. EXPERIMENTAL APPROACH: The pharmacological properties of two novel UT antagonists, GSK1440115 and GSK1562590, were compared using multiple bioassays. KEY RESULTS: GSK1440115 (pK(i)= 7.34-8.64 across species) and GSK1562590 (pK(i)= 9.14-9.66 across species) are high affinity ligands of mammalian recombinant (mouse, rat, cat, monkey, human) and native (SJRH30 cells) UT. Both compounds exhibited >100-fold selectivity for UT versus 87 distinct mammalian GPCR, enzyme, ion channel and neurotransmitter uptake targets. GSK1440115 showed competitive antagonism at UT in arteries from all species tested (pA(2)= 5.59-7.71). In contrast, GSK1562590 was an insurmountable UT antagonist in rat, cat and hUT transgenic mouse arteries (pK(b)= 8.93-10.12 across species), but a competitive antagonist in monkey arteries (pK(b)= 8.87-8.93). Likewise, GSK1562590 inhibited the hU-II-induced systemic pressor response in anaesthetized cats at a dose 10-fold lower than that of GSK1440115. The antagonistic effects of GSK1440115, but not GSK1562590, could be reversed by washout in rat isolated aorta. In ex vivo studies, GSK1562590 inhibited hU-II-induced contraction of rat aorta for at least 24 h following dosing. Dissociation of GSK1562590 binding was considerably slower at rat than monkey UT. CONCLUSIONS AND IMPLICATIONS: Whereas both GSK1440115 and GSK1562590 represent high-affinity/selective UT antagonists suitable for assessing the (patho)physiological role of U-II, only GSK1562590 exhibited sustained UT residence time and improved preclinical efficacy in vivo.

Palosuran inhibits binding to primate UT receptors in cell membranes but demonstrates differential activity in intact cells and vascular tissues.

BACKGROUND AND PURPOSE: The recent development of the UT ligand palosuran (1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulphate salt) has led to the proposition that urotensin-II (U-II) plays a significant pathological role in acute and chronic renal injury in the rat. EXPERIMENTAL APPROACH: In the present study, the pharmacological properties of palosuran were investigated further using a series of radioligand binding and functional bioassays. KEY RESULTS: Palosuran functioned as a 'primate-selective' UT ligand in recombinant cell membranes (monkey and human UT K(i) values of 4 +/- 1 and 5 +/- 1 nM), lacking appreciable affinity at other mammalian UT isoforms (rodent and feline K(i) values >1 microM). Paradoxically, however, palosuran lost significant (10- to 54-fold) affinity for native and recombinant human UT when radioligand binding was performed in intact cells (K(i) values of 50 +/- 3 and 276 +/- 67 nM). In accordance, palosuran also exhibited diminished activity in hUT (human urotensin-II receptor)-CHO (Chinese hamster ovary) cells (IC50 323 +/- 67 nM) and isolated arteries (K(b)>10 microM in rat aorta; K(b)>8.5 microM in cat arteries; K(b)>1.6 microM in monkey arteries; K(b) 2.2 +/- 0.6 microM in hUT transgenic mouse aorta). Relative to recombinant binding K(i) values, palosuran was subjected to a 392- to 690-fold reduction in functional activity in monkey isolated arteries. Such phenomena were peculiar to palosuran and were not apparent with an alternative chemotype, SB-657510 (2-bromo-N-[4-chloro-3-((R)-1-methyl-pyrrolidin-3-yloxy)-phenyl]-4,5-dimethoxybenzenesulphonamide HCl). CONCLUSIONS AND IMPLICATIONS: Collectively, such findings suggest that caution should be taken when interpreting data generated using palosuran. The loss of UT affinity/activity observed in intact cells and tissues cf. membranes offers a potential explanation for the disappointing clinical efficacy reported with palosuran in diabetic nephropathy patients. As such, the (patho)physiological significance of U-II in diabetic renal dysfunction remains uncertain.

Secretion of collagen by corneal epithelium. I. Morphology of the collagenous products produced by isolated epithelia grown on frozen-killed lens.

Corneal epithelium from 5-7-day old chick embryos was isolated with EDTA and grown in culture on frozen-killed lens as a substratum. Autoradiographs showed that in the presence of [(3)H]proline, the corneal epithelium synthesized and secreted onto the lens substratum, radioactive materials resistant to extraction by sodium hydroxide. The radioactive label was associated with newly formed striated collagen fibrils, large "sheets" of collagen, and basal lamina. The repeat period and interband pattern of the abundant new collagen sheets and fibrils was typical of "native" or so-called "mesenchymal" collagen. Collagen-like materials were observed in secretory (Golgi) vacuoles within the corneal cells and collagen fibrils within the intercellular canals (lateral interfaces) of the epithelium, as well as at the base of the cells. Both the granular endoplasmic reticulum and Golgi complexes were highly developed in the corneal epithelium. In the discussion, the role of cytoplasmic organelles in collagen secretion, the origin and structure of the basal lamina, and variations in collagen polymerization patterns in vitro are reviewed and evaluated. The morphogenetic significance of the synthesis and secretion of collagen by embryonic epithelium is appraised and the production of true native-striated collagen by epithelium is stressed.