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

Abstract

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 41 mM). 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 (IC 50 323 ± 67 nM) and isolated arteries (K b 410 mM in rat aorta; K b 48.5 mM in cat arteries; K b 41.6 mM in monkey arteries; K b 2.2±0.6 mM 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.

Role of urotensin II in health and disease

“…In a subsequent study in type 2 diabetics, palosuran demonstrated no clinically significant effects on ␤-cell secretory capacity and no effects on first-and secondphase insulin response (109). The reason for these disappointing results has been largely ascribed to a poor dosing regimen, as 125 mg of palosuran twice daily translates to a peak plasma concentration of Ͻ 260 nM, Ͼ 10-fold lower than the reported UT affinity for palosuran in mammals (8).…”

“…Thus, palosuran may help to alleviate portal hypertension (131). Despite the results, it was proposed that in the rat study on renal ischemia-reperfusion, only approximately half the dose necessary for efficacy was given (8), as a dose of 10 mg/(kg·h) translates to a plasma concentration of only 5 M (123). In addition, it should be noted that the binding affinity of palosuran in rats is very low (123).…”

Development of Filtration-Based Time-Resolved Fluorescence Assay for the High-Throughput Screening of Urotensin II Receptor Antagonist

“…These IC 50 values were very close to those from the radiolignad-binding assay (2.0 nM). 19 Excellent correlation coefficients were obtained in both assay methods (0.98 and 0.97 for FP-and TRF-based UT receptor binding assays, respectively) and the Hill slopes were evaluated as -1.3 for FP-based UT receptor binding assay and -1.7 for TRF-based binding assay. These results imply that current FP-and TRF-based UT receptor binding assays can mimic the classical detection results as in the radioligand receptor binding assay without numerous limitations of isotopic experiments.…”

“…For FP-based receptor binding assay, an FITC-labeled human recombinant UII (FITCGlu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val) was obtained from Phoenix Pharmaceuticals, Inc. (Burlingame, CA). As UT receptor selective reference compounds, 1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulfate salt (palosuran) 18 and 2-bromo-N-[4-chloro-3-((R)-1-methyl-pyrrolidin-3-yloxy)-phenyl]-4,5-dimethoxybenzenesulphonamide HCl (SB657510) 19 were synthesized in-house. Pluronic F-127 was purchased from Invitrogen (Carlsbad, CA) and 1 M MgCl 2 was obtained from Amresco, Inc. (Solon, OH).…”

The urotension II antagonist SB-710411 arrests fibrosis in CCL4 cirrhotic rats

“…However, the existence of marked differences between the prior investigation and the current study regarding the antagonist used (SB-710411 vs. palosuran), the experimental model (ccl 4 vs. bile duct ligation rats) and the experimental design (preventive effect for 8 weeks vs. therapeutic effect for 3 days) makes it difficult to identify the reason for this discrepancy. Moreover, responses to palosuran cannot be replicated with alternate uT antagonists, such as SB-710411 (41). Although drug efficacy cannot be directly compared because of variations in study parameters, it is evident that SB-710411 attenuated fibrosis in a fashion similar to other therapeutic modalities, including angiotensin-converting enzyme inhibitors.…”