The effect of dotinurad [(3,5-dichloro-4-hydroxyphenyl)(1,1dioxo-1,2-dihydro-3H-1l 6-1,3-benzothiazol-3-yl)methanone] was compared with that of commercially available uricosuric agents-namely, benzbromarone, lesinurad, and probenecid. Its effect on urate secretion transporters was evaluated using probe substrates for respective transporters. Dotinurad, benzbromarone, lesinurad, and probenecid inhibited urate transporter 1 (URAT1) with IC 50 values of 0.0372, 0.190, 30.0, and 165 mM, respectively. Dotinurad weakly inhibited ATP-binding cassette subfamily G member 2 (ABCG2), organic anion transporter 1 (OAT1), and OAT3, with IC 50 values of 4.16, 4.08, and 1.32 mM, respectively, indicating higher selectivity for URAT1. The hypouricemic effects of dotinurad and benzbromarone were evaluated in Cebus monkeys. Dotinurad, at doses of 1-30 mg/kg, concomitantly decreased plasma urate levels and increased fractional excretion of urate (FE UA) in a dose-dependent manner. On the contrary, benzbromarone, at a dose of 30 mg/kg, showed a modest effect on plasma urate levels. The inhibitory effect of dotinurad on urate secretion transporters was evaluated in Sprague-Dawley rats, with sulfasalazine and adefovir as probe substrates of ABCG2 and OAT1, respectively. Drugs, including febuxostat as a reference ABCG2 inhibitor, were administered orally before sulfasalazine or adefovir administration. Dotinurad had no effect on urate secretion transporters in vivo, whereas benzbromarone, lesinurad, probenecid, and febuxostat increased the plasma concentrations of probe substrates. These results suggested dotinurad is characterized as a selective urate reabsorption inhibitor (SURI), which is defined as a potent URAT1 inhibitor with minimal effect on urate secretion transporters, including ABCG2 and OAT1/3, because of its high efficacy in decreasing plasma urate levels compared with that of other uricosuric agents. SIGNIFICANCE STATEMENT Our study on the inhibitory effects on urate transport showed that dotinurad had higher selectivity for urate transporter 1 (URAT1) versus ATP-binding cassette subfamily G member 2 (ABCG2) and organic anion transporter (OAT) 1/3 compared to other uricosuric agents. In Cebus monkeys, dotinurad decreased plasma urate levels and increased fractional excretion of urate in a dose-dependent manner. To determine the inhibitory effect of dotinurad on urate secretion transporters, we studied the movement of substrates of ABCG2 and OAT1 in rats. Dotinurad had no effect on these transporters, whereas the other uricosuric agents increased the plasma concentrations of the substrates. These results suggested dotinurad as a potent and selective urate reabsorption inhibitor is characterized by increased efficacy with decreasing plasma urate levels. A part of the Cebus monkey studies in the work was previously presented at the 86th annual meeting of the Japanese Pharmacological Society (2013), Abstract number P2-143, Fukuoka, Japan.
Indoxyl sulfate (IS) accumulates in the body in chronic kidney disease (CKD). In the renal proximal tubules, IS excretion is mediated by OAT1/3 and ABCG2. These transporters are inhibited by some hypouricemic agents; OATs by probenecid and benzbromarone, ABCG2 by febuxostat and benzbromarone. Thus, we evaluated whether hypouricemic agents including dotinurad, a novel selective urate reabsorption inhibitor with minimal effect on OATs or ABCG2, affect IS clearance in rats. Intact and adenine-induced acute renal failure rats were orally administered hypouricemic agents, and both endogenous IS and exogenously administered stable isotope-labeled d4-IS in the plasma and kidney were measured. Our results demonstrated that OATs inhibitors, such as probenecid, suppress IS uptake into the kidney, leading to increased plasma IS concentration, whereas ABCG2 inhibitors, such as febuxostat, cause renal IS accumulation remarkably by suppressing its excretion in intact rats. The effects of these agents were reduced in adenine-induced acute renal failure rats, presumably due to substantial decrease in renal OAT1/3 and ABCG2 expression. Dotinurad did not significantly affected the clearance of IS under both conditions. Therefore, we suggest that hypouricemic agents that do not affect OATs and ABCG2 are effective therapeutic options for the treatment of hyperuricemia complicated by CKD.
Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.
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