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

Abstract

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.

Introduction

The xanthine oxidoreductase inhibitor 4-(5-pyridin-4-yl-1H-[1, 2, 4]triazol-3-yl)pyridine-2-carbonitrile (FYX-051) is a developing drug synthesized by Fuji Yakuhin Co., Ltd., that is expected to be used in the treatment of gout and hyperuricemia. With regard to the mode of action, Okamoto et al. (2004) have demonstrated that FYX-051 exerts potent xanthine oxidoreductase (XOR) inhibitory activities, resulting in decreased circulating uric acid levels via binding to the active site of molybdenum by covalent linkage, as in the case of allopurinol, which is a hypoxanthine analogue discovered by Elion et al. (1963).

Our previous midterm (thirteen-week) toxicity study of FYX-051 in rats (0.3, 1, and 3 mg/kg) and monkeys (10, 30, and 100 mg/kg) showed that xanthine-mediated nephropathy occurred at 1 mg/kg and more in rats, in contrast to no abnormality at doses of up to 100 mg/kg in monkeys (Shimo et al. 2005). Such observations have also been documented in XOR inhibitors such as allopurinol (Hitchings 1966) and TEI-6720 (Horiuchi et al. 1999). It has been postulated that species difference is attributable to the speed of purine metabolism (Hitchings 1966; Horiuchi et al. 1999). The present long-term toxicity studies of FYX-051 in rats and monkeys demonstrated the species difference in nephropathy was further marked. Therefore, we thought such a phenomenon could not be explained by purine metabolism alone, which urged us to clarify the underlying factors involved in the difference among species.

Taking these points into account, we attempted to clarify the possible factors involved in the difference in FYX-051–induced nephropathy between rats and monkeys. In addition, a comparison with humans was also performed concerning the same factors to discuss safety extrapolation of FYX-051 to humans.

Materials and Methods

Long-term Toxicity Studies of FYX-051

Twenty-six-week Dose Toxicity Study of FYX-051 in Rats

Animals: Male and female Crj:CD(SD)IGS rats, five weeks old, were purchased from Charles River Laboratories Japan (Shiga, Japan). Throughout the acclimatization and experimental periods, the animals were housed individually in wire mesh cages in an air-conditioned animal room (temperature 22°C ± 3°C; relative humidity 50% ± 20%; and light cycle twelve hours light/twelve hours dark). Animals without any abnormalities after a one-week acclimatization period were selected for the study. Pelleted diet (CR-LPF, Oriental Yeast, Tokyo, Japan) and tap water were made freely available throughout the study. Experimental procedures were approved by the Institutional Animal Care and Use Committee of Shin Nippon Biological Laboratories, Ltd.’s Drug Safety Research Laboratories.

Study Design: The rats were divided into four groups of fourteen males and fourteen females each. The animals received 0 (control, 0.5%methylcellulose [MC]), 0.04, 0.2, and 1 mg/kg of FYX-051 (Fukuju Seiyaku Co., Ltd., Toyama, Japan) for twenty-six weeks. The dosing was performed by oral gavage once daily, at a volume of 5 mL/kg. The dose levels were selected based on the thirteen-week dose toxicity study of FYX-051 in rats that received 0.3, 1, and 3 mg/kg FYX-051 in which nephropathy was seen at dosages of 1 mg/kg and more (Shimo et al. 2005).

Laboratory Investigations: The following examinations were performed: signs, body weight, and food consumption (dosing period); ophthalmology and urinalysis (before the initiation of dosing and at week 26 of dosing); hematology, blood chemistry, gross pathology, organ weights, and histopathology (at the end of dosing); and toxicokinetics (at the initiation and week 26 of dosing, by LC-MS/MS method). These examinations were conducted in accordance with the toxicity study guideline issued by the Japanese regulatory authorities.

Statistics: Means and standard deviations (SD) of numerical data were calculated. The difference between the control and treatment groups was analyzed by Dunnett’s test (at significance levels of 5%and 1%).

Fifty-two-week Dose Toxicity Study of FYX-051 in Monkeys

Animals: Three- to four-year-old male and female cynomolgus monkeys were purchased from Guangdong Scientific Instruments and Materials Import/Export Corporation (Guangzhou, China). Throughout the acclimatization and experimental periods, the animals were housed individually in stainless steel cages in an air conditioned animal room (room temperature 23°C ± 3°C; relative humidity 55% ± 20%; lighting cycle twelve hours light/twelve hours dark). After a two-week acclimatization period, the animals without any abnormal findings were selected for the study. Solid food (Teklad Global Certified 25%Protein Primate Diet, Harlan Sprague Dawley, UK) and tap water via automatic stainless steel nozzles were made freely available throughout the study. Experimental procedures were approved by the Institutional Animal Care and Use Committee of Shin Nippon Biological Laboratories, Ltd.’s Drug Safety Research Laboratories.

Study Design: The monkeys were divided into four groups of four males and four females each. The animals received 30, 100, and 300 mg/kg of FYX-051 for fifty-two weeks. The fourth group was the control group. A solution of FYX-051 suspended in 0.5%MC was administered by oral gavage once daily at a volume of 5 mL/kg. Additionally, two males and two females each were allocated to the control and 300 mg/kg FYX-051 groups to assess the reversibility of the toxicity during a four-week recovery period. The dose levels were selected based on the thirteen-week dose toxicity study of FYX-051 in monkeys, in which no toxicity was seen at dosages of up to 100 mg/kg. Therefore, to determine toxic symptoms, the highest dose for the present study was set at 300 mg/kg.

Laboratory Investigations: The following examinations were performed: signs, body weight, and food consumption (dosing and recovery period); ophthalmology, body temperature, electrocardiography, auditory reflex, urinalysis, hematology, and blood chemistry (before the initiation of dosing, at weeks 26 and 52 of dosing, and at week 4 of recovery); gross pathology, organ weights, and histopathology (at the end of dosing and recovery); and toxicokinetics (at the initiation, week 26, and the end of dosing). These examinations were conducted in accordance with the toxicity study guideline issued by the Japanese regulatory authorities.

Statistics: Statistical analysis was performed in the same manner as in the case of the twenty-six-week dose toxicity study of FYX-051 in rats.

Study on Possible Factors for Species Differences in Nephrotoxicity

Assay of Urinary Purine Metabolites in Rats, Monkeys, and Humans

Twenty-four-hour urine collected from three untreated male Crj:CD(SD)IGS rats, three male cynomolgus monkeys, and four male human volunteers was used. Urinary purine metabolites were determined by high performance liquid chromatography (HPLC), as in our previous study (Ashizawa et al. 2006). For the determination of hypoxanthine and xanthine levels, urine was diluted with 200 mM acetate buffer (pH 4.6). For uric acid level determination, 200 mM acetate buffer (pH 4.6) containing the internal standard allopurinol (Sigma-Aldrich, St. Louis, MO) was added to the urine. For allantoin level determination, 15 mM phosphate buffer, pH 2.0, containing the internal standard 1-methylnicotinamide chloride and acetonitrile were added to the urine. After the mixing and centrifugation of these samples, each supernatant was analyzed by HPLC. Hypoxanthine, xanthine, and uric acid levels were determined by using a Mightysil RP-18 Aqua column (4.6 mm i.d. × 250 mm, 5-μm particle size; Kanto Chemical, Tokyo, Japan). Hypoxanthine and xanthine were eluted by using acetate buffer (10 mM, pH 4.6) and acetonitrile as Solvents A and B, respectively. In the elution of uric acid, phosphate buffer (47 mM, pH 4.6) containing 0.05%tetrahy-drofuran and methanol were used as Solvents A and B, respectively. Detection of hypoxanthine and xanthine was at 260 nm, and that of uric acid was at 284 nm. Allantoin level was determined by using a Capcell Pak NH₂ UG80 column (4.6 mm i.d. × 250 mm, 5-μm particle size; Shiseido, Tokyo, Japan). Allantoin was eluted by using phosphate buffer (5 mM, pH 2.0) and acetonitrile as Solvents A and B, respectively. Detection was at 218 nm. All measurements were done on the basis of gradient flow. The column temperature was maintained at 35°C and the flow rate was 1 mL/min.

Assay of Urinary Xanthine Solubility in Rats, Monkeys, and Humans

Forty-eight-hour urine collected from four untreated male Crj:CD(SD)IGS rats and twenty-four-hour urine from three male cynomolgus monkeys and three human male volunteers as used. Urine from each animal was pooled and subjected to the xanthine solubility assay. Urinary xanthine solubility was determined as described previously (Shimo et al. 2005). In brief, xanthine was dissolved in the urine from each animal at pH 10 by the addition of sodium hydroxide solution. Thereafter, urine pH was adjusted to 5.0, 6.0, 7.0, 8.0, or 9.0 by the addition of hydrochloric acid solution. After incubation of the pH-adjusted urine for two hours at 37°C, the urine was filtered through a 0.45-μm pore size filter. An aliquot was diluted with water and used for the measurement of xanthine concentration. Xanthine was determined by HPLC.

Assays of Exposure Levels of FYX-051 in Rats, Monkeys, and Humans

Toxicokinetic data in rats and monkeys of FYX-051 were obtained from the twenty-six-week dose toxicity study and fifty-two-week dose toxicity study, respectively. In addition, the exposure level in humans was cited from a single-dose clinical trial, in which six volunteers were treated with 180 mg (approximately 3 mg/kg) of FYX-051.

Statistics

The amount of urinary purine metabolites was calculated by molar conversion and expressed as the sum of the four metabolites. Each value was expressed as the mean and SD.

Results

Long-term Toxicity Studies of FYX-051

Twenty-six-week Dose Toxicity Study of FYX-051 in Rats

One male receiving 1 mg/kg died at day 121 of dosing, with a reduction of body weight and decreased food consumption over a few days prior to death (Table 1). Pathological examination revealed severe nephritis, indicating the cause of death was renal failure. For surviving animals, one male receiving 1 mg/kg showed a reduction or decreased gain of body weight from week 21 of dosing. Laboratory investigations revealed yellow granular materials (proven to be xanthine crystals by chemical analysis [Shimo et al. 2005]) in urinary sediment at 0.2 mg/kg and more, and a significant increase of urinary volume and serum creatinine levels, an increase of blood urea nitrogen (BUN) level, and a significant decrease of urinary osmolarity in males receiving 1 mg/kg. Gross pathology revealed gross changes such as white discoloration, rough surface in the kidney, and yellowish-white granular materials in the kidney in the 1 mg/kg group, including yellowish-white granular materials in the kidney in a male that received 0.2 mg/kg. Histopathology revealed interstitial nephritis in the 1 mg/kg group, with a higher incidence and extent in males. These morphological changes were characterized by interstitial small round cell infiltration, increased interstitial connective tissues, dilatation, basophilia and epithelial necrosis of renal tubules and collecting ducts, and xanthine crystals and cell debris in renal tubules and collecting ducts (Figures 1 and 2). Yellowish-white granular materials in the kidney of a male that received 0.2 mg/kg were considered to be of no toxicological significance because no histological alterations were noted (see the third paragraph of the Discussion section). In the toxicokinetics, C_max and AUC_0–24h at the end of dosing tended to be higher than those at the start of dosing, and these levels increased with ascending dose levels in both males and females (Table 2).

Based on the above results, the no observed adverse effect level (NOAEL) in the present study was estimated to be 0.2 mg/kg.

Fifty-two-week Dose Toxicity Study of FYX-051 in Monkeys

In the fifty-two-week dose toxicity study, no animals died during the study period, nor were noticeable signs observed in all the treated groups. No treatment-related changes were seen in laboratory investigations such as general conditions, electrocardiography, ophthalmology, urinalysis, hematology, blood chemistry, gross pathology, and organ weight. In addition, histopathological examination revealed no remarkable alterations in full organs including the kidney (Figures 3 and 4). In the toxicokinetics, C_max and AUC_0–24h increased with ascending dose levels at both the start and the end of dosing, and there were no accumulation or sex differences (Table 2).

Based on the above results, the NOAEL of FYX-051 in this study was estimated to be more than 300 mg/kg.

Study on Possible Factors for Species Differences in Nephrotoxicity

Urinary Purine Metabolites in Rats, Monkeys, and Humans

The total amount of purine metabolites (hypoxanthine, xanthine, uric acid, and allantoin) in rats, monkeys, and humans was 1934.7 ± 385.0, 62.0 ± 26.0, and 52.8 ± 7.0 μmol/kg/day, respectively, which was approximately thirty-fold higher in rats compared to monkeys or humans (Table 3). The ratio of each animal to humans was 36.6 in rats and 1.2 in monkeys.

Urinary Xanthine Solubility in Rats, Monkeys, and Humans

Urinary xanthine solubility in each animal was remarkably increased with increases of pH in more than pH 8, in contrast to a slight increase within pH 5–7 (Table 4-1). Urinary pH used in the present assay was 6.7 for rats, 9.0 for monkeys, and 5.5 for humans. As the control urine pH in monkeys was high, urinary xanthine solubility was approximately six- and sevenfold higher in monkeys than in rats and humans, respectively (Table 4-2).

Exposure Levels in Rats, Monkeys, and Humans

The toxicokinetics data in the rat twenty-six-week and monkey fifty-two-week dose toxicity studies indicated that exposure levels were five-fold higher in rats than in monkeys, since the AUC_0–24h levels of rats given FYX-051 at 1 mg/kg corresponded to approximately one-sixth those of the monkey given FYX-051 at 30 mg/kg in comparison with their total averages of AUC_0–24h levels at the start and the end of dosing (Table 2). The AUC_0-t level in healthy male humans singly given approximately 3 mg/kg of FYX-051in the phase I trial was 2.82 μg·h/mL, which corresponded to approximately one-tenth that after administration of FYX-051 at 30 mg/kg to monkeys. Thus, these data indicated there was no difference in exposure levels between monkeys and humans.

Discussion

The present long-term toxicity study of FYX-051 demonstrated that nephropathy was seen at 1 mg/kg, despite intrarenal xanthine deposition at 0.2 mg/kg and more in the case of the twenty-six-week treatment in rats, whereas no changes were seen even at the highest dose (300 mg/kg) in the case of fifty-two-week treatment of monkeys. Such events were observed in our previous thirteen-week dose toxicity study in rats and monkeys (Shimo et al. 2005). Accordingly, the present toxicity testing reconfirms that rats are very susceptible to FYX-051–induced intrarenal xanthine deposition; this potency may be more than 1500-fold stronger in rats than in monkeys. Also, the extent of nephropathy in rats appears to have become more severe with prolongation of treatment period, but the dose at which renal changes occurred was invariable, implying there is a threshold between xanthine crystal deposition and renal tubular occlusion. In the study on the effects of allopurinol in relation to purine biosynthesis, Hitchings (1966) demonstrated that in small animals, the limiting factor with respect to the toxicity of allopurinol was crystallization of xanthine in the renal tubules, and such events were caused by differences of purine metabolic turnover; the ratio of each animal to humans was 7 in dogs and 40 in rats. Horiuchi et al. (1999) also showed not only renal xanthine calculus formation in rats given TEI-6720 as well as allopurinol, but species-specific differences on purine metabolic turnover; approximately twenty-fold faster in rats than in humans.

The above observations have encouraged researchers to postulate that the key factor of species difference on XOR inhibitor-induced nephropathy would be the rate of purine metabolism. In the case of either allopurinol or TEI-6720, the toxicological evaluation is speculated to have been made in rats or dogs, and thus it is likely that the researchers mentioned above could not notice a big difference on nephropathy between rats and monkeys, as proven by us. In light of the present study, the stringency of purine metabolism (thirty-fold difference between rats and monkeys) is not sufficient to account for marked species differences in intrarenal xanthine deposition (1500-fold or more difference between the two species), and thus we attempted to examine the underlying factors. First, the amount of urinary purine metabolites in terms of body weight was thirty-fold higher in rats than in monkeys, which is in good agreement with previous observations (Hitchings 1966; Horiuchi et al. 1999). Second, urinary xanthine solubility was six-fold higher in monkeys than in rats, indicating intrarenal deposition of xanthine is far easier in the latter than in the former. Third, toxicokinetics data, obtained from the rat twenty-six-week and monkey fifty-two-week dose toxicity studies, implied that exposure levels would be five-fold higher in rats than in monkeys. These three factors are likely to be deeply implicated in the difference of FYX-051–induced nephropathy between these two species. Therefore, it was considered that the present species difference was caused by the combined effects of the above three factors.

With regard to lack of intrarenal xanthine deposition in monkeys, we would comment as follows. The amount of urinary purine metabolites in monkeys was 62.0 μmol/kg/day. Even if all purines are converted to xanthine by FYX-051–induced complete inhibition of XOR, urinary xanthine concentration is estimated to be 47 mg/dL when the body weight and urine volume of a monkey is 5 kg and 100 mL/day, respectively. The calculated concentration is far below the estimated xanthine solubility (950 mg/dL at pH 9 in Table 4-2). Thus, it seems likely that intrarenal xanthine deposition would not occur in monkeys under any conditions.

The twenty-six-week dose toxicity study in rats, associated with inconsistency between intrarenal xanthine deposition at 0.2 mg/kg and more and nephropathy occurrence at 1 mg/kg, would provide us with the following pathomechanism for nephropathy. Treatment with FYX-051 produced increases in blood xanthine levels due to its XOR inhibitory activity, and as a result, intrarenal xanthine deposition occurred mainly during the process of urine concentration from distal tubules to collecting ducts. Where the amount of xanthine deposited exceeds the capacity of the kidney to excrete foreign materials, renal tubules and collecting ducts (specifically marked in distal tubules) are occluded by xanthine crystals, leading to interstitial nephritis accompanied by renal dysfunction, such as increased urinary volume and decreased urinary osmolarity.

A comparison of factors relevant to species differences between monkeys and humans demonstrated that urinary xanthine solubility in the former was seven-fold higher than in the latter, without any differences in the remaining two factors. The important thing is that neither xanthine crystals in urine nor serious side effects have been reported in the phase II trial of FYX-051, in which hyperuricemic patients received FYX-051 at dosages of 80 to 160 mg for ten weeks. In addition, it should be emphasized that in the monkey fifty-two-week dose toxicity study of FYX-051, no remarkable changes were noted in any laboratory investigations, even at the highest dose (300 mg/kg). Considering the points mentioned above and the estimated maximum clinical dose (3 mg/kg), safety to humans of FYX-051 seems plausible based on the monkey fifty-two-week dose toxicity study.

In conclusion, the present study suggested that species differences in nephropathy due to FYX-051 between rats and monkeys were induced by the combined effects of purine metabolism, urinary xanthine solubility, and exposure levels. Furthermore, the monkey fifty-two-week dose toxicity study would highlight a wide safety margin of FYX-051 in humans.