Simultaneous Treatment with Citrate Prevents Nephropathy Induced by FYX-051, a Xanthine Oxidoreductase Inhibitor, in Rats
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《毒物学科学杂志》
Research Laboratories 2, Fuji Yakuhin Co., Ltd., 636-1 Iidashinden, Nishi-ku, Saitama 331-0068, Japan
ABSTRACT
The possible mechanism of the underlying nephropathy found in the rat toxicity study of FYX-051, a xanthine oxidoreductase inhibitor, was investigated. Rats
Key Words: FYX-051; citrate; xanthine oxidoreductase inhibitor; xanthine crystals; nephropathy; rats.
INTRODUCTION
Gout is a type of acute arthritis that is induced by hyperuricemia and results in the crystallization of sodium urate (Fujimori, 2000). Recently, a high incidence of gout and hyperuricemia associated with hyperlipidemia, obesity, and hypertension have been demonstrated, and these complications are perceived as a risk factor for inducing mortality and ischemic heart disease (Freedman et al., 1995). It has also been shown that hyperuricemia increases the relative risk of cardiovascular or cerebrovascular diseases (Alderman et al., 1999; Tomita et al., 2000), and uric acid is reported to be an independent risk factor in the treatment of hypertension (Ward, 1998). Considering these factors, it has been recommended that asymptomatic hyperuricemic patients should receive treatment for decreasing blood uric acid levels (Nakamura, 2001). Indeed, patients are treated with a uric acid control drug to prevent recurrence of gout.
Xanthine oxidoreductase (XOR) catalyzes the last two reactions of purine catabolism-the hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Therefore, this enzyme is the target of drugs against gout and hyperuricemia, and various enzyme inhibitors have been developed. Allopurinol, a hypoxanthine analogue, was discovered by Elion et al. (1963) as an inhibitor of XOR more than 40 years ago and has been extensively used in the treatment of gout and hyperuricemia since that time. Fuji Yakuhin Co., Ltd., has synthesized a new XOR inhibitor FYX-051, 4-(5-pyridin-4-yl-1H-[1, 2, 4]triazol-3-yl)pyridine-2-carbonitrile. In contrast to allopurinol, FYX-051 does not have a purine-ring structure. This compound has more potent inhibitory effect on bovine milk XOR as compared to that by allopurinol, without any significant effects on other enzymes such as aldehyde oxidase and those metabolizing purines and pyrimidines.
Recently, X-ray crystal structure of XOR-FYX-051 complex has been determined by Okamoto et al. (2004). They have demonstrated that FYX-051 binds to the active site molybdenum by a covalent linkage, as in the case of allopurinol. Furthermore, the study indicated that the inhibition of the enzyme caused by FYX-051, which occurred through some interactions with amino acid residues, lasted for a long time when compared with that caused by allopurinol. This feature of enzyme inhibition by FYX-051 correlates closely with its hypouricemic effects in animals. With regard to hypouricemic effects in potassium oxonate-induced hyperuricemic rodent models, FYX-051 caused a dose-dependent reduction of serum urate concentrations. These effects of FYX-051 were approximately 30-fold more potent than those of allopurinol in rats. In yeast RNA-induced hyperuricemic chimpanzees, FYX-051 was shown to continuously reduce the serum urate level; this implies that FYX-051 is more effective than allopurinol.
The repeated toxicity of FYX-051 has been evaluated in rats, dogs, and monkeys. The results show that nephropathy, thought to occur due to xanthine deposition, was observed at a dosage of 1 mg/kg in rats in contrast to 100 mg/kg in dogs, and no abnormalities were observed at dosages of up to 100 mg/kg in monkeys (see referential data mentioned below). These observations, namely, the effects being found markedly in rodents when compared with non-rodents such as dogs or monkeys, have also been documented in the XOR inhibitor allopurinol (Hitchings, 1966; Isa et al., 1968). However, there is no definite evidence whether the cause of nephropathy would be due to the deposition of xanthine crystals alone because neither deliberate investigation for justifying this speculation nor the analysis of the materials deposited in the kidney has been carried out. In view of such a situation, we decided to perform an experiment to clarify the pathomechanism of the nephropathy. The most direct evidence for ensuring that the nephropathy in rats is due to the deposition of xanthine crystal would be the disappearance of the disorder in case of prevention of xanthine deposition. However, no such a study has ever been done because it is not easy to inhibit the XOR inhibitor-induced nephropathy in rats. At the same time, we believed that high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometer (LC-MS/MS) analyses were required to identify the entity of renal deposits.
Considering these issues, we performed the following mechanistic studies of FYX-051-induced nephropathy. In Experiment 1, rats were simultaneously treated with FYX-051 and citrate in order to elucidate the cause of nephropathy. In Experiment 2, renal deposits were analyzed to prove that these were xanthine crystals. In Experiment 3, urinary xanthine solubility was determined.
MATERIALS AND METHODS
Study on Simultaneous Treatment with Citrate (Experiment 1)
Animals and housing.
Five-week-old Crj:CD(SD)IGS male rats were purchased from Charles River Japan Inc. (Shiga, Japan). Throughout the acclimatization and experimental periods, the animals were housed individually in wire-mesh cages in an air conditioned animal room (room temperature, 22 ± 4°C; relative humidity, 60 ± 20%; lighting cycle, 12-h light/12-h dark). The animals without any abnormal findings after a one-week acclimatization period were selected for the present study. Pellet diet (CE-2, Clea Japan, Tokyo, Japan) and tap water via automatic stainless steel nozzles were made freely available throughout the study.
Test materials.
FYX-051 was synthesized by Fukuju Seiyaku (Toyama, Japan). Tri-sodium citrate 2-hydrate, tri-potassium citrate 1-hydrate, and citric acid 1-hydrate were purchased from Kishida Kagaku (Tokyo, Japan). The citrate used in the experiment was a mixture of its tri-sodium salt, tri-potassium salt, and free acid in a ratio of 2:2:1, calculated as anhydrous at molar base.
Experimental design.
The male rats were divided into five groups of eight animals each. The animals
Urinary pH, blood chemistry, toxicokinetics, autopsy, kidney weights, histopathology of urinary organ.
At week 4 (day 28) of administration, the urine was obtained from non-fasted animals to determine the pH using a pH meter (F-22, Horiba, Kyoto, Japan). After oral administration of FYX-051, the rats were transferred from their regular cages to metabolic cages for urine collection. During the 4-h urine collection, the rats were deprived of food but had free access to water and
Statistical analysis.
The mean and standard deviation of the blood chemical parameters, urinary pH, and kidney weights were calculated, and the difference between the control and treated groups was analyzed by the Dunnett's test (at significant levels of 5 and 1%). In addition, the incidence of renal histopathological lesions was analyzed by the Wilcoxon's test.
Analysis of Crystals in the Kidney (Experiment 2)
Collection of the renal deposits.
For analysis, samples were obtained from the yellowish-white deposits in the lumen of the renal pelvis of the SD rats treated orally with 3 mg/kg of FYX-051 for 13 weeks (see referential data mentioned below).
Assay of the renal deposits.
High performance liquid chromatography (HPLC) was used to determine the concentration of xanthine in the renal deposits. The collected deposits were dissolved in 25 mmol/l sodium hydroxide and chromatographed with a 2690 separation module (Waters, Milford, MA) using a Mightysil RP-18 Aqua column (4.6 mm i.d. x 250 mm, 5 μm particle size; Kanto Chemical, Tokyo, Japan) and eluted at a flow rate of 1.0 ml/min with 47 mmol/l sodium dihydrogenphosphate solution (pH 4.6). Detection was performed at 260 nm using 2487 detector (Waters, Milford, MA). Simultaneously, the eluate of xanthine fraction was collected and determined by a liquid chromatography-tandem mass spectrometer (LC-MS/MS). Detection was performed on API 4000 (Applied Biosystems, CA) and was operated by an infusion method. The eluted xanthine fraction was diluted with 10 mmol/l ammonium acetate solution and infused at a flow rate of 20 μl/min using a syringe pump model 11 (Harvard Apparatus, MA). Ionization was conducted using the TurboIon Spray and positive ion mode at 450°C. The precursor scan and product scan for m/z 153 and m/z 50–160, respectively were carried over the range based on collision energy for 5–130 eV (ramping parameter) using MCA scan mode. The HPLC retention time and product ion spectra of the analyte were compared with those of authentic xanthine (Sigma-Aldrich, St. Louis, MO).
Determination of Xanthine Solubility in Rat Urine (Experiment 3)
Urine collection.
Five-week-old Crj:CD(SD)IGS male rats were purchased from Charles River Japan (Shiga, Japan). After a two-week acclimatization period, four rats were transferred to metabolic cages for 48-h urine collection, during which the rats
Sample analysis.
For the measurement of xanthine solubility, urine was warmed to 37°C. Xanthine (Sigma-Aldrich, St. Louis, MO) was solubilized in urine 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 2 h at 37°C, the urine was filtered through a 0.45 μm pore size filter. An aliquot was diluted with water containing allopurinol as an internal standard and used for the measurement of xanthine concentration. Xanthine was determined by HPLC (pump, PU-980; auto sampler, AS-950; UV detector, UV-970, JASCO, Tokyo, Japan) by using a Mightysil RP-18 Aqua column (4.6 mm i.d. x 250 mm, 5 μm particle size; Kanto Chemical, Tokyo, Japan). Column temperature was maintained at 40°C, and xanthine detection was performed at 265 nm. Acetic acid solution (0.5%) was used as the mobile phase at a flow rate of 1.0 ml/min. At each pH, xanthine solubility was examined using urine with various concentrations of xanthine. The highest concentration of xanthine obtained at each pH was estimated as the xanthine solubility.
Thirteen-Week Dose Toxicity Study of FYX-051 in Various Animals (Referential Data)
Dogs
Animals and housing.
Nine- to ten-month-old male and female beagle dogs were obtained from the breeding facility of Shin Nippon Biomedical Laboratories (Kagoshima, Japan). Throughout the acclimatization and experimental periods, the animals were housed individually in stainless steel cages in an air conditioned animal room (room temperature, 23 ± 3°C; relative humidity, 55 ± 20%; lighting cycle, 12-h light/12-h dark). After a two-week acclimatization period, the animals without any abnormal findings were selected for the present study. Pellet diet (VE-10, Nippon Pet Food, Tokyo, Japan) and tap water via automatic stainless steel nozzles were made freely available throughout the study.
Experimental design.
The dogs were divided into four groups of three males and three females each. The animals
Laboratory investigations.
During the dosing and recovery periods, the parameters that were evaluated were as follows: signs, body weight, and food consumption. Before the initiation of dosing and at weeks 4 and 13 of dosing and week 4 of recovery, the following examinations were performed: ophthalmology, body temperature, electrocardiography, auditory reflex, urinalysis, hematology, blood chemistry, and toxicokinetics (by LC-MS/MS method). At the end of the dosing and recovery period, the following examinations were performed: gross pathology, organ weights, and histopathology. These examinations were conducted in accordance with the toxicity study guideline issued by the Japanese regulatory authorities.
Statistical analysis.
The mean and standard deviation of the parametric data were calculated, and the difference between the control and treated groups was analyzed by the Dunnett's test (at significant levels of 5 and 1%).
Monkeys
Animals and housing.
Three- to four-year-old male and female cynomolgus monkeys were purchased from Guangdong Scientific Instruments & Materials Import/Export Corporation (Guangzhou, China). The housing and quarantine conditions were the same as in the case of the dog study. Solid food (Teklad Global Certified 25% Protein Primate Diet, Harlan Sprague Dawley, U.K.) and tap water via automatic stainless steel nozzles were made freely available throughout the study.
Experimental design.
The experimental design for monkeys was set in the same manner as in the case of the dog study. However, justification for selection of the dose levels is as follows. In a two-week oral dose toxicity study of FYX-051 in monkeys, no toxicity was seen at dosages of up to 100 mg/kg. Therefore, to compare the toxicity between dogs and monkeys, the highest dose level for this study was set at 100 mg/kg.
Laboratory investigations.
The observation and examinations were performed in the same manner as in the case of the dog study.
Statistical analysis.
The statistical analysis was performed in the same manner as in the case of the dog study.
Rats
Animals and housing.
The animals that were purchased, the acclimatization, and selection procedure for the study were the same as described in Experiment 1. The housing conditions were as follows: room temperature, 22 ± 3°C; relative humidity, 50 ± 20%; lighting cycle, 12-h light/12-h dark. Pellet diet (CR-LPF, Oriental Yeast, Tokyo, Japan) and tap water via automatic stainless steel nozzles were made freely available throughout the study.
Experimental design.
The rats were divided into four groups of 10 males and 10 females each. The animals
Laboratory investigations.
During the dosing period, signs, body weight, and food consumption were recorded. Furthermore, the following examinations were performed: ophthalmology and urinalysis (before the initiation of dosing and at weeks 4 and 13 of dosing), hematology, blood chemistry, gross pathology, organ weights, and histopathology (at the end of dosing), and toxicokinetics (by LC-MS/MS method). These examinations were conducted in accordance with the toxicity study guideline issued by the Japanese regulatory authorities.
Statistical analysis.
The statistical analysis was performed in the same manner as in the case of the dog study.
RESULTS
Study on Simultaneous Treatment with Citrate (Experiment 1)
The pH of the urine was 7.83 ± 0.26, 7.22 ± 0.34, 6.86 ± 0.11, 7.96 ± 0.22, and 7.94 ± 0.10 in the control (citrate), 1 mg/kg FYX-051, 3 mg/kg FYX-051, 1 mg/kg FYX-051 + citrate, and 3 mg/kg FYX-051 + citrate groups, respectively; the pH was higher by approximately 1 in the simultaneous treatment with citrate groups than the FYX-051 alone groups (Table 1). Blood chemistry revealed a significant increase in the creatinine and BUN levels (0.75 ± 0.14 and 50 ± 8 mg/dl, respectively) in the 3 mg/kg FYX-051 group as compared to the control group values (0.31 ± 0.03 and 19 ± 4 mg/dl, respectively) (Table 1).
Relative kidney weights were 0.87 ± 0.06, 1.01 ± 0.27, 1.70 ± 0.32, 0.86 ± 0.04, and 0.87 ± 0.07 in the control, 1 mg/kg FYX-051, 3 mg/kg FYX-051, 1 mg/kg FYX-051 + citrate, and 3 mg/kg FYX-051 + citrate groups, respectively; the 3 mg/kg FYX-051 alone group showed a significant increase in kidney weights when compared with the control (Table 1). Treatment with citrate exerted no remarkable effect on the toxicokinetic profile of FYX-051 (Table 1). Autopsy revealed gross changes, such as yellowish-white foci and rough surface in the kidney and yellowish-white granular deposits on the cut surface of the kidney in the FYX-051 alone groups, and one incidence of a yellowish-white macula in the 3 mg/kg FYX-051 + citrate group.
Histopathological examination revealed minimal to moderate and moderate to severe interstitial nephritis in six of the eight rats and in all the eight rats in the 1 and 3 mg/kg FYX-051 alone groups, respectively, and extensive lesions were observed throughout the cortex and medulla (Table 2; Figs. 1A, 2A, and 3A). 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, crystals and cell debris in renal tubules and collecting ducts, crystals in the renal pelvic lumen, and papillary or pelvic epithelial hyperplasia. The presence of crystal deposits was remarkable in the distal tubules. In contrast, in the simultaneous treatment with citrate groups, no renal alterations, except minimal interstitial nephritis in one of the eight rats in the 3 mg/kg FYX-051 + citrate group were seen (Table 2; Figs. 1B, 2B, and 3B).
With regard to the urinary tracts, no remarkable changes were observed in the ureters and urinary bladder in any of the groups, including the control (citrate alone) group.
Analysis of Crystals in the Kidney (Experiment 2)
Chromatogram at 260 nm detected a main peak of the renal deposits; the peak showed the same retention time as that of xanthine (Fig. 4). Quantitative analysis of the main peak (similar to that of xanthine) revealed that 85.5% of the renal deposits were composed of xanthine. On comparison of the MS/MS spectrum of main constituent in renal deposits with that of authentic xanthine, the main fragment and parent ions were detected for m/z 64, 110, 136, and 153 (parent ion [M+H]+) and these corresponded with both samples (Fig. 5).
Xanthine Solubility in Rat Urine (Experiment 3)
Xanthine solubility in rat urine was pH-dependent (Table 3). Within a pH range of 5–7, xanthine solubility slightly increased; 155 mg/dl at pH 5 and 167 mg/dl at pH 7. At a pH greater than 8, xanthine solubility remarkably increased, i.e., xanthine solubility at pH 8 and 9 was 2.0 and 3.9 times higher than that at pH 7, respectively.
Thirteen-Week Dose Toxicity Study of FYX-051 in Various Animals (Referential Data)
Dogs.
No animals died during the study period, and no remarkable symptoms were observed, except transient minimal reduction of body weights at week 1 of dosing in one male receiving 100 mg/kg FYX-051 (Table 4). Urinalysis revealed the presence of minute yellow granular materials in sediments, which were suggestive of xanthine crystals, but no abnormality was detected in the other parameters. At autopsy, yellowish-white granular materials were present on the cut surface of the kidney in the 100 mg/kg group, and one female of the same group exhibited a large calculus in the left pelvic lumen. Histopathological examination revealed epithelial hyperplasia of the papilla and pelvis due to foreign materials (xanthine crystals) in the pelvic lumen at dosages of 30 mg/kg FYX-051 and above. This was seen unilaterally and focally, with minimal severity and low incidence at 30 mg/kg FYX-051, while the finding appeared to be slightly stronger at 100 mg/kg. Accordingly, this change was considered to be of little toxicological significance because of the localized nature of hyperplasia and absence of renal functional disorder. On the other hand, one female dog mentioned above exhibited a tendency toward elevated serum creatinine levels and showed changes in the tissues around the pelvis due to formation of a large calculus indicative of physical irritation, and alterations of renal distal tubules. The distal tubular changes were also considered to be the result of repeated stimulation due to the xanthine deposited in the distal tubule under the condition of urinary excretion disorder. Therefore, a series of renal changes observed in this female were regarded to be a renal functional disorder, although these changes were induced by secondary effects of intrarenal xanthine deposits. At the end of the four-week recovery period, no histopathological alterations were seen in the 100 mg/kg FYX-051 group, despite the presence of yellowish-white granular materials in the pelvic lumen. In general, in the toxicokinetics study, Cmax and AUC levels increased with the dose in both males and females.
Based on the above results, the no observed adverse effect level (NOAEL) of FYX-051 in this study was estimated to be 30 mg/kg.
Monkeys.
No animals died during the study period, and no remarkable changes were observed in the general conditions, including signs and body weights (Table 5). Treatment-related changes were not observed during clinical examinations. In addition, no abnormalities were noted in the pathological examination of all the organs and tissues. In the toxicokinetics study, Cmax and AUC levels increased with the dose in both males and females.
Based on the above results, the NOAEL of FYX-051 in this study was estimated to be greater than 100 mg/kg.
Rats.
No animals died during the study period. In males of the 3 mg/kg FYX-051 group, body weight gains were inhibited at week 1 of dosing. Blood chemistry revealed an increase in BUN and creatinine levels in the 3 mg/kg FYX-051 group. Autopsy revealed gross changes, such as yellowish-white foci, rough surface, and yellowish-white granular materials on the cut surface of the kidney, in groups that
Based on the above results, the NOAEL of FYX-051 in this study was estimated to be 0.3 mg/kg.
DISCUSSION
It is known that treatment with XOR inhibitors in rodents induces striking intrarenal xanthine deposition (Hitchings, 1966; Horiuchi et al., 1999). However, these reports do not provide a definite evidence of nephropathy being caused by xanthine deposition alone or of the renal deposits being composed of xanthine. Based on the result that the retention time of the main peak of the test sample was the same as that of xanthine, the present analysis by HPLC showed that the renal deposits in the rats could be composed of xanthine crystals. In addition, LC-MS/MS analysis demonstrated a correspondence in the spectrum between authentic xanthine and the renal deposits.
Thus far, no study has demonstrated the successful amelioration of XOR inhibitor-induced nephropathy in rats by prevention of xanthine deposition. It may be possible that on account of the large amount of xanthine excretion in rat urine, it was difficult to prevent xanthine deposition in the kidney. In order to minimize the potential hazard of xanthinuria in humans during allopurinol administration, the alkalinization of urine and increase in the fluid intake are recommended (Klinenberg et al., 1965). Based on this information, we examined the effects of the urine alkalinizing agent citrate with a large amount of water on the nephropathy caused by FYX-051 administration in rats. The treatment described above, without affecting the toxicokinetic profile of FYX-051, showed that urinary pH increased from 7 to approximately 8; this elevation was attributable to rapid increase in xanthine solubility in the urine. As a result, intrarenal xanthine deposition was reduced, leading to disappearance of renal alterations comprising small round cell infiltration, dilatation and basophilic changes of renal tubules, and so forth. These results provided us with the definite evidence that a series of renal alterations were caused by the deposition of xanthine crystals during the process of concentrating the urine and showed that nephropathy could not be caused by the direct action of this agent.
Citrate was used as a mixture of its sodium salt, potassium salt, and free acid in a ratio of 2:2:1 at molar base. It has been known that citrate is metabolized via the TCA cycle to yield bicarbonate that exerts its urine alkalinizing effect. In our preliminary experiment, the use of free citric acid alone lessened this effect, probably because some amount of unchanged citric acid is excreted into the urine, resulting in a depression of its urine alkalinizing effect. Administration of sodium salt alone is not appropriate because it forces the kidneys to excrete a large amount of sodium. As a result, drastic disappearance of renal lesions induced by FYX-051 is made possible by the administration of citrate as a mixture of its sodium and potassium salts, without any associated adverse effects.
The solubility of xanthine in urine obtained in the present study is higher than that in the previous report (Klinenberg et al., 1965) in which the xanthine solubility was 5 mg/dl at pH 5 and 13 mg/dl at pH 7. The discrepancy in the solubility between the two studies is probably attributable to the difference in the method used. In the previous study, xanthine solubility was determined by simply dissolving xanthine in urine. On the other hand, in the present study, xanthine was dissolved under alkaline conditions, and the urine pH was adjusted. In consideration of the fact that xanthine is likely to get deposited during the process of urine condensation that occurs in the distal tubules and collecting ducts in the kidney, it is reasonable to consider that the maximum concentration of xanthine that remains dissolved in urine is of greater significance. In fact, the dissolved xanthine concentration in urine from rats administered 1–3 mg/kg FYX-051 was approximately 100 mg/dl (data not shown). From these results, we conclude that the xanthine solubility obtained in the present study is more appropriate than the previous values as an index of xanthine deposition in the kidney of animals treated with XOR inhibitors.
The pathomechanism of nephropathy could be speculated as follows. Treatment with FYX-051 induced high blood xanthine levels due to its pharmacological activity, and consequently, renal xanthine deposition occurred mainly during the process of urine-condensation from distal tubules to collecting ducts. When the amount of deposited xanthine exceeds the capacity of the kidney to excrete foreign materials, renal tubules and collecting ducts (particularly remarkable in distal tubules) are occluded by these deposits, leading to secondary interstitial nephritis that is characterized by inflammatory cell infiltration, epithelial basophilia or necrosis and dilatation of the renal tubules and collecting ducts, increased interstitial connective tissue, and cell debris in the renal tubules and collecting ducts in extensive areas over the cortex and medulla. This speculation concerning the occurrence of renal changes would be supported by the 13-week dose toxicity study in dogs; renal xanthine deposition occurred in both rats and dogs, but the severity was considerably weaker in the latter than in the former. Considering the findings that xanthine crystals were markedly scattered in the distal tubules, the distal tubules appear to be the main site of crystal deposition. Accordingly, the present results suggest that there is a clear relation between crystal deposition and condensing magnitude of urine. Furthermore, papillary or pelvic epithelial hyperplasia is also likely to be due to the physical irritation caused by the xanthine calculi formed in the renal pelvic lumen. Such renal lesions are considered to be a common phenomenon in treatment with XOR inhibitors.
With regard to the effects of sodium citrate in the lower urinary tracts, Fukushima et al. (1986) have demonstrated that sodium citrate exerts promoting effects on urinary bladder carcinogenesis in the experiment with 32-week dietary administration of a basal diet containing 5% sodium citrate to F344 male rats following a four-week initiation with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water. However, no epithelial alterations were observed in the group without a BBN supplement. Similarly, in the present study, no histopathological changes were observed in the ureter and urinary bladder tissues. While there was a relationship between the elevation of urinary pH and proliferative changes in the urothelium, it is likely that administration of sodium citrate alone would not affect the urothelium. Taken together, the present findings showing consistency with the observation of Fukushima et al. would be of appropriate results.
With regard to species difference in nephrotoxicity, the urinary xanthine solubility assay indicated that renal deposits of xanthine would occur more easily in rats because the value was increased in a pH-dependent fashion, and urinary pH was lower in rats than in dogs or monkeys (see Tables 1, 4, and 5). Toxicokinetics data of each animal suggested that exposure levels would be higher in rats than in dogs or monkeys because the AUC value of rats administered FYX-051 at a dosage of 3 mg/kg was similar to those of dogs and monkeys administered FYX-051 at a dosage of 10 mg/kg. Therefore, these results may illustrate the fact that severe nephropathy occurred in rats when compared with dogs or monkeys.
With regard to the safety extrapolation in humans, it has been suggested there is an extremely low possibility that nephropathy might be induced in humans by treatment with FYX-051. This can be explained based on the following fact that the amount of purine metabolite excreted daily are far higher in rats than in humans (Hitchings, 1966; Horiuchi et al., 1999); this data has been confirmed by us. Regarding the xanthine deposited in the urinary tract or renal pelvis during the clinical use of allopurinol, there have been no documented cases with the exception of a special case in which the rate of urate formation was greatly increased, such as that observed in Lesch-Nyhan syndrome with associated purine metabolism abnormality (Brock et al., 1983; Greene et al., 1969; Kranen et al., 1985; Mizuno et al., 1976; Ogawa et al., 1985). In contrast, some studies reported that large doses of allopurinol produce stones in the urinary tract that result from oxypurinol (main metabolite) in humans (Landgrebe et al., 1975; Stote et al., 1980). In case of FYX-051, the possibility of metabolite crystallization in humans is likely to be extremely low, considering the following facts. Other compounds, except xanthine, were not detected in the present analysis of crystals deposited in the kidney, and highly soluble N-glucuronides could be estimated to be the main metabolites of FYX-051.
In conclusion, the present study demonstrated 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.
ACKNOWLEDGMENTS
We would like to thank Prof. Kunitoshi Mitsumori, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, for his advice. We are deeply indebted to Dr. Hiroshi Tokado, Department of Safety Research, Shin Nippon Biomedical Laboratories, Ltd., for analysis of renal deposits from the rats and toxicological evaluation. We also wish to thank Mr. Kazuhiko Oba, Research Laboratories 2, Fuji Yakuhin Co., Ltd., for his excellent technical support. Conflict of interest: none declared.
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ABSTRACT
The possible mechanism of the underlying nephropathy found in the rat toxicity study of FYX-051, a xanthine oxidoreductase inhibitor, was investigated. Rats
Key Words: FYX-051; citrate; xanthine oxidoreductase inhibitor; xanthine crystals; nephropathy; rats.
INTRODUCTION
Gout is a type of acute arthritis that is induced by hyperuricemia and results in the crystallization of sodium urate (Fujimori, 2000). Recently, a high incidence of gout and hyperuricemia associated with hyperlipidemia, obesity, and hypertension have been demonstrated, and these complications are perceived as a risk factor for inducing mortality and ischemic heart disease (Freedman et al., 1995). It has also been shown that hyperuricemia increases the relative risk of cardiovascular or cerebrovascular diseases (Alderman et al., 1999; Tomita et al., 2000), and uric acid is reported to be an independent risk factor in the treatment of hypertension (Ward, 1998). Considering these factors, it has been recommended that asymptomatic hyperuricemic patients should receive treatment for decreasing blood uric acid levels (Nakamura, 2001). Indeed, patients are treated with a uric acid control drug to prevent recurrence of gout.
Xanthine oxidoreductase (XOR) catalyzes the last two reactions of purine catabolism-the hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Therefore, this enzyme is the target of drugs against gout and hyperuricemia, and various enzyme inhibitors have been developed. Allopurinol, a hypoxanthine analogue, was discovered by Elion et al. (1963) as an inhibitor of XOR more than 40 years ago and has been extensively used in the treatment of gout and hyperuricemia since that time. Fuji Yakuhin Co., Ltd., has synthesized a new XOR inhibitor FYX-051, 4-(5-pyridin-4-yl-1H-[1, 2, 4]triazol-3-yl)pyridine-2-carbonitrile. In contrast to allopurinol, FYX-051 does not have a purine-ring structure. This compound has more potent inhibitory effect on bovine milk XOR as compared to that by allopurinol, without any significant effects on other enzymes such as aldehyde oxidase and those metabolizing purines and pyrimidines.
Recently, X-ray crystal structure of XOR-FYX-051 complex has been determined by Okamoto et al. (2004). They have demonstrated that FYX-051 binds to the active site molybdenum by a covalent linkage, as in the case of allopurinol. Furthermore, the study indicated that the inhibition of the enzyme caused by FYX-051, which occurred through some interactions with amino acid residues, lasted for a long time when compared with that caused by allopurinol. This feature of enzyme inhibition by FYX-051 correlates closely with its hypouricemic effects in animals. With regard to hypouricemic effects in potassium oxonate-induced hyperuricemic rodent models, FYX-051 caused a dose-dependent reduction of serum urate concentrations. These effects of FYX-051 were approximately 30-fold more potent than those of allopurinol in rats. In yeast RNA-induced hyperuricemic chimpanzees, FYX-051 was shown to continuously reduce the serum urate level; this implies that FYX-051 is more effective than allopurinol.
The repeated toxicity of FYX-051 has been evaluated in rats, dogs, and monkeys. The results show that nephropathy, thought to occur due to xanthine deposition, was observed at a dosage of 1 mg/kg in rats in contrast to 100 mg/kg in dogs, and no abnormalities were observed at dosages of up to 100 mg/kg in monkeys (see referential data mentioned below). These observations, namely, the effects being found markedly in rodents when compared with non-rodents such as dogs or monkeys, have also been documented in the XOR inhibitor allopurinol (Hitchings, 1966; Isa et al., 1968). However, there is no definite evidence whether the cause of nephropathy would be due to the deposition of xanthine crystals alone because neither deliberate investigation for justifying this speculation nor the analysis of the materials deposited in the kidney has been carried out. In view of such a situation, we decided to perform an experiment to clarify the pathomechanism of the nephropathy. The most direct evidence for ensuring that the nephropathy in rats is due to the deposition of xanthine crystal would be the disappearance of the disorder in case of prevention of xanthine deposition. However, no such a study has ever been done because it is not easy to inhibit the XOR inhibitor-induced nephropathy in rats. At the same time, we believed that high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometer (LC-MS/MS) analyses were required to identify the entity of renal deposits.
Considering these issues, we performed the following mechanistic studies of FYX-051-induced nephropathy. In Experiment 1, rats were simultaneously treated with FYX-051 and citrate in order to elucidate the cause of nephropathy. In Experiment 2, renal deposits were analyzed to prove that these were xanthine crystals. In Experiment 3, urinary xanthine solubility was determined.
MATERIALS AND METHODS
Study on Simultaneous Treatment with Citrate (Experiment 1)
Animals and housing.
Five-week-old Crj:CD(SD)IGS male rats were purchased from Charles River Japan Inc. (Shiga, Japan). Throughout the acclimatization and experimental periods, the animals were housed individually in wire-mesh cages in an air conditioned animal room (room temperature, 22 ± 4°C; relative humidity, 60 ± 20%; lighting cycle, 12-h light/12-h dark). The animals without any abnormal findings after a one-week acclimatization period were selected for the present study. Pellet diet (CE-2, Clea Japan, Tokyo, Japan) and tap water via automatic stainless steel nozzles were made freely available throughout the study.
Test materials.
FYX-051 was synthesized by Fukuju Seiyaku (Toyama, Japan). Tri-sodium citrate 2-hydrate, tri-potassium citrate 1-hydrate, and citric acid 1-hydrate were purchased from Kishida Kagaku (Tokyo, Japan). The citrate used in the experiment was a mixture of its tri-sodium salt, tri-potassium salt, and free acid in a ratio of 2:2:1, calculated as anhydrous at molar base.
Experimental design.
The male rats were divided into five groups of eight animals each. The animals
Urinary pH, blood chemistry, toxicokinetics, autopsy, kidney weights, histopathology of urinary organ.
At week 4 (day 28) of administration, the urine was obtained from non-fasted animals to determine the pH using a pH meter (F-22, Horiba, Kyoto, Japan). After oral administration of FYX-051, the rats were transferred from their regular cages to metabolic cages for urine collection. During the 4-h urine collection, the rats were deprived of food but had free access to water and
Statistical analysis.
The mean and standard deviation of the blood chemical parameters, urinary pH, and kidney weights were calculated, and the difference between the control and treated groups was analyzed by the Dunnett's test (at significant levels of 5 and 1%). In addition, the incidence of renal histopathological lesions was analyzed by the Wilcoxon's test.
Analysis of Crystals in the Kidney (Experiment 2)
Collection of the renal deposits.
For analysis, samples were obtained from the yellowish-white deposits in the lumen of the renal pelvis of the SD rats treated orally with 3 mg/kg of FYX-051 for 13 weeks (see referential data mentioned below).
Assay of the renal deposits.
High performance liquid chromatography (HPLC) was used to determine the concentration of xanthine in the renal deposits. The collected deposits were dissolved in 25 mmol/l sodium hydroxide and chromatographed with a 2690 separation module (Waters, Milford, MA) using a Mightysil RP-18 Aqua column (4.6 mm i.d. x 250 mm, 5 μm particle size; Kanto Chemical, Tokyo, Japan) and eluted at a flow rate of 1.0 ml/min with 47 mmol/l sodium dihydrogenphosphate solution (pH 4.6). Detection was performed at 260 nm using 2487 detector (Waters, Milford, MA). Simultaneously, the eluate of xanthine fraction was collected and determined by a liquid chromatography-tandem mass spectrometer (LC-MS/MS). Detection was performed on API 4000 (Applied Biosystems, CA) and was operated by an infusion method. The eluted xanthine fraction was diluted with 10 mmol/l ammonium acetate solution and infused at a flow rate of 20 μl/min using a syringe pump model 11 (Harvard Apparatus, MA). Ionization was conducted using the TurboIon Spray and positive ion mode at 450°C. The precursor scan and product scan for m/z 153 and m/z 50–160, respectively were carried over the range based on collision energy for 5–130 eV (ramping parameter) using MCA scan mode. The HPLC retention time and product ion spectra of the analyte were compared with those of authentic xanthine (Sigma-Aldrich, St. Louis, MO).
Determination of Xanthine Solubility in Rat Urine (Experiment 3)
Urine collection.
Five-week-old Crj:CD(SD)IGS male rats were purchased from Charles River Japan (Shiga, Japan). After a two-week acclimatization period, four rats were transferred to metabolic cages for 48-h urine collection, during which the rats
Sample analysis.
For the measurement of xanthine solubility, urine was warmed to 37°C. Xanthine (Sigma-Aldrich, St. Louis, MO) was solubilized in urine 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 2 h at 37°C, the urine was filtered through a 0.45 μm pore size filter. An aliquot was diluted with water containing allopurinol as an internal standard and used for the measurement of xanthine concentration. Xanthine was determined by HPLC (pump, PU-980; auto sampler, AS-950; UV detector, UV-970, JASCO, Tokyo, Japan) by using a Mightysil RP-18 Aqua column (4.6 mm i.d. x 250 mm, 5 μm particle size; Kanto Chemical, Tokyo, Japan). Column temperature was maintained at 40°C, and xanthine detection was performed at 265 nm. Acetic acid solution (0.5%) was used as the mobile phase at a flow rate of 1.0 ml/min. At each pH, xanthine solubility was examined using urine with various concentrations of xanthine. The highest concentration of xanthine obtained at each pH was estimated as the xanthine solubility.
Thirteen-Week Dose Toxicity Study of FYX-051 in Various Animals (Referential Data)
Dogs
Animals and housing.
Nine- to ten-month-old male and female beagle dogs were obtained from the breeding facility of Shin Nippon Biomedical Laboratories (Kagoshima, Japan). Throughout the acclimatization and experimental periods, the animals were housed individually in stainless steel cages in an air conditioned animal room (room temperature, 23 ± 3°C; relative humidity, 55 ± 20%; lighting cycle, 12-h light/12-h dark). After a two-week acclimatization period, the animals without any abnormal findings were selected for the present study. Pellet diet (VE-10, Nippon Pet Food, Tokyo, Japan) and tap water via automatic stainless steel nozzles were made freely available throughout the study.
Experimental design.
The dogs were divided into four groups of three males and three females each. The animals
Laboratory investigations.
During the dosing and recovery periods, the parameters that were evaluated were as follows: signs, body weight, and food consumption. Before the initiation of dosing and at weeks 4 and 13 of dosing and week 4 of recovery, the following examinations were performed: ophthalmology, body temperature, electrocardiography, auditory reflex, urinalysis, hematology, blood chemistry, and toxicokinetics (by LC-MS/MS method). At the end of the dosing and recovery period, the following examinations were performed: gross pathology, organ weights, and histopathology. These examinations were conducted in accordance with the toxicity study guideline issued by the Japanese regulatory authorities.
Statistical analysis.
The mean and standard deviation of the parametric data were calculated, and the difference between the control and treated groups was analyzed by the Dunnett's test (at significant levels of 5 and 1%).
Monkeys
Animals and housing.
Three- to four-year-old male and female cynomolgus monkeys were purchased from Guangdong Scientific Instruments & Materials Import/Export Corporation (Guangzhou, China). The housing and quarantine conditions were the same as in the case of the dog study. Solid food (Teklad Global Certified 25% Protein Primate Diet, Harlan Sprague Dawley, U.K.) and tap water via automatic stainless steel nozzles were made freely available throughout the study.
Experimental design.
The experimental design for monkeys was set in the same manner as in the case of the dog study. However, justification for selection of the dose levels is as follows. In a two-week oral dose toxicity study of FYX-051 in monkeys, no toxicity was seen at dosages of up to 100 mg/kg. Therefore, to compare the toxicity between dogs and monkeys, the highest dose level for this study was set at 100 mg/kg.
Laboratory investigations.
The observation and examinations were performed in the same manner as in the case of the dog study.
Statistical analysis.
The statistical analysis was performed in the same manner as in the case of the dog study.
Rats
Animals and housing.
The animals that were purchased, the acclimatization, and selection procedure for the study were the same as described in Experiment 1. The housing conditions were as follows: room temperature, 22 ± 3°C; relative humidity, 50 ± 20%; lighting cycle, 12-h light/12-h dark. Pellet diet (CR-LPF, Oriental Yeast, Tokyo, Japan) and tap water via automatic stainless steel nozzles were made freely available throughout the study.
Experimental design.
The rats were divided into four groups of 10 males and 10 females each. The animals
Laboratory investigations.
During the dosing period, signs, body weight, and food consumption were recorded. Furthermore, the following examinations were performed: ophthalmology and urinalysis (before the initiation of dosing and at weeks 4 and 13 of dosing), hematology, blood chemistry, gross pathology, organ weights, and histopathology (at the end of dosing), and toxicokinetics (by LC-MS/MS method). These examinations were conducted in accordance with the toxicity study guideline issued by the Japanese regulatory authorities.
Statistical analysis.
The statistical analysis was performed in the same manner as in the case of the dog study.
RESULTS
Study on Simultaneous Treatment with Citrate (Experiment 1)
The pH of the urine was 7.83 ± 0.26, 7.22 ± 0.34, 6.86 ± 0.11, 7.96 ± 0.22, and 7.94 ± 0.10 in the control (citrate), 1 mg/kg FYX-051, 3 mg/kg FYX-051, 1 mg/kg FYX-051 + citrate, and 3 mg/kg FYX-051 + citrate groups, respectively; the pH was higher by approximately 1 in the simultaneous treatment with citrate groups than the FYX-051 alone groups (Table 1). Blood chemistry revealed a significant increase in the creatinine and BUN levels (0.75 ± 0.14 and 50 ± 8 mg/dl, respectively) in the 3 mg/kg FYX-051 group as compared to the control group values (0.31 ± 0.03 and 19 ± 4 mg/dl, respectively) (Table 1).
Relative kidney weights were 0.87 ± 0.06, 1.01 ± 0.27, 1.70 ± 0.32, 0.86 ± 0.04, and 0.87 ± 0.07 in the control, 1 mg/kg FYX-051, 3 mg/kg FYX-051, 1 mg/kg FYX-051 + citrate, and 3 mg/kg FYX-051 + citrate groups, respectively; the 3 mg/kg FYX-051 alone group showed a significant increase in kidney weights when compared with the control (Table 1). Treatment with citrate exerted no remarkable effect on the toxicokinetic profile of FYX-051 (Table 1). Autopsy revealed gross changes, such as yellowish-white foci and rough surface in the kidney and yellowish-white granular deposits on the cut surface of the kidney in the FYX-051 alone groups, and one incidence of a yellowish-white macula in the 3 mg/kg FYX-051 + citrate group.
Histopathological examination revealed minimal to moderate and moderate to severe interstitial nephritis in six of the eight rats and in all the eight rats in the 1 and 3 mg/kg FYX-051 alone groups, respectively, and extensive lesions were observed throughout the cortex and medulla (Table 2; Figs. 1A, 2A, and 3A). 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, crystals and cell debris in renal tubules and collecting ducts, crystals in the renal pelvic lumen, and papillary or pelvic epithelial hyperplasia. The presence of crystal deposits was remarkable in the distal tubules. In contrast, in the simultaneous treatment with citrate groups, no renal alterations, except minimal interstitial nephritis in one of the eight rats in the 3 mg/kg FYX-051 + citrate group were seen (Table 2; Figs. 1B, 2B, and 3B).
With regard to the urinary tracts, no remarkable changes were observed in the ureters and urinary bladder in any of the groups, including the control (citrate alone) group.
Analysis of Crystals in the Kidney (Experiment 2)
Chromatogram at 260 nm detected a main peak of the renal deposits; the peak showed the same retention time as that of xanthine (Fig. 4). Quantitative analysis of the main peak (similar to that of xanthine) revealed that 85.5% of the renal deposits were composed of xanthine. On comparison of the MS/MS spectrum of main constituent in renal deposits with that of authentic xanthine, the main fragment and parent ions were detected for m/z 64, 110, 136, and 153 (parent ion [M+H]+) and these corresponded with both samples (Fig. 5).
Xanthine Solubility in Rat Urine (Experiment 3)
Xanthine solubility in rat urine was pH-dependent (Table 3). Within a pH range of 5–7, xanthine solubility slightly increased; 155 mg/dl at pH 5 and 167 mg/dl at pH 7. At a pH greater than 8, xanthine solubility remarkably increased, i.e., xanthine solubility at pH 8 and 9 was 2.0 and 3.9 times higher than that at pH 7, respectively.
Thirteen-Week Dose Toxicity Study of FYX-051 in Various Animals (Referential Data)
Dogs.
No animals died during the study period, and no remarkable symptoms were observed, except transient minimal reduction of body weights at week 1 of dosing in one male receiving 100 mg/kg FYX-051 (Table 4). Urinalysis revealed the presence of minute yellow granular materials in sediments, which were suggestive of xanthine crystals, but no abnormality was detected in the other parameters. At autopsy, yellowish-white granular materials were present on the cut surface of the kidney in the 100 mg/kg group, and one female of the same group exhibited a large calculus in the left pelvic lumen. Histopathological examination revealed epithelial hyperplasia of the papilla and pelvis due to foreign materials (xanthine crystals) in the pelvic lumen at dosages of 30 mg/kg FYX-051 and above. This was seen unilaterally and focally, with minimal severity and low incidence at 30 mg/kg FYX-051, while the finding appeared to be slightly stronger at 100 mg/kg. Accordingly, this change was considered to be of little toxicological significance because of the localized nature of hyperplasia and absence of renal functional disorder. On the other hand, one female dog mentioned above exhibited a tendency toward elevated serum creatinine levels and showed changes in the tissues around the pelvis due to formation of a large calculus indicative of physical irritation, and alterations of renal distal tubules. The distal tubular changes were also considered to be the result of repeated stimulation due to the xanthine deposited in the distal tubule under the condition of urinary excretion disorder. Therefore, a series of renal changes observed in this female were regarded to be a renal functional disorder, although these changes were induced by secondary effects of intrarenal xanthine deposits. At the end of the four-week recovery period, no histopathological alterations were seen in the 100 mg/kg FYX-051 group, despite the presence of yellowish-white granular materials in the pelvic lumen. In general, in the toxicokinetics study, Cmax and AUC levels increased with the dose in both males and females.
Based on the above results, the no observed adverse effect level (NOAEL) of FYX-051 in this study was estimated to be 30 mg/kg.
Monkeys.
No animals died during the study period, and no remarkable changes were observed in the general conditions, including signs and body weights (Table 5). Treatment-related changes were not observed during clinical examinations. In addition, no abnormalities were noted in the pathological examination of all the organs and tissues. In the toxicokinetics study, Cmax and AUC levels increased with the dose in both males and females.
Based on the above results, the NOAEL of FYX-051 in this study was estimated to be greater than 100 mg/kg.
Rats.
No animals died during the study period. In males of the 3 mg/kg FYX-051 group, body weight gains were inhibited at week 1 of dosing. Blood chemistry revealed an increase in BUN and creatinine levels in the 3 mg/kg FYX-051 group. Autopsy revealed gross changes, such as yellowish-white foci, rough surface, and yellowish-white granular materials on the cut surface of the kidney, in groups that
Based on the above results, the NOAEL of FYX-051 in this study was estimated to be 0.3 mg/kg.
DISCUSSION
It is known that treatment with XOR inhibitors in rodents induces striking intrarenal xanthine deposition (Hitchings, 1966; Horiuchi et al., 1999). However, these reports do not provide a definite evidence of nephropathy being caused by xanthine deposition alone or of the renal deposits being composed of xanthine. Based on the result that the retention time of the main peak of the test sample was the same as that of xanthine, the present analysis by HPLC showed that the renal deposits in the rats could be composed of xanthine crystals. In addition, LC-MS/MS analysis demonstrated a correspondence in the spectrum between authentic xanthine and the renal deposits.
Thus far, no study has demonstrated the successful amelioration of XOR inhibitor-induced nephropathy in rats by prevention of xanthine deposition. It may be possible that on account of the large amount of xanthine excretion in rat urine, it was difficult to prevent xanthine deposition in the kidney. In order to minimize the potential hazard of xanthinuria in humans during allopurinol administration, the alkalinization of urine and increase in the fluid intake are recommended (Klinenberg et al., 1965). Based on this information, we examined the effects of the urine alkalinizing agent citrate with a large amount of water on the nephropathy caused by FYX-051 administration in rats. The treatment described above, without affecting the toxicokinetic profile of FYX-051, showed that urinary pH increased from 7 to approximately 8; this elevation was attributable to rapid increase in xanthine solubility in the urine. As a result, intrarenal xanthine deposition was reduced, leading to disappearance of renal alterations comprising small round cell infiltration, dilatation and basophilic changes of renal tubules, and so forth. These results provided us with the definite evidence that a series of renal alterations were caused by the deposition of xanthine crystals during the process of concentrating the urine and showed that nephropathy could not be caused by the direct action of this agent.
Citrate was used as a mixture of its sodium salt, potassium salt, and free acid in a ratio of 2:2:1 at molar base. It has been known that citrate is metabolized via the TCA cycle to yield bicarbonate that exerts its urine alkalinizing effect. In our preliminary experiment, the use of free citric acid alone lessened this effect, probably because some amount of unchanged citric acid is excreted into the urine, resulting in a depression of its urine alkalinizing effect. Administration of sodium salt alone is not appropriate because it forces the kidneys to excrete a large amount of sodium. As a result, drastic disappearance of renal lesions induced by FYX-051 is made possible by the administration of citrate as a mixture of its sodium and potassium salts, without any associated adverse effects.
The solubility of xanthine in urine obtained in the present study is higher than that in the previous report (Klinenberg et al., 1965) in which the xanthine solubility was 5 mg/dl at pH 5 and 13 mg/dl at pH 7. The discrepancy in the solubility between the two studies is probably attributable to the difference in the method used. In the previous study, xanthine solubility was determined by simply dissolving xanthine in urine. On the other hand, in the present study, xanthine was dissolved under alkaline conditions, and the urine pH was adjusted. In consideration of the fact that xanthine is likely to get deposited during the process of urine condensation that occurs in the distal tubules and collecting ducts in the kidney, it is reasonable to consider that the maximum concentration of xanthine that remains dissolved in urine is of greater significance. In fact, the dissolved xanthine concentration in urine from rats administered 1–3 mg/kg FYX-051 was approximately 100 mg/dl (data not shown). From these results, we conclude that the xanthine solubility obtained in the present study is more appropriate than the previous values as an index of xanthine deposition in the kidney of animals treated with XOR inhibitors.
The pathomechanism of nephropathy could be speculated as follows. Treatment with FYX-051 induced high blood xanthine levels due to its pharmacological activity, and consequently, renal xanthine deposition occurred mainly during the process of urine-condensation from distal tubules to collecting ducts. When the amount of deposited xanthine exceeds the capacity of the kidney to excrete foreign materials, renal tubules and collecting ducts (particularly remarkable in distal tubules) are occluded by these deposits, leading to secondary interstitial nephritis that is characterized by inflammatory cell infiltration, epithelial basophilia or necrosis and dilatation of the renal tubules and collecting ducts, increased interstitial connective tissue, and cell debris in the renal tubules and collecting ducts in extensive areas over the cortex and medulla. This speculation concerning the occurrence of renal changes would be supported by the 13-week dose toxicity study in dogs; renal xanthine deposition occurred in both rats and dogs, but the severity was considerably weaker in the latter than in the former. Considering the findings that xanthine crystals were markedly scattered in the distal tubules, the distal tubules appear to be the main site of crystal deposition. Accordingly, the present results suggest that there is a clear relation between crystal deposition and condensing magnitude of urine. Furthermore, papillary or pelvic epithelial hyperplasia is also likely to be due to the physical irritation caused by the xanthine calculi formed in the renal pelvic lumen. Such renal lesions are considered to be a common phenomenon in treatment with XOR inhibitors.
With regard to the effects of sodium citrate in the lower urinary tracts, Fukushima et al. (1986) have demonstrated that sodium citrate exerts promoting effects on urinary bladder carcinogenesis in the experiment with 32-week dietary administration of a basal diet containing 5% sodium citrate to F344 male rats following a four-week initiation with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water. However, no epithelial alterations were observed in the group without a BBN supplement. Similarly, in the present study, no histopathological changes were observed in the ureter and urinary bladder tissues. While there was a relationship between the elevation of urinary pH and proliferative changes in the urothelium, it is likely that administration of sodium citrate alone would not affect the urothelium. Taken together, the present findings showing consistency with the observation of Fukushima et al. would be of appropriate results.
With regard to species difference in nephrotoxicity, the urinary xanthine solubility assay indicated that renal deposits of xanthine would occur more easily in rats because the value was increased in a pH-dependent fashion, and urinary pH was lower in rats than in dogs or monkeys (see Tables 1, 4, and 5). Toxicokinetics data of each animal suggested that exposure levels would be higher in rats than in dogs or monkeys because the AUC value of rats administered FYX-051 at a dosage of 3 mg/kg was similar to those of dogs and monkeys administered FYX-051 at a dosage of 10 mg/kg. Therefore, these results may illustrate the fact that severe nephropathy occurred in rats when compared with dogs or monkeys.
With regard to the safety extrapolation in humans, it has been suggested there is an extremely low possibility that nephropathy might be induced in humans by treatment with FYX-051. This can be explained based on the following fact that the amount of purine metabolite excreted daily are far higher in rats than in humans (Hitchings, 1966; Horiuchi et al., 1999); this data has been confirmed by us. Regarding the xanthine deposited in the urinary tract or renal pelvis during the clinical use of allopurinol, there have been no documented cases with the exception of a special case in which the rate of urate formation was greatly increased, such as that observed in Lesch-Nyhan syndrome with associated purine metabolism abnormality (Brock et al., 1983; Greene et al., 1969; Kranen et al., 1985; Mizuno et al., 1976; Ogawa et al., 1985). In contrast, some studies reported that large doses of allopurinol produce stones in the urinary tract that result from oxypurinol (main metabolite) in humans (Landgrebe et al., 1975; Stote et al., 1980). In case of FYX-051, the possibility of metabolite crystallization in humans is likely to be extremely low, considering the following facts. Other compounds, except xanthine, were not detected in the present analysis of crystals deposited in the kidney, and highly soluble N-glucuronides could be estimated to be the main metabolites of FYX-051.
In conclusion, the present study demonstrated 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.
ACKNOWLEDGMENTS
We would like to thank Prof. Kunitoshi Mitsumori, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, for his advice. We are deeply indebted to Dr. Hiroshi Tokado, Department of Safety Research, Shin Nippon Biomedical Laboratories, Ltd., for analysis of renal deposits from the rats and toxicological evaluation. We also wish to thank Mr. Kazuhiko Oba, Research Laboratories 2, Fuji Yakuhin Co., Ltd., for his excellent technical support. Conflict of interest: none declared.
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