A New Uricosuric , S-8666, in Rats and Chimpanzees

Yukio YONETANI, Kazumi IWAKI, Toshihiro SHINOSAKI, Atsuko KAWASE-HANAFUSA, Hiroshi HARADA and Arthur A. van ES* ShionogiResearch Laboratories, Shionogi & Co., Ltd., Fukushima-ku,Osaka 553, Japan *Primate Center TNO , Lange Kleiweg151, 2288 GJ Rijswijk,The Netherlands AcceptedJanuary 6, 1987

Abstract-5 Dimethylsulfamoyl-6,7-dichloro-2,3-dihydrobenzofuran-2-carboxylic acid (S-8666) was studied as a possible new uricosuric diuretic agent using rats and chimpanzees. Various new compounds belonging to the 5-sulfamoyl-6,7 dichloro-2,3-dihydrobenzofuran-2-carboxylic acids were clearly diuretic with uricosuric activity in intraperitoneally oxonate-treated rats. S-8666 was chosen as a favorable candidate because its uricosuric activity due to the effects of tubular transport of were apparently more marked than those of known uricosuric agents such as probenecid, benzbromarone, and indacrinone in oxonate treated rats. S-8666 was also uricosuric in rats not given urate oxidase inhibitor. The diuretic effect of S-8666 in oxonate-treated rats was as high-ceilinged as that of , while those of tienilic acid, indacrinone and a known compound of a 5-carbonyl-6,7-dichloro-2,3-dihydrobenzofuran-2-carboxylic acid were rather low-ceilinged. These uricosuric and diuretic activities of S-8666 were manifested by two enantiomers, of which the (+)-enantiomer displayed predominantly uricosuric activity and the (-)-enantiomer, diuretic activity like furosemide. The new compound was also uricosuric and diuretic in chimpanzees, although the potency of the uricosuric activity was similar to that of probenecid and less than that of indacrinone. Thus, it seems that S-8666 is a different type of uricosuric diuretic from known agents which have already been tried in humans.

Diuretic and various loop excretion of uric acid. To help alleviate this have been generally utilized as problem, we recently devised a practical useful antihypertensive agents, but the method employing a commonly used experi hyperuricemic effect incident to their major mental animal, the rat, which was treated effects has sometimes necessitated their with an inhibitor of urate oxidase, potassium withdrawal (1-3). Uricosuric (generally used oxonate (11). Rats are different from primates to mean 'hyperuricosuric') diuretics have in how they metabolize uric acid to allantoin been studied over the past decade to im in the liver, but the net flux of uric acid in prove diuretic antihypertensives. the renal tubules is clearly reabsorptive (12). At first, tienilic acid (4) was considered to Therefore, the animals have sometimes been be an excellent uricosuric diuretic, which used to support the uricosuric activity of could lower the blood level of uric acid test compounds (13, 14). However, the during medication, but its hepatotoxicity results do not always demonstrate the effect forced it to be banned from clinical use (5). of test compounds on the renal clearance of This accelerated further studies on a new uric acid under experimental conditions using generation of diuretic antihypertensives (6 animals displaying both hepatic metabolism 10), but their development as drugs has been and renal excretion of uric acid, and hence delayed because the evaluation of uricosuric the animals are not feasible for evaluating the activity requires tests on primates, due to uricosuric activity of new compounds. On species differences in the metabolism and the other hand, oxonate-treated rats also had a reabsorptive net flux of uric acid in the renal collected. Plasma was separated immediately tubules and responded well to various known after the blood collection using a Beckman agents which had already been demonstrated Microfuge. Plasma concentrations of uric to have effects on uric acid excretion in acid and inulin paired with those in the urine humans, although all responses were not sample were assessed as the average of their always identical to those in primates. In values at the beginning and end of the urine addition, an experiment using rats is much collection. Test compounds were adminis easier than that using primates. It thus seems tered intraperitoneally just after the first urine that the clearance experiment using oxonate collection as a suspension in 1 % gum arabic treated rats is feasible for the first screening solution. Experiments using oxonate of favorable candidates from various new untreated rats were done according to the compounds. In the present study, 5-dimethyl method described previously (15). Fourteen sulfamoyl-6,7-dichloro-2,3-dihydrobenzo week-old male SIc-Wistar rats were infused fu ran -2 carboxyl ic acid (S-8666) was with 0.26% sodium pentobarbital-4% studied as a favorable candidate for a -1.5% inulin-0.25% sodium chloride uricosuric diuretic after being chosen from solution at a flow rate of 0.03 ml/min after a screening with the animals, and then it was the cannulation. After the equilibration for confirmed to also be uricosuric and diuretic 60 min, continuous 20-min urine samples in chimpanzees*. were collected four times, and blood was collected at the midpoint of each urine Materials and Methods collection period. In these experiments, uric Chemicals: Probenecid (Sigma), furose acid was determined by the fluorometric mide (Sigma) and furosemide solution for method, basically according to the procedure injection (LASIX, Hoechst) were com of Sum] et al. (16), while inulin was estimated mercial preparations. The other compounds according to the method of Vurek and Pegram used were prepared in our laboratories. (17). Experiments in rats: Uricosuric activity in Data are given as the mean±standard oxonate-treated rats was determined by the error, and the significance of the difference method described previously (11); ten-week from the level before dosing was evaluated old male SIc-Wistar rats which had received by Student's t-test. 250 mg/kg potassium oxonate i.p. were The abbreviations used here are UuaV, cannulated in the right femoral artery, left urine-excreted amounts of uric acid; Fua, femoral vein and urinary bladder under glomerular filtered amounts of uric acid; anesthesia with sodium pentobarbital within FEua, fractional excretion of uric acid; and 2 hr after dosing with oxonate. The same dose Cin, inulin clearance. of oxonate, i.p.; 2 ml/kg of 60% urethane, Experiments in chimpanzees: Three 10 s.c.; and 4 ml/kg of 15% inulin, s.c., were year-old male laboratory-born chimpanzees given just 2 hr after the first administration (Pan troglocytes) weighing 55 to 65 kg were of oxonate, and then the animals were infused used. Clearance experiments were performed with 4% mannitol-1.5% inulin-0.9% sodium basically according to the commonly used chloride solution at the flow rate of 0.1 ml/ procedure under anesthesia (18); the animals min on a hot plate at 30°C. After the were anesthetized by the premedication of equilibration for 40 min, 0.2 ml of arterial ketamine hydrochloride, 10 mg/kg, 1.m., and blood was collected six times at 20-min atropine sulfate, 0.05 mg/kg, i.m., and then intervals, and five 20-min urine samples were the introductory and maintenance anesthesia with oxygen-nitrous oxide-halothane mixture. * The clearance experiments using chimpanzees The clearance experiments were started with

were performed in the Netherlands Organization an intravenous infusion of Ringer-lactate for Applied Scientific Research, TNO, supervised solution at a flow rate of 3 ml/min, a bolus by Dr. A.A. van Es and directed by Dr. P.M.C.A. administration of inulin, 50 mg/kg, i.v., and van Eerd under the sponsorship of Shionogi an intravenous infusion of 1 mg/kg/min Research Laboratories. inulin in saline as a sustaining dose. Blood and urine samples were collected every 30 treated rats ranged from 0.5 to 0.7, which min. After equilibration for 120 min, the test was apparently higher than those in humans compound dissolved into 1.26% sodium and chimpanzees, and meant that the net bicarbonate solution was administered orally flux of uric acid in the renal tubules in the by a stomach tube; and thereafter, the animals was definitely reabsorptive. Therefore, infusion volume of the Ringer-lactate solution it should be possible to evaluate the increase was adjusted to the urine flow to prevent of the FEua value in the animals due to volume loss in the animals after the adminis inhibition of the tubular transport of uric tration of diuretic agents. acid. The uricosuric and diuretic effects of Determination of S-8666 and the metab various compounds which have been well olite, 5 monomethylsulfamoyl-6,7-dichloro known for their effects on uric acid excretion 2,3 dihydrobenzofuran 2 carboxylic acid: in primates are summarized in Table 1. Part of the plasma and urine samples in the Experiments on each drug were performed clearance studies using chimpanzees was with 8 to 10 animals, of which part of the used to determine the concentrations of S experimental results had been reported 8666 and the metabolite to confirm the previously (11). The increases of urine reliability of the clearance experiments. The excreted amounts of uric acid (UuaV), plasma sample (0.5 ml) was mixed and glomerular-filtered amounts of uric acid shaken well with 1.0 ml of 1 N NaOH and (Fua), FEua and urine volume were calculated 2.0 ml of dichloromethane containing 20 /cg using the mean values in each experimental of 5-(pyrrolidin-1-yl-sulfonyl)-6,7-dichloro period, when the values were significantly 2,3-dihydrobenzofuran-2-carboxylic acid as different from the level before dosing with an internal standard. the drug at P<0.05, and then the increases After centrifuging, 1.0 ml of the aqueous were shown as the average value for 80 min phase was mixed and shaken with 1.0 ml after the dosing. The high doses of test 2 N HCI and 4.0 ml ethyl acetate; then an drugs except for benzbromarone increased aliquot of the organic phase was evaporated the UuaV value with a rising Fua value in the to dryness at reduced pressure. The urine animals. Such an increase of UuaV was also sample, 2.0 ml, was mixed and shaken with brought about by furosemide, which is not 0.5 ml 1 N NaOH and 2.0 ml dichloromethane. a uricosuric drug. The typical uricosuric Next, 1.0 ml of the aqueous phase was drug probenecid clearly elevated the FEua mixed with 0.5 ml 2 N HCI and 2.0 ml ethyl value in the animals. Benzbromarone and acetate containing 100 /cg of the same tienilic acid also increased the FEua value, internal standard compound. The residue but the activities were obviously lower. On from the organic phase was dissolved in the other hand, indacrinone and a known 5 200 ,ul of developing solvent [11/9 (V/V) carbonyl dihydrobenzofurancarboxylic acid mixture of methanol and a phosphate buffer derivative did not increase the FEua value in containing 10 mM of Na2HPO4, 30 mM of the animals, in spite of being markedly NaH2PO4 and 5 mM of PIC-A reagent (Waters uricosuric in chimpanzees (6, 7). Thus, Associates)]; then a portion (1-10 ,ul) was oxonate-treated rats produced two different used for analysis by an H PLC method types of uricosuric responses: an increase of employing a Nucleosil 5C18 column UuaV due to higher glomerular-filtered (Chemco), in which S-8666 and the me amounts of uric acid and elevation of the tabolite were analyzed at 254 nm at the flow FEua value due to inhibition of the tubular rate of 1.0 ml/min. reabsorption. Among the new compounds, those of a Results series of 5-sulfamoyl-6,7-dichloro-2,3-di Uricosuric and diuretic activities of hydrobenzofuran 2-carboxylic acids were various compounds in oxonate-treated rats: markedly uricosuric, causing a rise of the As described previously (11), the average FEua value and acting as a diuretic in the value of fractional excretion of uric acid animals as shown in Table 2. (FEua) in many experiments using oxonate These new compounds were compared Table 1. Uricosuric and diuretic effects of known compounds in oxonate-treated rats

Each experiment was done using 8-10 animals; then the increases of UuaV, Fua, FEua and urine volume were calculated as the average values for 80 min after dosing in the clearance experiment. -: Unchanged.

Table 2. Uricosuric and diuretic effects of 5-sulfamoyl-6,7-dichloro-2,3-dihydrobenzofuran-2 carboxylic acids in oxonate-treated rats

Test compounds were given at the dose of 50 mg/kg, i.p. Data are shown as described in the footnote of Table 1. as to their utility as diuretic antihypertensives effect on the urine volume and uric acid by the tests of their natriuretic and anti excretion in oxonate-treated rats, but doses hypertensive activities and acute toxicity of more than 10 mg/kg, i.p., were apparently (data not shown). S-8666 was chosen as diuretic with uricosuric activity also ap the most favorable candidate. pearing as shown in Table 3. Uricosuric and diuretic effects of S-8666 The uricosuric response at 10 mg/kg was in rats: S-8666 at 1 mg/kg, i.p., had no due to enhancement of the FEua value, while Table 3. Uricosuric and diuretic effects of S-8666 in oxonate-treated rats

Data represent the mean±standard error, and the number of animals is in parentheses. The average increases of UuaV, Fua, FEua and urine volume are 0.040, -, 0.232 and 0.26 at the dose of 10 mg/kg, and 0.107, 0.092, 0.212 and 0.42 at the dose of 50 mg/kg, respectively. *,**: Significantly different from the level before dosing at P<0.05 and 0.01, respectively.

Data represent the mean±standard error, and the number of animals is in parentheses. *,**: Significantly different from the level before dosing at P<0.05 and 0.01, respectively. the 50 mg/kg dose involved a rise of the Fua enantiomer being markedly diuretic like value. The diuretic effect of S-8666 was a furosemide (Table 4). high-ceiling one as in the case of furosemide. Next, the uricosuric effect of S-8666 was Known uricosuric diuretics such as tienilic evaluated using rats without oxonate. As acid, indacrinone and a 5-carbonyl dihydro reported previously (15), a high dose of benzofurancarboxylic acid derivative were probenecid by intravenous administration also diuretic in the animals, but the activities increased the FEua value in the rats without had lower ceiling levels than furosemide urate oxidase inhibitor. S-8666 also elevated (Table 1). the FEua value in a similar experiment as S-8666 is composed of two enantiomers, shown in Table 5, while furosemide decreased with the (+)-enantiomer producing a the FEua value. response with an increase in the FEua value Uricosuric and diuretic effects of S-8666 in oxonate-treated rats and the (-) in chimpanzees: Two chimpanzees which Table 5. Uricosuric and diuretic effects of S-8666 and furosemide in rats without urate oxidase inhibitor

Furosemide was used as a commercial preparation 'LASIX', and S-8666 was prepared as the sodium salt. Drugs were dissolved in saline; then administered intravenously as a bolus from the tail vein. Data represent the meanfstandard error, and the number of animals is in parentheses. *,**: Significantly different from the level before dosing at P<0.05 and 0.01, respectively.

Fig. 1. Diuretic and saluretic effects of S-8666 in chimpanzees. A and B are for chimpanzee iden tification. S-8666 was administered at the dose of 10 mg/kg, p.o., at zero time. received S-8666, 10 mg/kg, p.o., showed three animals, the uricosuric potency of S obviously enhanced diuresis and saluresis 8666 was similar to that of probenecid and (Fig. 1), and also uric acid excretion after less than that of indacrinone, as seen in Fig. 3. the dosing (Fig. 2). Although a generalization On the other hand, indacrinone and cannot be made from the data from two or furosemide were markedly diuretic in chim Fig. 2. Uricosuric effect of S-8666 in chimpanzees. A and B are for chimpanzee identification. S-8666 was administered at the dose of 10 mg/kg, p.o., at zero time.

Fig. 3. Uricosuric effects of probenecid and indacrinone in chimpanzees. A, B and C are-for chimpanzee identification. The normal level of FEua was assessed using the values before dosing in all experiments. panzees (data not shown). The diuretic and indacrinone and furosemide in chimpanzees. saluretic effects of S-8666, 10 mg/kg, p.o., were less than those of 5 mg/kg indacrinone Discussion and 5 mg/kg furosemide, p.o. Thus, S-8666 Various nonhuman mammalian species showed uricosuric activity like probenecid have been studied to characterize uricosuric with a milder diuresis than those of drugs (12), but only a few primates have been useful for such studies. De Rougemont rats was high-ceilinged like that of et al. (19) proved that the net flux of uric furosemide, while indacrinone was less acid in the renal tubules of rats is also diuretic and low-ceilinged. Thus, S-8666 reabsorptive under constant venous infusion should be classified as a different type of of a urate oxidase inhibitor, potassium uricosuric diuretic from indacrinone, and oxonate, although no evidence was presented also from tienilic acid which has no on the effects of uricosuric agents in the enantiomer. animals. We also recognized a similar level of The uricosuric activity of S-8666 was FEua in intraperitoneally oxonate-treated also recognized in oxonate-untreated rats rats, in which various known compounds in the intravenous administration of its high produce uricosuric responses (11). The dose which markedly decreased the FEua value in the oxonate-treated rats was glomerular filtration rate. Furosemide obviously higher than those in humans and decreased the FEua value under a similar chimpanzees, and the animals produced diuretic condition. Such effects of S-8666 two different types of uricosuric responses, and furosemide, however, were not observed that is, an increase of UuaV with a rise of at a lesser dose without the decrease of Fua together with elevation of plasma uric glomerular filtration rate and by intraperi acid level and an increase of UuaV that was toneal dosing (data not shown). Ac dependent upon the elevation of FEua. The cordingly, although the rats without urate rise of plasma uric acid might be due to oxidase inhibitor can be used to characterize various causes not connected with uricosuric uricosuric activity as demonstrated in the drugs. If this is true, the former response can studies on tienilic acid (13) and indacrinone not be used to evaluate uricosuric activity on (14), oxonate-treated rats are better for new compounds, even if known uricosuric evaluating the uricosuric activity of new drugs such as probenecid and indacrinone compounds. had such a response. On the contrary, the The present study using oxonate-treated increase of FEua in oxonate-treated rats rats offered a new candidate for uricosuric should mean that the test compound inhibits diuretics. However, the final conclusion on the tubular reabsorption of uric acid . whether the oxonate-treated rats are useful However, the response might be modified for studying uricosuric drugs should be by the activity of the test compound on decided only confirming that the new inhibiting tubular secretion of uric acid more candidate is also uricosuric in primates such than in primates, because of the high FEua as chimpanzees and humans. Tests using value before dosing. We, therefore, presume chimpanzees were done for this purpose in that the tubular transport function on uric TNO. The experiments were performed as a acid in oxonate-treated rats differs from blind test on compounds; then the reliability those in primates, and thus the uricosuric of the experimental results was judged first responses of various compounds in the by whether a reasonable glomerular filtration animals may not always be identical to those rate (inulin clearance: 70-130 ml/min) was in primates. However, as experiments using maintained during the experiments. Also, primates are not always easy to perform, the data of the animals which had received oxonate-treated rats are useful for screening S-8666 were also confirmed by measuring some candidates for uricosuric agents . the levels of S-8666 and the metabolite in Probenecid increased the FEua value in plasma and urine samples. As indicated in oxonate-treated rats most obviously among Fig. 4, the two chimpanzees which were the known compounds, but S-8666 uricosuric and diuretic by the administration elevated the FEua value even more than of S-8666, 10 mg/kg, p.o., showed an probenecid. The new compound is composed appreciable concentration of S-8666 in the of two enantiomers, which predominantly plasma and urine, and a slight amount of the display either uricosuric or diuretic activity, monomethylsulfamoyl metabolite in the urine. as found with indacrinone. However, the Thus, the two animals were carefully diuretic effect of S-8666 in oxonate-treated studied, and S-8666 was found to be uri Fig. 4. Concentrations of S-8666 and its metabolite in plasma and urine samples of chimpanzees. A and B are for chimpanzee identification. S-8666 was administered at the dose of 10 mg/kg, p.o., at zero time. cosuric and diuretic also in chimpanzees. details on uricosuric activity of S-8666 have However, the uricosuric and diuretic activities not been examined in chimpanzees as yet, were quantitatively less than those of the experimental results using oxonate indacrinone which had been already demon treated rats support the hypothesis that the strated to be highly uricosuric and diuretic in activity of S-8666 is nearer to that of chimpanzees (6), in spite of its not displaying probenecid, rather than that of indacrinone. these qualities in oxonate-treated rats. Thus, the new agent should be further Furosemide was also found to be as potently studied to find its utility as a new drug for diuretic as indacrinone in chimpamzees, humans, independent of its lesser uricosuric although the activity in oxonate-treated rats effect in chimpanzees than that of indacrinone. was near that of S-8666. Although the causes In the present report, the uricosuric and for the different responses among the tested diuretic activities were focused on the animals are not clear, these differences sug character of S-8666. We now have infor gest that S-8666 should be classified as a mation on its toxicity and antihypertensive different type of diuretic from indacrinone activity in experimental animals. The new and furosemide. As reported by Tobert et al. candidate displayed markedly low toxicity in (20), the markedly diuretic effect of experimental animals, and the antihyper indacrinone made its uricosuric activity tensive activity in the hypertensive rats ineffective for preventing the incidence of established with deoxycorticosterone acetate hyperuricemia. The utility as a practical and sodium chloride was two or three times uricosuric diuretic should be considered not higher than that of tienilic acid. These data only from the potency of uricosuric and will be reported elsewhere. Thus, S-8666 is diuretic activities, but also from the well a new class of uricosuric diuretic that is balanced activities on hyperuricosuria and effective in rats and chimpanzees. Further diuresis to produce an iso or hypouricemic studies are needed to evaluate the utility as a effect during the medication. Uricosuric useful drug in humans. responses in chimpanzees have been well Acknowledgment: We wish to thank to Dr. investigated by Fanelli et al. (18), who also P.M.C.A. van Eerd (Primate Center TNO), Dr. J.A.P. demonstrated a qualitative difference in the Stroes (Stichting Samenwerking Delftse Zieken uricosuric characteristics between pro huizen), Dr. Yoshio Harada (Shionogi Research benecid and indacrinone (21). Though the Laboratories) and Dr. Taichiro Komeno (Shionogi Research Laboratories) for help with the experi f]-1,2-benzisoxazole-6-carboxylic acids. J. 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