European Journal of Pharmacology, 241 (1993) 183-188
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
Hypouricemic effect of the novel xanthine oxidase inhibitor, TEI-6720, in rodents Y o s h i o O s a d a , M a s a h i r o T s u c h i m o t o , Hisashi F u k u s h i m a , K a t s u s h i T a k a h a s h i , Shiro K o n d o , M a s a i c h i H a s e g a w a a n d Keiji K o m o r i y a Teijin Institute for Bio-medical Research, Asahigaoka 4-3-2, Hino, Tokyo 191, Japan
Received 25 February 1993,revised MS received 21 June 1993, accepted 6 July 1993
We investigated the xanthine oxidase/xanthine dehydrogenase inhibitory activity and hypouricemic effect of a newly synthesized xanthine oxidase/xanthine dehydrogenase inhibitor, TEI-6720, 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid, and compared its effects with those of allopurinol in rodents. TEI-6720 was found to inhibit bovine milk xanthine oxidase, and mouse liver and rat liver xanthine oxidase/xanthine dehydrogenase with IC50 values of 1.4, 1.8 and 2.2 nM, respectively. On bovine milk xanthine oxidase, TEI-6720 exhibited mixed-type inhibition and the K i value was 0.7 nM. TEI-6720 displayed prolonged urate-lowering activity in normal mice and rats. We evaluated the hypouricemic effect of TEI-6720 on hyperuricemia induced by the uricase inhibitor, potassium oxonate (250 mg/kg s.c., 1 h before the test drugs), and measured the total molarity of both serum allantoin and urate in rats. Oral TEI-6720 and allopurinol had a hypouricemic effect 2 h after their administration to oxonate-pretreated rats with ED50 values of 1.5 and 5.0 mg/kg, respectively. Both compounds also reduced the combined molarity of uric acid and allantoin in rats. The ED5o values of TEI-6720 and allopurinol were 2.1 and 6.9 mg/kg p.o., respectively. These results suggest that TEI-6720 may be useful for the treatment of hyperuricemia. TEI-6720; Allopurinol; Xanthine oxidase/xanthine dehydrogenase; Uric acid; Hyperuricemia
1. Introduction Hyperuricemia has long been considered the most important risk factor for the onset of gout (Hall et al., 1967; Campion et al., 1987). The development of hyperuricemia may be due to an excessive rate of uric acid production, a decrease in renal excretion of uric acid, or a combination of both (Nugent and Tyler, 1959). Urate-lowering therapy to prevent the onset of gout has been performed for some time. Two types of drugs are used to treat hyperuricemia, uricosuric agents and xanthine o x i d a s e / x a n t h i n e dehydrogenase inhibitors. Uricosuric agents, e.g. probenecid or benzbromarone, block uric acid reabsorption at the luminal membrane of the renal proximal tubules (Sinclair and Fox, 1975). These drugs have certain disadvantages with respect to clinical application, i,e. the necessity of alkalinizing the urine and the inability to use them in patients with impaired renal function. Allopurinol is
Correspondence to: Keiji Komoriya,Teijin Institute for Bio-medical Research, Asahigaoka 4-3-2, Hino, Tokyo, 191, Japan. Tel. 81-42586-8251, fax 81-425-87-5516.
the only commercially available drug of the second type, xanthine oxidase/xanthine dehydrogenase inhibitors, and first became available about 30 years ago (Rundles e.t al., 1963). Patients classified as overproducing uric acid represent about 10% of the gouty population. Allopurinol is indicated when uricosuric drugs fail to reduce serum urate to below 7.0 mg per dl or when patients are intolerant of uricosuric agents, as well as in gout patients with hyperuricemia associated with increased uric acid production and with renal insufficiency (Kelley and Schumacher, 1993). Allopurinol gives rise to certain severe adverse effects, such as hepatitis, nephropathy and allergic reactions (Hande et al., 1984). Although xanthine oxidase/xanthine dehydrogenase inhibitory activity has been discovered in newly synthesized compounds (Wortmann et al., 1985; Sato et al., 1991) and previously known compounds (Bindoli et al., 1985; Hall et al., 1990), none of these are currently used to treat hyperuricemia. We now report that TEI-6720, 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid (fig. 1), displays mixed-type inhibition of bovine milk xanthine oxidase and has a hypouricemic effect more potent than that of allopurinol in rodents.
Nc o2H Fig. 1. Chemical structure of TEI-6720: 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid.
2. Materials and methods
2.1. Animals Male ICR strain mice (6-8 weeks old) and male Sprague-Dawley strain rats (4-5 weeks old) were purchased from Charles River Japan, Inc. (Kanagawa, Japan). They were allowed one week to adapt to their environment before being used. All the animals were kept in an air-conditioned room and given standard chow and water ad libitum for the duration of the study.
2.2. Materials The following materials were used: TEI-6720 synthesized in our own laboratory, allopurinol, xanthine, nicotinamide adenine dinucleotide, bovine milk xanthine oxidase (Sigma Chemicals Co., St. Louis, MO), potassium oxonate (Aldrich Chemical Co., Milwaukee, WI), uric acid, allantoin and the Uric acid-Test Wako Kit (Wako Pure Chemical Industries Ltd., Osaka, Japan).
2.3. Xanthine oxidase assay Bovine milk xanthine oxidase (1 m U / m l ) was incubated with 15 ~M xanthine in the presence and absence of the test compound at 25°C. Uric acid formation was determined by absorbance at 292 nm using a Hitachi U-3200 or a Beckman DU-3200 spectrophotometer, and the IC50 values of the test compounds were determined. The initial rate was calculated from the linear portion of each reaction, from 0.5 to 2.5 min in most cases. K i values were calculated by the replot method. Mouse and rat liver xanthine oxidase/ xanthine dehydrogenase was used in some experiments. Mouse or rat liver was homogenized in 50 mM phosphate buffer (pH 7.4) and centrifuged at 100000 × g for 1 h at 4°C, and the supernatant was used as the liver xanthine oxidase/xanthine dehydrogenase. In these experiments, the concentration of the liver xan-
thine oxidase/xanthine dehydrogenase was adjusted to approximately the same 1 m U / m l as bovine milk xanthine oxidase, and the reaction buffer contained 200 g M nicotinamide adenine dinucleotide as the electron acceptor for the xanthine dehydrogenase reaction.
2.4. Hypouricemic effect in mice and rats The test compounds were administered orally, and blood was taken from the abdominal aorta 1 to 8 h after administration. The blood was allowed to clot for 0.5-1 h at room temperature and then centrifuged. The sera were stored at -20° C until assayed. In some experiments, rats were s.c. injected with the uricase inhibitor, potassium oxonate (250 mg/kg), 1 h before drug administration in order to inhibit uricase and increase serum urate levels.
2.4.1. Measurement of uric acid In most experiments, the phosphotungstic acid method (Uric acid-Test Wako Kit) was used (Henry et al., 1957), because the uricase-peroxidase method cannot be utilized for sera from potassium oxonate-treated rats. 2.4.2. Measurement of uric acid and allantoin by HPLC Pooled sera from five rats in each group were mixed with the same volume of 0.6 M perchloric acid. After centrifugation at 2 000 × g for 10 min at 4°C, 0.7 ml of the supernatant was neutralized with approximately 0.31-0.33 ml of 1.0 M sodium hydroxide, and the total volume was adjusted to 1.4 ml with distilled water. Serum allantoin levels were measured by using highperformance liquid chromatography (HPLC). Separation was performed using a column packed with polyhydroxyalkyl methacrylate gel, Shodex OH pak KB802.5, and a 0.1 M K2HPO 4 (pH 8.4) mobile phase. A pre-column (Shodex OH pak B-800P) was used to protect the analytical column. The flow rate was 0.8 ml/min. Allantoin and uric acid levels were measured by absorbances at 210 and 292 nm, respectively. 2.5. Test drugs All test drugs were dissolved in dimethylsulfoxide (DMSO) and diluted with 50 mM sodium phosphate buffer (pH 7.4) for the in vitro experiments. The final concentration of DMSO in the reaction solution was 0.1%. For in vivo experiments, drugs were suspended in 5% gum Arabic solution at a constant volume of 5 m l / kg for rats and 10 m l / kg for mice.
2.6 Statistics The data from the in vivo experiments are expressed as means + S.E.M., and the results were statistically evaluated using the Dunnett t-test.
3.1. Inhibition of the various xanthine oxidase /xanthine
dehydrogenases by TEI-6720 3000
TEI-6720 inhibited bovine milk xanthine oxidase, and mouse liver and rat liver xanthine oxidase/ xanthine dehydrogenase. The IC50 values or K i values for TEI-6720 or allopurinol are shown in table 1. The inhibitory activity of TEI-6720 was much greater than that of allopurinol. Figure 2 shows the Hanes-Woolf plot for inhibition of the bovine milk xanthine oxidase by TEI-6720. TE16720 appears to possess mixed-type inhibitory activity.
3.2 Hypouricemic effect of TEI-6720 in normal mice As shown in fig. 3, orally administered TEI-6720 decreased serum urate levels in normal mice. This effect was more potent and longer lasting than that of allopurinol. The hypouricemic effect of TEI-6720, but not of allopurinol, was still observed 6 h after administration. The EDs0 values of TEI-6720 and allopurinol 2 h after administration were 0.7 and 2.7 mg/kg, respectively (data not shown).
3.3. Hypouricemic effect of TEI-6720 in normal rats Figure 4 shows the time course of the urate-lowering activity of TEI-6720 in rats. TEI-6720 was as potent as allopurinol 1 to 2 h after administration, and its duration of action was slightly longer than that of allopurinol.
3.4. Hypouricemic effect of TEI-6720 in potassium oxonate-pretreated rats The purine metabolism of rodents is different from that of humans. In rodents uric acid is metabolized further into allantoin by uricase, while in humans, who lack uricase, the final metabolite of purine is uric acid. We, therefore, evaluated the hypouricemic effect of
[Xanthine] (/z M) Fig. 2. Kinetic analysis of inhibition of bovine milk xanthine oxidase by TEI-6720. The Hanes-Woolf plot shows mixed-type inhibition by TEI-6720. Xanthine (in final concentrations of 5, 7.5, 10, 12.5 and 15 /zM) and 1 m U / m l xanthine oxidase were incubated with or without TEI-6720. The initial rate of each reaction was determined on the basis of the rate of increase in optical density at 292 nm from 0.5 to 2.5 min. Closed circles (o), triangles ( • ) , and squares (11) represent the control, TEI-6720 (0.5 nM) and TEI-6720 (1.0 nM), respectively. The data represent the means-t- S.E.M. of three different reactions.
TEI-6720 on hyperuricemia induced by the uricase inhibitor, potassium oxonate, and measured urate and allantoin levels in normal rats in order to estimate the effect of the test drugs in humans. S.c. injection of potassium oxonate (250 mg/kg) caused a marked increase in serum urate levels, and this increase was prolonged up to 5 h after the injection (data not shown). As shown in fig. 5, TEI-6720 also had a hypouricemic effect in oxonate-pretreated rats. The ED50 values of TEI-6720 and allopurinol 2 h after the dose were 1.5 and 5.0 mg/kg, respectively.
to Q) >
TABLE 1 Summary of xanthine oxidase/xanthine dehydrogenase inhibition by TEI-6720 and ailopurinol. Xanthine oxidase/ xanthine dehydrogenase
K i (nM)
Bovine milk Mouse liver Rat liver Bovine milk
1.4 1.8 2.2 0.7
1700 380 1 100 280
Type of inhibition
IC50 (nM) IC50 (nM) IC50 (nM)
Inhibition type was determined on the basis of Hanes-Woolf plots. K i values were calculated by the replot method,
Time after administration (h) Fig. 3. Effect of TEI-6720 and allopurinol on serum urate levels in mice. Blood was collected by cardiac puncture 2 and 6 h after administration. Serum uric acid levels were determined by the phosphotungustic acid method. Black, gray and shaded bars represent the control, TEI-6720 (1 mg/kg) and allopurinol (1 mg/kg), respectively. The data represent the means :t: S.E.M. for five animals. * P < 0.05, • * P < 0.01: significantly different from the control (Dunnett t-test).
E 2.0. ¢n
> (D ._J o)
to ¢D >
Dose (mg/kg p.o.)
Time after administration (h)
Fig. 5. Effect of TEI-6720 and allopurinol on serum urate levels in rats pretreated with the uricase inhibitor, potassium oxonate. Rats were treated with potassium oxonate (250 mg/kg) 1 h before administration of the test drugs. Blood was sampled from the abdominal aorta 2 h after administration. The data represent the means + S.E.M. for five animals. * P < 0,05, * * P < 0.01, * * * P < 0.001: significantly different from the control (Dunnett t-test).
~b) AIIopurinol v
Time after administration (h) Fig. 4. Time course of the effect of TEI-6720 and allopurinol on serum urate levels in rats. Blood was collected from the abdominal aorta 1 to 8 h after administration. Graphs (a) and (b) represent the effects of TEI-6720 and aUopurinol, respectively. Crosses ( x ), circles (e) squares ( • ) and triangles ( • ) represent values after doses of 0, 1, 3 and 10 m g / k g , respectively. The data represent the means:l: S.E.M. for five animals. * P < 0.05, * * P < 0.01, * * * P < 0.001: significantly different from the control (Dunnett t-test).
available xanthine oxidase/xanthine dehydrogenase inhibitor for the treatment of hyperuricemia and gout. Xanthine oxidase reversibly converts between reduced and oxidized forms. Allopurinol is metabolized to oxipurinol by xanthine oxidase/xanthine dehydrogenase. By tightly binding to the reduced form, oxipurinol is a true dead-end inhibitor of xanthine oxidase (Spector, 1977, 1988), and is eliminated from the kidney in the same manner as uric acid (Elion et al., 1968). Oxipurinol clearance is affected by the glomerular filtration rate and uric acid clearance, i.e. the half-life of oxipurinol is very long in patients with renal insufficiency, and allopurinol sometimes causes severe adverse effects in such patients (Elion et al., 1968; Hande et al., 1984; Murrell and Rapeport, 1986; Cameron and Simmons, 1987). Although several xanthine oxidase/xanthine dehydrogenase inhibitors have been described (Wort-
3.5. Serum urate- and allantoin-lowering activity of TE16720 in normal rats As shown in fig. 6, TEI-6720 reduced the total molarity of urate and allantoin 2 h after the dose. This effect was about three times more potent than that of allopurinol; the EDs0 values of TEI-6720 and allopurinol were 2.1 and 6.9 mg/kg, respectively. These results seem to be consistent with the hypouricemic potency of these drugs in the oxonate-pretreated rats.
400  I
_.m E 2 loo ¢D
In man, uric acid is formed primarily by the xanthine oxidase/xanthine dehydrogenase-catalyzed oxidation of hypoxanthine and xanthine. For many years, allopurinol has been used as the only commercially
Dose (mg/kg p.o.) Control
Fig. 6. Effect of TEI-6720 and allopurinol on serum urate and allantoin levels in rats. Pooled sera from five rats in each group were assayed by HPLC. Black and gray bars represent the concentrations of urate and aUantoin, respectively.
mann et al., 1985; Bindoli et al., 1985; Hall et al., 1990; Sato et al., 1991), none of them are currently being used clinically. For this reason we evaluated newly synthesized compounds, and in the course of our evaluations discovered a potent xanthine oxidase/xanthine dehydrogenase inhibitor, TEI-6720. TEI-6720 inhibited xanthine oxidase/xanthine dehydrogenase much more potently than allopurinol (table 1). In the case of bovine milk xanthine oxidase, TEI-6720 exhibited mixed-type inhibition (fig. 2), whereas allopurinol displayed competitive inhibition under the same assay conditions (data not shown). TEI-6720 reduced the serum uric acid level. This effect was more potent and enduring than that of allopurinol in mice (fig. 3). TEI-6720 also displayed a hypouricemic effect approximately as potent and enduring as that of allopurinol in rats (fig. 4). Although TEI-6720 exhibited greater potency in vitro, the hypouricemic effect of TEI-6720 was comparable to that of allopurinol in vivo. This may be explained by the hypouricemic effect of allopurinol in vivo being actually produced by its metabolite, oxipurinol. Oxipurinol binds to the reduced form of xanthine oxidase with a dissociation constant (K d) value of 0.5 nM (Spector, 1977). The net in vitro activity of both compounds appears to be comparable, because the K i value of TEI-6720 is approximately equal to the K d value of oxipurinol. Since, in rodents, uric acid is further metabolized into allantoin by uricase, both compounds must be evaluated in animals treated with a uricase inhibitor, or allantoin levels must be determined in normal animals, in order to estimate the urate-lowering activity of the compounds in humans. TEI-6720 reduced the serum uric acid levels more potently than allopurinol in both these animal models (fig. 5). In contrast to its effects in normal rats, allopurinol had a weak hypouricemic effect in these models. These findings suggest that the net in vivo activity of allopurinol may be overestimated in normal rats. The precise mechanism for this phenomenon is unclear, but an explanation can be attempted. First, since allopurinol may enhance the conversion of uric acid into allantoin, we tried to determine whether bovine kidney uricase activity is affected by TEI-6720 or allopurinol. Although neither compound had any effect in vitro (data not shown), an enhancing effect of allopurinol on the conversion to allantoin in vivo could not be ruled out. Second, allopurinol may inhibit allantoin excretion by the kidney. This explanation seems unlikely to apply, because allopurinol displayed almost the same activity in both of the above models. In any event, the activity of xanthine oxidase inhibitors in rats must be compared cautiously. These data indicate that TEI-6720 may possess clinical efficacy in humans. Allopurinol is a purine derivative, and its metabo-
lite, oxipurinol, is eliminated from the kidney in the same manner as uric acid (Elion et al., 1968). Allopurinol sometimes causes severe adverse effects in patients with renal insufficiency (Hande et al., 1984; Cameron and Simmons, 1987), because the half-life of oxipurinol is very long in such patients. Moreover, both allopurinol and oxipurinol are known to undergo conversion to their corresponding nucleotides in vivo (Nelson et al., 1973). These adverse effects may be avoidable, since TEI-6720 is not a purine derivative (fig. 1). In conclusion, in rodents, the newly synthesized xanthine oxidase inhibitor, TEI-6720, is more potent than allopurinol both in vitro and in vivo. This compound may become an alternative effective drug for use in the treatment of hyperuricemia and gout.
Acknowledgement We gratefully acknowledge the excellent technical assistance of Akiko Matsuzawa-Kojima and Yumiko Urabe.
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