Xanthine crystals induced by topiroxostat, a xanthine oxidoreductase inhibitor, in rats, cause transitional cell tumors.

The present study was performed to elucidate the underlying mechanism of transitional cell tumors found in the carcinogenicity testing of topiroxostat, a xanthine oxidoreductase inhibitor, in which topiroxostat was orally given to F344 rats at 0.3, 1, and 3 mg/kg for 2 years. In the urinary bladder, transitional cell papillomas and/or carcinomas were seen in males receiving 0.3, 1, and 3 mg/kg (1/49, 3/49, and 10/50, respectively). In the kidney, transitional cell papillomas and/or carcinomas in the pelvis were seen in 2/50 males and 1/50 females receiving 3 mg/kg. In the mechanistic study by 52-week oral treatment with topiroxostat at 3 mg/kg to F344 male rats, with and without citrate, simple and papillary transitional cell hyperplasias of the urinary bladder epithelium were observed in 5/17 in the topiroxostat-alone treatment group, along with xanthine-induced nephropathy, in contrast to neither xanthine crystals nor lesions in urinary organs by co-treatment group with citrate. As for sex differences of urinary bladder tumors, the BrdU labeling index for epithelial cells of the urinary bladder by 5-week oral treatment with topiroxostat at 10 mg/kg to F344 rats was increased in males only, showing consistency with histopathological findings. Therefore, the present study indicates that transitional cell tumors induced by topiroxostat in rats were due to physical stimulation to transitional cells of xanthine crystals/calculi and provides that other factors were not implicated in this tumorigenesis. Furthermore, the present study suggests that such tumors do not predict for humans since topiroxostat-induced xanthine deposition is a rodent-specific event.

Study on toxicological aspects of crystal-mediated nephrotoxicity induced by FYX-051, a xanthine oxidoreductase inhibitor, in rats.

To clarify the toxicological aspects of crystal-mediated nephrotoxicity, we performed analysis concerning the correlation between representative kidney-related parameters and renal histopathology, using the individual data obtained from the 4-week toxicity studies of FYX-051, a xanthine oxidoreductase inhibitor, by oral administration at 1 and 3 mg/kg to Sprague-Dawley (SD) rats and at 3 and 10 mg/kg to F344 rats. In SD rats, the correlation coefficient on histopathology between the right and left kidneys was 0.7826 and remained within a lower range of strong correlation (range: ±0.7 ∼ ±0.9). The correlation coefficient between body-weight gains, urinary volume, osmolarity, serum blood urea nitrogen (BUN), creatinine, and relative kidney weights and renal histopathology was -0.6648, 0.7896, -0.7751, 0.8195, 0.8479, and 0.8969, respectively, showing a strong correlation, except a moderate correlation in body-weight gains (range: ±0.4 ∼ ±0.7). In F344 rats, the correlation coefficient on histopathology between the right and left kidneys was 0.8637, remaining within an upper range of strong correlation. The correlation coefficient between the above parameters and renal histopathology was -0.8175, 0.8616, -0.9045, 0.9010, 0.8991, and 0.9524, respectively, showing an extremely strong correlation in urinary osmolarity, serum BUN, and relative kidney weights (range: ±0.9 ∼ ±1.0). Therefore, the present study suggests that FYX-051-induced nephrotoxicity may occur with more inconsistency in the degree of nephropathy between the right and left kidneys in SD rats than in F344 rats, which would explain the above outcomes.

Establishment of simultaneous treatment model with citrate for preventing nephropathy induced by FYX-051, a xanthine oxidoreductase inhibitor, in rats.

As a precedent study for elucidating the mechanism of possible urinary bladder carcinogenesis due to xanthine crystals induced by FYX-051, a xanthine oxidoreductase inhibitor, we have determined the experimental conditions suitable for the 52-week simultaneous treatment with citrate in F344 rats. Simultaneous treatment with citrate and FYX-051 produced both increased urinary citrate excretion and suppression of urinary xanthine deposition at around 4 hours after a single dosing, but these effects disappeared 2 hours later, indicating a lack of the durability of citrate effects. Next, we carried out a 7-day simultaneous treatment study by two daily treatments, that is, FYX-051 (6 mg/kg) and citrate (2,000 mg/kg), followed by citrate-alone treatment, under the conditions of selected dosing intervals, the second dose of citrate, and dosing volume. As a result, the dosing interval of citrate was found to be optimal at 4 hours, but not at 3 or 5 hours, because this treatment completely inhibited intrarenal xanthine deposition. The dose of citrate for the second treatment and the dosing volume were found to be sufficient at 1,500 mg/kg and 10 mL/kg, respectively. Subsequently, a 4-week study by simultaneous treatment at 3 mg/kg of FYX-051 and citrate (2,000 mg/kg) + citrate (1,500 mg/kg), under the improved conditions, revealed that renal lesions could be drastically inhibited. Thus, the present study demonstrated that the interval of two citrate treatments is pivotal and indicated that the improved model would be useful for the mechanistic study of FYX-051-induced urinary bladder carcinogenesis because of an easier treatment method than our previous model.

Study on species differences in nephropathy induced by FYX-051, a xanthine oxidoreductase inhibitor.

To clarify the toxicological aspects of FYX-051, a xanthine oxidoreductase inhibitor, which is currently being developed as a therapeutic agent against gout and hyperuricemia, we performed the study focused on species differences in FYX-051-induced nephropathy. In the repeated toxicology testing by oral administration, nephropathy was seen at 1 mg/kg and more in rats and at 100 mg/kg in dogs, in contrast to no toxicity even at the practical maximum dose (300 mg/kg) in monkeys. The HPLC and LC-MS/MS analyses of intrarenal deposits in dogs have proven that the entity was xanthine. The study on dose dependency of pharmacokinetics, pharmacodynamics, urinary xanthine excretion, and kidney xanthine content by oral administration at 0.3, 1, and 3 mg/kg to rats revealed the involvement of xanthine in the occurrence of nephropathy, thus suggesting that plasma concentrations of FYX-051 can contribute to species differences. Regarding the possible factors of species differences, the daily urinary excretion of total purine metabolites was 30.5- and 6.3-fold greater in rats and dogs, respectively, than in monkeys. Urinary xanthine solubility was 2.3- and 6.3-fold higher in dogs and monkeys, respectively, than in rats. Plasma concentrations of FYX-051 were fivefold higher in rats than in dogs and monkeys, without differences between the latter two species. Therefore, the present study indicated that species differences in nephropathy were produced by the combined effects of purine metabolism, urinary xanthine solubility, and plasma concentrations of FYX-051.

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.

Strain differences in the responsiveness between Sprague-Dawley and Fischer rats to nephropathy induced by FYX-051, a xanthine oxidoreductase inhibitor.

To determine a rat strain appropriate for carcinogenicity testing of FYX-051, a xanthine oxidoreductase inhibitor, we performed a 4-week oral toxicity study by administering 0.3, 1 and 3 mg/kg, and 1, 3 and 10 mg/kg of FYX-051 to male Sprague-Dawley (SD) and Fischer (F344) rats, respectively. Histopathology revealed that the degree of FYX-051-induced nephropathy was 3-fold stronger in SD rats than in F344 rats. Our previous study demonstrated that the key factor of species differences in FYX-051-induced nephropathy is purine metabolism. This observation led us to examine the involvement of purine metabolism in differences among two strains of rats. However, purine metabolism was proven not to be implicated as an important factor. Subsequently, other factors responsible for the strain differences were examined. FYX-051-induced increases in plasma xanthine concentrations were higher in SD rats than in F344 rats, suggesting more remarkable effects on pharmacodynamics in the former than the latter. Urinary volume was greater in F344 rats administered 10 mg/kg of FYX-051 (6.8 ml/h/kg) than in SD rats administered 3 mg/kg of FYX-051 (5.0 ml/h/kg), implying easier xanthine excretion in the former. Urinary xanthine solubility was 55 mg/dl in F344 rats aged 6 weeks, in contrast to 38 mg/dl in SD rats of the same age. Also, there were no significant differences in exposure levels at the same dose between SD and F344 rats. The outcomes of exposure levels and renal histopathology in both rats suggest the possibility that F344 rats could be exposed to a 3-fold higher amount of drug than SD rats in a carcinogenicity bioassay. The present study, therefore, suggested that strain differences of nephrotoxicity were caused by the combined effects of pharmacodynamics, xanthine excretion capacity, and urinary xanthine solubility. Furthermore, these results indicate that F344 rats would be a suitable strain for the carcinogenicity study of FYX-051.

Study on the mechanism of photosensitive dermatitis caused by ketoprofen in the guinea pig.

To investigate the mechanism on photosensitive dermatitis caused by ketoprofen (KP) in humans, the following experiments were performed by topical application on guinea pigs. The phototoxicity study involving treatment with 10% solution of KP, its enantiomers (R-KP and S-KP), loxoprofen, and flurbiprofen revealed no phototoxic reactions. In the photoallergenicity study, KP and its enantiomers (0.5-2% solution) induced skin reaction at all dosages; however, loxoprofen and flurbiprofen (1-5% solution) did not induce such a photoallergenic reaction. These results suggest that the chemical structure of the benzophenone chromophore in KP would be one of the important factors for induction of the photoallergy since both loxoprofen and flurbiprofen do not possess this structure and hence lack photoallergenic potential. Furthermore, to assess time profiles of KP concentration in the skin and plasma, guinea pigs received a repeated topical application of R-KP and S-KP at a dosage of 40 mg/kg over a period of 3 days. Plasma KP concentrations were extremely low as compared to skin KP concentrations and were not detected at 72 h after the final dosing. At 24 h after the final dosing, KP concentrations in the skin with R-KP and S-KP treatment were 187.4 and 254.7 microg/g, respectively, and their half-lives were 80.5 and 84.4 h, respectively. KP concentrations at 336 h after final dosing were 11.3 microg/g for R-KP and 15.7 microg/g for S-KP treatment. The acylglycerol-combined KP concentrations at 336 h were 2% or less as compared to KP concentrations with R-KP and S-KP treatment. There were no differences in KP concentrations in the skin between R-KP and S-KP and in combined KP concentrations between the enantiomers. The present study indicates that photosensitive dermatitis after topical application of KP in humans, caused by photoallergenicity and not phototoxicity, can be reproduced in the animal testing, and suggests that the skin reaction may be caused by the long period of retention of KP in the skin.

Simultaneous treatment with citrate prevents nephropathy induced by FYX-051, a xanthine oxidoreductase inhibitor, in rats.

The possible mechanism of the underlying nephropathy found in the rat toxicity study of FYX-051, a xanthine oxidoreductase inhibitor, was investigated. Rats received oral treatment of either 1 or 3 mg/kg of FYX-051, with and without citrate for four weeks to elucidate whether nephropathy could be caused by materials deposited in the kidney. Furthermore, analysis of the renal deposits in rats was also performed. Consequently, interstitial nephritis comprising interstitial inflammatory cell infiltration, dilatation, basophilia and epithelial necrosis of renal tubules and collecting ducts, deposits in renal tubules and collecting ducts, and so forth was seen in six of the eight rats and in all eight rats in the 1 and 3 mg/kg FYX-051 alone groups, respectively, with the intensity in the 3 mg/kg group being moderate to severe. In the simultaneous treatment with citrate group, however, no alterations were observed in the kidney, except for minimal interstitial nephritis in one instance in the 3 mg/kg FYX-051 + citrate group along with an increased urinary pH, leading to an increase in xanthine solubility. Analysis of intrarenal deposits showed that the entity would be composed of xanthine crystals. The present study, therefore, showed that nephropathy in rats occurring after the administration of FYX-051 was a secondary change caused by xanthine crystals being deposited in the kidney, and no other causes could be implicated in this kidney lesion.