Takashi Iwanaga

INVOLVEMENT OF URIC ACID TRANSPORTER IN INCREASED RENAL CLEARANCE OF THE XANTHINE OXIDASE INHIBITOR OXYPURINOL INDUCED BY A URICOSURIC AGENT, BENZBROMARONE

Benzbromarone has been reported to increase the renal clearance of oxypurinol, an active metabolite of allopurinol. We examined the renal transport of oxypurinol to determine whether such a change in renal clearance could be explained by altered transporter-mediated reabsorption. Since the first step of reabsorption takes place at the renal epithelial apical membrane, we focused on membrane transporters. Benzbromarone is an inhibitor of reabsorption of uric acid mediated by the uric acid transporter (URAT) URAT1 (SLC22A12), which is expressed at the apical membrane of proximal tubular cells in humans. Uptake of oxypurinol by Xenopus oocytes injected with complementary RNA of URAT1 was significantly higher than that by water-injected oocytes, and the uptake was saturable, with a Km of about 800 μM. Moreover, benzbromarone inhibited the oxypurinol uptake by URAT1 at concentrations as low as 0.01 μM. The uptake of oxypurinol by another organic anion transporter (OAT), OAT4 (SLC22A11), which is also expressed at the apical membrane of proximal tubular epithelial cells, was negligible, whereas the uptake of [3H]estrone-3-sulfate by OAT4 was significantly inhibited by oxypurinol. Furthermore, neither the transport activity of organic cation/carnitine transporter (OCTN) 1 nor OCTN2 was affected by oxypurinol or benzbromarone. These results indicate that URAT1 is involved in renal reabsorption of oxypurinol, and the increment of renal clearance of oxypurinol upon concomitant administration of benzbromarone could be due to drug interaction at URAT1.

Concentration-dependent mode of interaction of angiotensin II receptor blockers with uric acid transporter.

Serum uric acid (SUA) is currently recognized as a risk factor for cardiovascular disease. It has been reported that an angiotensin II receptor blocker (ARB), losartan, decreases SUA level, whereas other ARBs, such as candesartan, have no lowering effect. Because the renal uric acid transporter (URAT1) is an important factor controlling the SUA level, we examined the involvement of URAT1 in those differential effects of various ARBs on SUA level at clinically relevant concentrations. This study was done by using URAT1-expressing Xenopus oocytes. Losartan, pratosartan, and telmisartan exhibited cis-inhibitory effects on the uptake of uric acid by URAT1, whereas at higher concentrations, only telmisartan did, and these ARBs reduced the uptake in competitive inhibition kinetics. On the other hand, candesartan, EXP3174 [2-n-butyl-4-chloro-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yI)methyl]imidazole-5-carboxylic acid] (a major metabolite of losartan), olmesartan, and valsartan were not inhibitory. Preloading of those ARBs in the oocytes enhanced the URAT1-mediated uric acid uptake, showing a trans-stimulatory effect. The present study is a first demonstration of the differential effects of ARBs on URAT1 that some ARBs are both cis-inhibitory and trans-stimulatory, depending on concentration, whereas others exhibit either a trans-stimulatory or cis-inhibitory effect alone, which could explain the clinically observed differential effects of ARBs on SUA level. Furthermore, it was found that such differential effects of ARBs on URAT1 could be predicted from the partial chemical structures of ARBs, which will be useful information for the appropriate use and development of ARBs without an increase of SUA.

Discovery of 3-(3-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole, FYX-051-a xanthine oxidoreductase inhibitor for the treatment of hyperuricemia

Our previous study identified 2-[2-(2-methoxyethoxy)ethoxy]-5-[5-(2-methyl-4-pyridyl)-1H-[1,2,4] triazol-3-yl]benzonitrile (2)[corrected]as a safe and potent xanthine oxidoreductase (XOR) inhibitor for the treatment of hyperuricemia. Here, we synthesized a series of 3,5-dipyridyl-1,2,4-triazole derivatives and, in particular, examined their in vivo activity in lowering the serum uric acid levels in rats. As a result, we identified 3-(2-cyano-4-pyridyl)-5-(4-pyridyl)-1,2,4-triazole (FYX-051, compound 39) [corrected] to be one of the most potent XOR inhibitors; it exhibited an extremely potent in vivo activity, weak CYP3A4-inhibitory activity and a better pharmacokinetic profile than compound 2. Compound 39 is currently being evaluated in a phase 2 clinical trial.

Design, synthesis, and pharmacological and pharmacokinetic evaluation of 3-phenyl-5-pyridyl-1,2,4-triazole derivatives as xanthine oxidoreductase inhibitors

In an effort to find a potent xanthine oxidoreductase (XO) inhibitor, we discovered the best compound 2-[2-(2-methoxy-ethoxy)-ethoxy]-5-[5-(2-methyl-pyridin-4-yl)-1H-[1,2,4]triazol-3-yl]-benzonitrile 28. Here, we describe the following: (1) the design, synthesis, and structure-activity relationship of a series of 3-phenyl-5-pyridyl-1,2,4-triazole derivatives by in vitro studies of XO inhibitory activity in bovine milk and in vivo studies of serum uric acid (UA) reductive activity in rats, (2) a drug interaction study by a cytochrome P450 3A4 (CYP3A4) assay, and (3) a pharmacokinetic (PK) study. Compound 28 exhibits potent XO inhibitory activity, serum UA-lowering activity in rats, weak CYP3A4 inhibitory activity, and moderate PK profile.

Metabolic profile of FYX-051 (4-(5-pyridin-4-yl-1h-[1,2,4]triazol-3-yl)pyridine-2-carbonitrile) in the rat, dog, monkey, and human: identification of N-glucuronides and N-glucosides.

FYX-051, 4-(5-pyridin-4-yl-1H-[1,2,4]triazol-3-yl)pyridine-2-carbonitrile, is a novel xanthine oxidoreductase inhibitor that can be used for the treatment of gout and hyperuricemia. We examined the metabolism of FYX-051 in rats, dogs, monkeys, and human volunteers after the p.o. administration of this inhibitor. The main metabolites in urine were pyridine N-oxide in rats, triazole N-glucoside in dogs, and triazole N-glucuronide in monkeys and humans, respectively. Furthermore, N-glucuronidation and N-glucosidation were characterized by two types of conjugation: triazole N1- and N2-glucuronidation and N1- and N2-glucosidation, respectively. N1- and N2-glucuronidation was observed in each species, whereas N1- and N2-glucosidation was mainly observed in dogs. With regard to the position of conjugation, N1-conjugation was predominant; this resulted in a considerably higher amount of N1-conjugate in each species than N2-conjugate. The present results indicate that the conjugation reaction observed in FYX-051 metabolism is unique, i.e., N-glucuronidation and N-glucosidation occur at the same position of the triazole ring, resulting in the generation of four different conjugates in mammals. In addition, a urinary profile of FYX-051 metabolites in monkeys and humans was relatively similar; triazole N-glucuronides were mainly excreted in urine.

Effects of topiroxostat and febuxostat on urinary albumin excretion and plasma xanthine oxidoreductase activity in db/db mice.

Topiroxostat, a xanthine oxidoreductase (XOR) inhibitor, has been shown to decrease the urinary albumin-to-creatinine ratio compared with placebo in hyperuricemic patients with stage 3 chronic kidney disease. Thus, we aimed to ascertain the albuminuria-lowering effect of topiroxostat in diabetic mouse. Db/db mice were fed standard diets with or without topiroxostat (0.1, 0.3, 1, and 3mg/kg/day) and febuxostat (0.1, 0.3, and 1mg/kg/day) for four weeks. Urinary albumin and purine bodies levels, XOR activities, and drug concentrations in the liver, kidney, and plasma were measured. Moreover, the XOR inhibitory activity of each XOR inhibitor was evaluated with or without an exogenous protein in vitro. Topiroxostat decreased dose-dependently the urinary albumin excretion, but febuxostat did not show such a tendency. Treatment with topiroxostat inhibited plasma XOR activity with dose-dependent increase in plasma purine levels, which was not observed by febuxostat. Pharmacokinetic/pharmacodynamic analysis revealed that topiroxostat and febuxostat concentration in each tissue showed a good correlation with both the hypouricemic effect and plasma drug concentration, whereas the change in albuminuria correlated neither with the change in uric acid nor with drug concentration in plasma. However, the change in urinary albumin and plasma XOR activity showed good correlation in topiroxostat group. The 50% inhibitory concentration (IC50 value) of febuxostat against plasma XOR in vitro was 12-fold higher than that of topiroxostat, and increased by approximately 13-fold by interfering with an exogenous protein. Topiroxostat caused reduced urinary albumin excretion, in which potent inhibition of the plasma XOR activity might be involved.

Characterization of N-glucuronidation of 4-(5-pyridin-4-yl-1H-[1,2,4]triazol-3-yl) pyridine-2-carbonitrile (FYX-051): a new xanthine oxidoreductase inhibitor.

In humans, orally administered 4-(5-pyridin-4-yl-1H-[1,2,4]triazol-3-yl) pyridine-2-carbonitrile (FYX-051) is excreted mainly as triazole N1- and N2-glucuronides in urine. It is important to determine the enzyme(s) that catalyze the metabolism of a new drug to estimate individual differences and/or drug-drug interactions. Therefore, the characterization and mechanism of these glucuronidations were investigated using human liver microsomes (HLMs), human intestinal microsomes (HIMs), and recombinant human UDP-glucuronosyltransferase (UGT) isoforms to determine the UGT isoform(s) responsible for FYX-051 N1- and N2-glucuronidation. FYX-051 was metabolized to its N1- and N2-glucuronide forms by HLMs, and their Km values were 64.1 and 72.7 μM, respectively; however, FYX-051 was scarcely metabolized to its glucuronides by HIMs. Furthermore, among the recombinant human UGT isoforms, UGT1A1, UGT1A7, and UGT1A9 catalyzed the N1- and N2-glucuronidation of FYX-051. To estimate their contribution to FYX-051 glucuronidation, inhibition analysis with pooled HLMs was performed. Mefenamic acid, a UGT1A9 inhibitor, decreased FYX-051 N1- and N2-glucuronosyltransferase activities, whereas bilirubin, a UGT1A1 inhibitor, did not affect these activities. Furthermore, in the experiment using microsomes from eight human livers, the N1- and N2-glucuronidation activity of FYX-051 was found to significantly correlate with the glucuronidation activity of propofol, a specific substrate of UGT1A9 (N1: r2 = 0.868, p < 0.01; N2: r2 = 0.775, p < 0.01). These results strongly suggested that the N1- and N2-glucuronidation of FYX-051 is catalyzed mainly by UGT1A9 in human livers.

FYX-051, a Xanthine Oxidoreductase Inhibitor, Induces Nephropathy in Rats, but not in Monkeys

The present studies were performed to investigate the possible mechanism of marked species differences on nephropathy found in the long-term toxicity study of FYX-051, a xanthine oxidoreductase inhibitor. In the twenty-six-week dose toxicity study in the rat, in which FYX-051 was administered by oral gavage at 0.04, 0.2, and 1 mg/kg, xanthine-mediated nephropathy was seen only at 1 mg/kg, despite the presence of xanthine crystals in urine at 0.2 mg/kg and more; however, in the fifty-two-week dose toxicity study in the monkey, in which FYX-051 was administered by oral gavage at 30, 100, and 300 mg/kg, no toxicities were seen, even at 300 mg/kg. These outcomes showed there would be 1500-fold or more differences in the mode of intrarenal xanthine deposition between rats and monkeys. Thus we performed the mechanistic study, and the following outcomes were obtained. First, the amount of urinary purine metabolites was thirty-fold higher in rats than in monkeys. Second, urinary xanthine solubility was sixfold higher in monkeys than in rats. Third, exposure levels of FYX-051 were five-fold higher in rats than in monkeys. Therefore, the present study indicated that the combined effects of purine metabolism, urinary xanthine solubility, and toxicokinetics would contribute to species differences in nephropathy, that is, absence of xanthine-mediated nephropathy in monkeys even at the highest dose of FYX-051.

Uricosuric agents decrease the plasma urate level in rats by concomitant treatment with topiroxostat, a novel xanthine oxidoreductase inhibitor

The aim of this study was to establish the rat model for evaluating hypouricemic effects by some uricosuric agents.