Biological activities of metabolites of 6-chloro-5-cyclohexylindan-1-carboxylic acid (TAI-284: anti-inflammatory agent).

The pharmacological activities of five metabolites of an anti-inflammatory compound, 6-chloro-5-cyclohexylindan-1-carboxylic acid (TAI-284) were investi gated and compared with TAI-284 and phenylbutazone. The metabolite IIb (cis 3'-hydroxy-cyclohexyl, &beta;-form) was almost equivalent to TAI-284 regarding anti inflammatory activity as estimated by the carrageenin edema test, and was more active than TAI-284 in the analgesic activity estimated by the phenylquinone writh ing test and determination of the ulcerogenic activity. The metabolites IIa (cis-4' hydroxy-cyclohexyl) and IV (cis-3'-hydroxy-cyclohexyl, &alpha;-form) had about half the anti-inflammatory activity and one third to one quarter the analgesic activity of TAI 284, however the degree of ulcerogenicity was considerably lower than that of TAI 284. 1 (4'-oxo-cyclohexyl) was less active, and III (trans-4'-hydroxy-cyclohexyl) showed the weakest activity comparable to that of phenylbutazone. Antipyretic ac tivities of all five metabolites were much lower than those of TAI-284. As the phar macological activities of metabolites varied greatly according to the position of the hydroxyl group on the cyclohexane ring of TAI-284, it is feasible that the distribution pattern and affinity of the metabolites for receptors could be modified. Anti-inflammatory drugs adminikiered at a high dosage or for a long period of time often induce gastro-intestinal disorders. TAI-284 with potent anti-inflammatory, anal gesic and antipyretic activities (1) produced at high doses penetrating ulcers in rat je junum and ileum. The toxicological properties (2) and detailed features of the ulcer de velopment following administration of the compound have been previously described (3, 4). It was observed that a phenobarbital pretreatment of animals reduced the toxicity of TAI-284 without altering the anti-inflammatory activity. As phenobarbital is known to be an enzyme inducer in drug metabolism (5), the metabolic mechanisms presumably play a role in the action of this drug. Recently, Aspinall reported that spironolactone pre treatment exerted no effect on the anti-inflammatory activity of indomethacin but mark edly suppressed ulcerogenic activity in adjuvant arthritis of rats (6). He suggested that such a differential effect of spironolactone may be explained by the difference between the minimum effective doses of indomethacin exerting the two actions, the same of which is indeed worthy of consideration. The present report deals with the anti-inflammatory, analgesic, antipyretic and ulcer ogenic activities of five metabolites of TAI-284 in comparison with the parent compound and phenylbutazone, and characteristic properties of the metabolites are discussed. Metabolites of TAI-284, shown in Table 1, were isolated from the perfusate of isolated rat livers by Kanai et al. in our Biological Research Laboratories (7), and also chemically synthesized by Kishimoto et al. in our Chemical Research Laboratories. Male SD-JCL rats and ICR-JCL mice were used. Test agents were suspended with 4 % gum acacia in water and administered p.o. in 1 nil per 100 g body weight to rats, and in 0.2 ml per 10 g body weight to mice. All experiments were carried out following a double-blind schedule. 1. Anti-inflammatory test TABLE 1. Chemical structures of TAI-284 and its metabolites * : IIb and IV are diastereoisorneric with each other . (Cited from Kanai et al.) Following the method of Winter et al. (8), 0.05 ml of 1 % carrageenin suspension in physiological saline was injected s.c. into the plantar side of the hind paw of male rats weighing 190 ± 10 g, and 3 hr later the edema developed was estimated. Test agents were administered p.o. one hr before the injection of carrageenin. 2. Analgesic test Mice weighing 20±2 g were used for the phenylquinone writhing syndrome test. Following the procedure of Siegmund et al. (9), 0.02% phenylquinone dissolved in water by adding 5 % ethanol was injected i.p. in 0.1 ml per 10 g body weight to induce writhing and stretching responses. The frequency of those responses was counted for 20 min after the injection. Test agents were administered 30 min before the phenylquinone injection. 3. Antipyretic test Following the method of Winder et al. (10), rats weighing 210+ 10 g were made feb rile by a s.c. injection of 15% baker's yeast suspended in physiological saline in I ml per 100g body weight. The animals were fasted but allowed free access to drinking water until 16 hr later, after which rectal temperature was estimated three times at one-hr in tervals. Oral administration of test agents thereafter was followed by hourly estimations of rectal temperature for 5 hr. 4. Ulcerogenicity Rats weighing 140-1-10g were sacrificed 6 hr after the oral administration of test agents. Gastric mucosal ulcers as well as those in the small intestine and other areas of digestive tract were counted and the ulcer area measured. RESULTS 1. Anti-inflanunatoj_v activity FIG. 1. Comparative anti-inflammatory activity of TAI-284 and its metabolites in carrageenin edema test in rats. The activity of metabolites of TAI-284 against carrageenin edema in rats is shown in Fig. 1. The activity of metabolite 111) was equivalent to that of TAI-284. Metabolites 1, IIa and IV were significantly effective at 3 to 12 mg/kg, indicating the relative activities of one half to one fifth of those of TAI-284. Metabolite III displayed much less activity. The analgesic activity in the phenylquinone writhing test is presented as the ED50 in Table 2. Metabolite llb, the most potent anti-inflammatory one, was about 4 times TABLE 2. ED50's of TAI-284 and its metabolites in phenylquinone writhing syndrome test in mice C.L. : Confidence limits 2. Analgesic activity Fic. 2. Antipyretic activity of TAI-284 and its metabolites in febrile rats injected with Baker's yeast. 0 : P<0.05, P<0.01 as analgesic as TAI-284. Metabolites 1, Ila and IV had considerably less analgesic ac tivity than did TAI-284 but were nevertheless more active than phenylbutazone. Me tabolite 111, the least active of all, was observed to have only one twelfth the activity of TAI-284. 3. Antipyretic activity In contrast to the anti-inflammatory and analgesic actions, antipyretic activities of the five metabolites were much lower than those of TAI-284 as shown in Fig. 2. Metabo lite lib which had the strongest antipyretic activity proved to be only one third as potent as TAI-284. Metabolite I at 12.5 mg/kg, IV at 12.5 mg/kg, III at 25 mg/kg and IIa at 25 mg/kg were equivalent to TAI-284 at 0.3 mg/kg or to phenylbutazone at 25 mg/kg. 4. Ulcerogenicity TAI-284 at 10 mg/kg p.o. produced quite a number of small round ulcers, mostly about 1 mm in diameter, in the jejunum and ileum, however, none were found in the stomach, duodenum and large intestine (Table 3). Ulcers induced by metabolites I, IIa, III and IV showed a similar distribution pattern, but were significantly less in number than when TAI-284 had been administered. Metabolite lib was more potent than TAI-284 regarding the ulcerogenicity factor. TABLI; 3. Comparative ulcerogenicity of TAI-284 and its metabolites in rats ** : P<0 .01 as compared with TAI-284.