The Journal of Veterinary Medical Science

Transcription

1 Advance Publication The Journal of Veterinary Medical Science Accepted Date: Jun 0 J-STAGE Advance Published Date: 0 Jul 0

2 FULL PAPER Pharmacology Comparison of Gastrointestinal Adverse Effects of Ketoprofen between Adult and Young Cats 0 Kenji TAKATA ) *, Yoshiaki HIKASA ) and Hiroshi SATOH ) ) Laboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Koyama-Minami -0, Tottori 0-, and ) Department of Pharmacology and Experimental Therapeutics, Division of Pathological Sciences, Kyoto Pharmaceutical University, Misasagi-Nakauchicho, Yamashinaku, Kyoto 0-, Japan CORRESPONDENCE TO: TAKATA, K., Laboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Koyama-Minami -0, Tottori 0-, Japan Tel.: ()--, Fax: ()-- k_takatavet@yahoo.co.jp 0 Running head: EFFECTS OF KETOPROFEN ON ADULT AND YOUNG CATS

3 0 0 ABSTRACT. This study elucidated differences in predisposition to the gastrointestinal adverse effects of ketoprofen between young and adult cats. Ketoprofen was administered subcutaneously (.0 mg/kg, s.c.) once a day for days. The animals were sacrificed hr after final injection to allow examination of gastrointestinal mucosal lesions. Ketoprofen caused gastric lesions in adult cats (> months) but not in young cats (< months). Ketoprofen caused more severe small intestinal lesions in adult cats than in young cats. In the study of prevention of lipopolysaccharide (LPS)-induced hyperthermia using ketoprofen, young and adult cats of both sexes were administered LPS (0. μg/kg, intravenously), and body temperature was measured hr later. Ketoprofen was administered subcutaneously 0 min before LPS injection. LPS-induced hyperthermia was almost completely inhibited by pretreatment with ketoprofen in both adult and young cats. In the pharmacokinetics of ketoprofen, plasma concentrations were analyzed by high-performance liquid chromatography. No significant differences were observed in plasma concentrations of two mirror-image R( ) and S(+) ketoprofen between young and adult cats from 0. hr after injection. As observed in a previous study using flunixin, the degree of gastrointestinal damage was unrelated to plasma concentrations of ketoprofen. The results of this study demonstrated that ketoprofen is safer for use in young cats than in adult cats from the viewpoint of gastrointestinal adverse effects. KEY WORDS: gastrointestinal effect, ketoprofen, lipopolysaccharide, non-steroidal anti-inflammatory drug, pharmacokinetics

4 0 0 INTRODUCTION Non-steroidal anti-inflammatory drugs (NSAIDs) are the most widely used analgesics in veterinary and human medicine. Ketoprofen is a member of the -arylpropionic acid subgroup of carboxylic acid NSAIDs. It is a chiral molecule. Similar to other members of this subgroup (e.g., carprofen and vedaprofen), it contains a single asymmetric carbon atom and therefore exists in two, mirror-image, enantiomeric forms: R( ) and S(+) ketoprofen. In most species, NSAIDs are very effective in alleviating acute and chronic pain. For example, NSAIDs are often the first drugs used in treating pain caused by osteoarthritis in veterinary medicine []. Ketoprofen is also used for pain treatment. In an analgesic study of cats, the single-observer visual analog score (VAS) for pain and the need for intervention were significantly lower in the ketoprofen group than in the meperidine and buprenorphine groups []. However, NSAIDs have been known to cause some adverse effects, including gastrointestinal side effects, in veterinary medicine. In rats, even newly developed NSAIDs have been reported to cause gastrointestinal lesions because of inhibition of prostaglandin synthesis [, ]. A previous study in cats has also reported that flunixin causes intestinal adverse effects []. However, no studies have been published about gastrointestinal side effects of NSAIDs other than flunixin in cats. Most NSAIDs are cleared from the body through hepatic metabolism primarily comprising glucuronidation followed by excretion of the resultant polar metabolites via bile and/or urine. Ketoprofen bioavailability in cats has been reported as % and % for S(+) and R( ) ketoprofen, respectively []. However, this value of bioavailability for S(+) ketoprofen may be an overestimate because of inversion of R( ) to S(+) ketoprofen. Nevertheless, the percentage absorption of R( ) and S(+) ketoprofen seems relatively high []. For many NSAIDs, glucuronidation is an important pathway. For example,

5 0 ketoprofen metabolism is dominated by glucuronidation reactions in dogs []. However, many studies have reported that glucuronidation in cats is limited [,,, 0,, ]. Therefore, NSAIDs may induce more severe adverse effects in cats than in other species. In human beings, NSAIDs are widely used in children. However, adverse gastrointestinal or renal events from short-term use of ibuprofen or acetaminophen appear to be quite rare in children []. Vasopressin plays a role in microcirculation and may be responsible for resistance to gastric mucosal lesions []. In vasopressin-deficient rats, gastric lesions caused by indomethacin have been reported to be significantly more severe in older rats than in younger rats [0]. These findings suggest that age-dependent severity of indomethacin-induced gastric injury is not related to vasopressin activity. In addition, intestinal adverse effects caused by flunixin are more severe in adult cats than in young cats []. However, few studies have compared the effects of NSAIDs between young and adult cats. The veterinary medicine market utilizes many NSAIDs including ketoprofen. Flunixin, which was the focus of our previous study, is not approved for use in cats. On the other hand, ketoprofen is approved for use in cats in Europe, Australasia, Canada, and Japan. However, no studies have investigated the therapeutic and adverse effects of ketoprofen in cats. The purpose of the present study was to elucidate differences in therapeutic and adverse effects of ketoprofen between adult and young cats. 0 MATERIALS AND METHODS Animals: This study compared the effects of ketoprofen administration (ketoprofen,.0 mg/kg, subcutaneous administration) between young and adult cats in terms of antipyretic, pharmacokinetic, and gastrointestinal adverse effects. All of cats were obtained by

6 0 0 self-breeding in the laboratory animal facilities of Tottori University (pharmacological laboratory, department of veterinary medicine). Young (< months, kg body weight, ) and adult (> months, > kg body weight) cats of both sexes were used ( animals in each group). The cats were kept in the experimental room for more than > days before the start of the experiment. Three to four cats were housed together in a cage (PEC-0; IRIS Ohyama Inc., Sendai, Japan). During the experiments, the animals were housed individually at an ambient temperature of ± C with a - hr light/dark cycle, with the (lights were being switched on at 0:00). All of cats received commercially available dry cat food. Water was allowed ad libitum. Experiments were conducted between 0:0 and :00. Study protocols were approved by the Animal Research Committee of Tottori University, Tottori, Japan. Drugs: Ketoprofen (% Ketofen, Dainippon Sumitomo Pharmaceutical Co. Ltd., Osaka, Japan), xylazine (Ceractal, % solution, Bayer, Osaka, Japan), sodium pentobarbital (Nembutal, Dainippon Sumitomo Pharmaceutical Co. Ltd.), and lipopolysaccharide (LPS, Lot no.ojc, W. E. Coli, Wako Pure Chemical Industries Ltd., Osaka, Japan) were used in this study. Prevention of LPS-induced hyperthermia using ketoprofen: The experiment consisted of treatment groups in both young and adult cats. Four cats were assigned to each of the treatment groups ( males and females in each group). For induction of pyrexia, LPS (0. μg/kg) was administered intravenously (i.v.) at 0 hr. Ketoprofen was administered subcutaneously (s.c.) 0. hr before LPS injection. In both young and adult cats, the control group was administered 0. ml/kg physiological saline solution (PSS, s.c. and i.v.). PSS was injected before LPS in the LPS group. Cats in the other groups received ketoprofen (.0 mg/kg, s.c.) and LPS (0. μg/kg, i.v.). These groups will hereafter be referred to as

7 0 0 CONT, LPS, and KP. Body temperature was measured 0. hr before and 0, 0.,,,,, and hr after LPS injection. Examination of gastrointestinal lesions: This experiment consisted of non-injected (young and adult) and ketoprofen-injected (young and adult) animals. In this design, each group was composed of males and females. To determine gastrointestinal lesions, ketoprofen (.0 mg/kg, s.c.) was administered once a day after the morning meal for days. In all groups, animals were sacrificed using xylazine (.0.0 mg/kg, s.c.) and sodium pentobarbital (0 mg/kg, i.v.) hr after the final injection of ketoprofen. The stomach and small intestine were excised as follows. Stomachs were opened along the greater curvature. Small intestines were opened on the antimesenteric side. These organs were then immersed in,000 ml saline. Thirty min later, visible mucosal lesions in the stomach and small intestine were examined by stereomicroscopy. This method has been used in the study of rats []. The percentage of the lesion area was calculated as 00% of total small intestine area. Lesion rate (%) = (lesion area in small intestine / total of small intestine area ) 00. Analysis of pharmacokinetics of ketoprofen: ketoprofen (.0 mg/kg, s.c.) was administered to young ( males, females) and adult cats ( males, females). The pharmacokinetics of this NSAID were analyzed by high-performance liquid chromatography (HPLC). HPLC analysis was performed using a model L-000 instrument (Hitachi Co. Ltd., Tokyo, Japan). For pharmacokinetic analysis, blood samples were collected from the jugular vein 0, 0.,,, and hr after NSAID injection. Heparin was used as an anticoagulant. Plasma was separated, stored at 0 C for week, and analyzed by HPLC. Chromatographic separations were performed on Bioptic AV (. mm ID 0 mm L; GL Sciences Inc., Tokyo, Japan). The mobile phase was composed of 0 mm phosphate buffer (ph.)/acetonitrile (:, v/v). The solution was filtered through a

8 0.-μm membrane prior to use. The flow rate was.0 ml/min, and column temperature was maintained at 0 C. The channel on the UV detector was configured at nm. The volume injection was 0 μl. Each sample was analyzed once. 0 Statistical analysis: All values were expressed as means and standard errors. In the data for body temperature, one-way analysis of variance for repeated measures was used to examine time effects within each group and the combined effect of all groups at each time point. Least significant difference (LSD) test was used to compare means for those values that showed significant differences. Other data were subjected to analysis of variance. When F values were not significant, differences between groups were analyzed by Student s t-test. When F values were significant, Wilcoxon Mann Whitney test was used for statistical evaluation. The level of significance in all tests was set at P < RESULTS Prevention of LPS-induced hyperthermia using ketoprofen: As shown in Fig., in young cats, mean body temperature before LPS injection was. ± 0.0 C (n = ). In the LPS group, body temperatures at,,, and hr were significantly higher than those at 0. hr (P < 0.0). After LPS injection, body temperatures increased and reached a maximum hr after injection (.0 ± 0. C). Body temperature decreased after the peak of LPS-induced hyperthermia at hr. Body temperature was significantly higher in the LPS group than in the CONT group at 0.,,,, and hr (P < 0.0 or 0.0). In the KP group, body temperatures did not change significantly at each elapsed time than at 0. hr. In the KP group, body temperatures and hr after LPS injection were significantly lower than those in the LPS group (P < 0.0). Thus, ketoprofen suppressed LPS-induced hyperthermia in young cats.

9 0 0 As shown in Fig., in adult cats, mean body temperature in the LPS group was significantly (P < 0.0 or 0.0) higher at,,, and hr than at 0. hr. Body temperature increased to a maximum (. ± 0. C) hr after LPS injection and then decreased gradually. Body temperature was significantly (P < 0.0 or 0.0) higher at,,, and hr in the LPS group than the CONT group. In the KP group, body temperatures did not change significantly at each elapsed time than at 0. hr. Thus, ketoprofen suppressed LPS-induced hyperthermia in adult cats. Gastrointestinal side effects: In both adult and young cats, lesions were observed in the duodenum and lower small intestine following repeated doses of ketoprofen. In the stomach, some lesions were observed in adult cats but not in young cats. As shown in Fig., the area (cm ) of lesions caused by ketoprofen in the small intestine was smaller in young cats than in adult cats. In the duodenum, the lesion frequency rate in young cats (0%) was lower than that in adult cats (%). No significant difference was observed in the lesion area in the duodenum between young and adult cats. In addition, the area (%) of small intestine occupied by lesions was lower in young cats (0. ± 0.%) than in adult cats (0. ± 0.0%) (Fig. ). In both adult and young cats in the non-injected groups, no gastrointestinal lesions were observed. Pharmacokinetics of ketoprofen: Figures and show the pharmacokinetics of R( ) and S(+) ketoprofen, respectively. In both adult and young cats, blood levels of ketoprofen reached a maximum 0. hr after injection (Figs. and ). One hr after injection, ketoprofen concentrations in both adult and young cats decreased to approximately 0% of the maximum and then decreased gradually. The area under the curve (AUC) of the plasma concentration in young cats was similar to that in adult cats. AUC of S(+) ketoprofen in young and adult cats was. ±. (μg hr/ml) and. ±

10 . (μg h/ml), respectively. AUC of R( ) ketoprofen in young and adult cats was 0. ± 0.0 (μg hr/ml) and 0. ±. (μg hr/ml), respectively. No significant difference in AUCs was observed between adult and young cats. 0 0 DISCUSSION Cyclooxygenase (COX) has been recognized as the principal catalyst in the synthesis of prostanoids from arachidonic acid. COX has two isoforms, COX- and COX- [, ]. COX- is constitutively expressed in most cells, while COX- is induced in inflammatory conditions. Much effort has gone into developing NSAIDs that selectively inhibit COX- rather than COX- so as not to affect the homeostatic functions of prostanoids preferentially synthesized by COX-, and in particular to reduce gastrointestinal bleeding caused by COX- inhibition [, ]. In an in vitro study, ketoprofen demonstrated lower COX- selectivity in comparison with ibuprofen and piroxicam []. LPS from gram-negative bacteria stimulates host defense cells to release several endogenous pyrogens. Many studies show that LPS-induced fever is mediated by a number of endogenous pyrogenic cytokines [,, ], which are transported to the thermoregulatory center in the preoptic area. They stimulate the production of COX--dependent prostaglandin (PG) E, the putative final mediator of the febrile response []. The present study demonstrated that LPS caused hyperthermia in both young and adult cats and that ketoprofen suppressed LPS-induced hyperthermia. This study also indicated that ketoprofen significantly suppresses hyperthermia in cats. Ketoprofen may thus be useful in the treatment of bacterial febrile conditions in veterinary practice, except in cats

11 0 0 0 because of its adverse effects. The analgesic effects of ketoprofen were not monitored in this study. However, ketoprofen significantly suppressed LPS-induced hyperthermia hr after LPS injection. Therefore, ketoprofen may show analgesic effects from hr after injection. A further study is required to confirm this possibility. In the present study, in both adult and young cats, lesions were observed in the duodenum and lower small intestine following repeated doses of ketoprofen. In the stomach, some lesions were observed in adult cats but not in young cats. Because this discrepancy could be a result of the difference in the small intestinal area between adult ( ±. cm ) and young cats ( ±. cm ), the percentage of eroded tissue relative to the surface area of the intestinal lumen was calculated. As shown in Fig., in adult cats, the eroded area was 0. ± 0.0% of the total surface area, while in young cats, the eroded area was 0. ± 0.%. These results of lower values for the percentage of lesion-eroded tissue differed from the results using flunixin []. However, in our previous study using flunixin, the mucosal lesion area was more severely damaged than that in this study. Therefore, gastrointestinal lesions caused by ketoprofen administration may be milder than those caused by flunixin administration. These differences may be related to the selectivity of COX- and COX-. Possible reasons for these differences in erosion between adult and young cats are as follows. First, plasma concentrations of both enantiomeric forms of ketoprofen were determined. HPLC analysis showed that pharmacokinetic data of both drugs were similar for young and adult cats. In pharmacokinetic studies of ketoprofen, T max of S(+) and R( ) ketoprofen after oral administration has been reported to be approximately 0. and 0. hr, respectively, in cats []. In our study, T max of both ketoprofen enantiomers after injection was 0. hr in both adult and young cats. The elimination half-life of R( ) and

12 0 0 S(+) ketoprofen was approximately and. hr, respectively, in both young and adult cats. Therefore, our study demonstrated no difference in plasma concentrations of ketoprofen between young and adult cats. Significant age-based differences in bile acids, enterobacteria, and mucosal defense may also play a role in progression of gastrointestinal lesions caused by ketoprofen administration, as suggested previously []. Alterations in the intestinal glycocalyx have been reported in rats after indomethacin administration, suggesting that changes in epithelial mucin content may contribute to NSAID-induced deleterious effects on bowel. Inflammation and ulceration are the ultimate result, once the mucosal barrier has been disrupted by the local and systematic effects of damaging therapeutic agents []. Intraluminal factors including bacteria may play a key role in the initiation of damage in NSAID-induced mucosal erosions. On the other hand, indomethacin administration has been reported to induce an increase in bacterial counts in the mucosa []. One study suggested that bacterial flora may play a role in the pathogenesis of NSAID-induced bowel injury. That study demonstrated that antimicrobials attenuated NSAID-induced enteropathy in rats []. In pigs, one study reported that microbial flora in feces changed with age []. From these observations, the possibility arises that changes in gastrointestinal bacterial counts or bacterial flora may be responsible for the gastrointestinal adverse effects. These factors may be among the causes of variation in ketoprofen-induced erosion between young and adult cats. Another recent study documented that soluble dietary fibers can protect cats from indomethacin-induced small intestinal lesions; dietary fibers act same as endogenous mucin []. Dietary fibers may also influence enterobacteria. The other possibility is that differences in age influence the activity or quantity of COX. In rats, COX- protein was

13 0 reduced in the cortex of old rats than of young rats of both sexes []. And then, in this study, gastrointestinal lesion may not be relation to differences in sexes, too. In conclusion, this study demonstrated that ketoprofen suppressed LPS-induced hyperthermia in both young and adult cats and that ketoprofen-induced small intestinal lesions were fewer and smaller in young cats than in adult cats. This difference between young and adult cats in gastrointestinal adverse effects was unrelated to plasma concentrations of ketoprofen. In the present study, because ketoprofen caused severe gastrointestinal adverse effects in adult cats, it may not be suitable for use in adult cats. Although the essential causes of differences in reactions are unknown, the present results suggest that ketoprofen may be safer for use in young cats than in adult cats. These results are similar to those of our previous study using flunixin in cats [].Thus, some NSAIDs may be used more safely in young cats than in adult cats from the viewpoint of intestinal adverse effects. 0 REFERENCES. Alessandro, M., Cristina, P., Elena, G., Elvira, S., Daniela, G., Silvia, B., Simone, B., Valentina, V. and Gabriella, C. 00. Intestinal effects of nonselective and selective

14 0 0 cyclooxygenase inhibitors in the rat. Eur. J. Pharmacol. : 0.. Basivireddy, J., Jacob, M., Ramamoorthy, P. and Balasubramanian, K. A. 00. Alteration in the intestinal glycocalyx and bacterial flora in response to oral indomethacine. Int. J. Biochem. Cell Biol. :.. Berde, C.B. and Sethna, N.F. 00. Analgesics for the treatment of pain in children. N Engl J Med. :0-0.. Blatteis, C.M., Li, S., Li, Z., Feleder, C. and Perlik, V. 00. Cytokines, PGE and endotoxic fever: a re-assessment. Prostaglandins & other Lipid Mediators. :-.. Bishai, I., Dinarello, C. A. and Coceani, F.. Prostaglandin formation in feline cerebral microvessels: effect of endotoxin and interleukin-. Can. J. Physiol. Pharmacol. : 0.. Cout, M. H. and Greenblatt, D. J Molecular genetic basis for deficient acetaminophen glucuronidation by cats: UGTA is a pseudogene, and evidence for reduced diversity of expressed hepatic UGTA isoforms. Pharmacogenetics 0:.. Cronstein, B. M. 00. Cyclooxygenase- selective inhibitors: translating pharmacology into clinical utility. Clev. Clin. J. Med. :.. Cryer, B. and Feldman, M.. Cyclooxygenase- and cyclooxygenase- selectivity of widely used nonsteroidal anti-inflammatory drugs. Am. J. Med. 0:.. Davis, L. E. and Westfall, B. A.. Species differences in biotransformation and excretion of salicylate. Am. J. Vet. Res. :. 0. Dóra Z. and Ludmila F. 00. Age-dependent role of vasopressin in susceptibility of gastric mucosa to indomethacin-induced injury. Regulatory Peptides :.

15 0 0. Frank, E. and Landgraf, R. 00. The vasopressin system from antidiuresis to psychopathology. Eur. J. Pharmacol. :.. Filaretova, L. Bagaeva, T. and Makara, G.B. 00. Aggravation of nonsteroidal antiinflammatory drug gastropathy by glucocorticoid deficiency or blockade of glucocorticoid receptors in rats. Life Sci. :.. Jernigan, A. D.. Idiosyncrasies of feline drug metabolism. In: th Annual Kal Kan Symposium for the Treatment of Small Animal Diseases. Kal Kan, Vernon, CA, USA... Kim, H. B., Borewicz, K., White, B. A., Singer, R. S., Sreevatsan, S., Tu, Z. J. and Isaacson, R. E. 0. Longitudinal investigation of the age-related bacterial diversity in the feces of commercial pigs. Vet. Microbiol. :.. Lees, P., Taylor, P. M., Landoni, F. M., Arifah, A. K. and Waters, C. 00. Ketoprofen in the cat: pharmacodynamics and chiral pharmacokinetics. Vet. J. :.. McCann, M. E., Rickes, E. L., Hora, D. F., Cunningham, P. K., Zhang, D., Brideau, C. and Black, W. C. and Hickey, G. J. 00. In vitro effects and in vivo efficacy of a novel cyclooxygenase- inhibitor in cats with lipopolysaccharide-induced pyrexia, Am. J. Vet. Res. :.. Menozzi, A., Pozzoli, C., Giovannini, E., Solenghi, E., Grandi, D., Bonardi, S., Bertini, S., Vasina, V. and Coruzzi, G. 00. Intestinal effect of nonselective and selective cyclooxygenase inhibitor in the rat. Eur. J. Pharmacol. : 0.. Rechel, J. K. 00. Diagnosis and Treatment of Osteoarthritis. Top. Companion Anim. Med. : 0.. Ren, Y., Walker, C., Loose-Mitchel, D. S., Deng, J., Ruan, K. H. and Kulmacz, R. J.

16 0 0. Topology of prostaglandin H synthase- in the endoplasmic reticulum membrane. Arch. Biochem. Biophys. : Robinson, D. and Williams, R. T.. Do cats form glucuronides? Biochem. J. :.. Said, C., Marie-Helence, M., Jacques, M., Pierre, L., Nathalie, D., Francoise, L., Zaid, A. and Alain, N.. High-performance liquid chromatographic enantioselective assay for the measurement of ketoprofen glucuronidation by liver microsomes. J. Chromatography B :.. Sanguino, E., Roglans, N., Alegret, M., Sanchez, R. M., Vazquez, C.M. and Laguna, J. C. 00. Prevention of age-related changes in rat cortex transcription factor activator protein- by hypolipidemic drugs. Biochem. Pharmacol. :.. Satoh, H. Hara, T. Murakawa, D. Matsuura, M. and Takata, K. 00. Soluble dietary fiber protects against nonsteroidal anti-inflammatory drug-induced damage to the small intestine in cats. Dig. Dis. Sci. :.. Savides, M., Oehme, F. and Nash, S.. The toxicity and biotransformation of single doses of acetaminophen in dogs and cats. Toxicol. Appl. Pharmacol. :.. Slingsby, L. S. and Waterman-Pearson, A. E.. Comparison of pethidine, buprenorphine and ketoprofen for postoperative analgesia after ovariohysterectomy in the cat. Vet. Rec. :.. Takata, K., Hikasa, Y. and Satoh, H. 0. Therapeutic and adverse effects of flunixin-meglumine in adult and young cats. J. Vet. Med. Sci. :.. Takeuchi, K., Tanaka, A., Kato, S., Amagase, K. and Satoh, H. 00. Roles of COX inhibition in pathogenesis of NSAID-induced small intestinal damage. J. Physol. Phamacol. :.

17 . Yeh, S., Chernov, H. and Woods. L.. Metabolism of morphine by cats. J. Pharm. Sci. 0:. 0 Legends for Figures Fig.. Effect of ketoprofen on hyperthermia induced by LPS in young cats. LPS (0. μg/kg body weight) was injected i.v. at 0 hr. Ketoprofen was administered s.c. at 0. hr before LPS injection. The control group was received 0. ml/kg physiological saline solution (PSS, s.c. and i.v.). The LPS group was injected PSS before LPS injection. Those groups are referred to as CONT, LPS and KP. Data show the mean value and S.E. of cats. At 0.,, and hr after LPS injection, body temperature was significantly higher than CONT. At and hr after LPS injection, ketoprofen significantly suppressed hyperthermia induced by LPS. Body temperature in the KP group was not significantly different from the CONT group. * P<0.0; ** P<0.0, significantly different from body temperature at -0. hr value. # P<0.0; ##P<0.0, significantly different from the CONT group. P<0.0, significantly different from the LPS group. 0 Fig.. Effect of ketoprofen on hyperthermia induced by LPS in adult cats. LPS (0. μg/kg body weight) was injected i.v. at 0 hr. Ketoprofen was administered s.c. at 0. hr before LPS injection. The control group was received 0. ml/kg physiological saline solution (PSS, s.c. and i.v.). The LPS group was injected PSS before LPS injection. Those groups are referred to as CONT, LPS and KP. Data show the mean value and S.E. of cats. At,, and hr after LPS injection, body temperature was significantly higher than CONT. At, and hr after LPS injection, ketoprofen significantly

18 suppressed hyperthermia induced by LPS. Body temperature in the KP group was not significantly different from the CONT group. * P<0.0, significantly different from body temperature at -0. hr value. # P<0.0; ##P<0.0, significantly different from the CONT group. P<0.0; P<0.0, significantly different from the LPS group. Fig.. Gastrointestinal lesions induced by ketoprofen in adult and young cats. 0 Ketoprofen (.0 mg/kg body weight s.c.) was administered for days; once a day after a morning meal. The animals were sacrificed hr after the final dose of ketoprofen, and gastrointestinal mucosal lesions were examined. Data show the mean value and S.E. of cats. In young cats, very few gastrointestinal lesions were produced by ketoprofen. * P<0.0; ** P<0.0, significantly different from adult cats. Fig.. Percentages of small intestinal lesion area induced by ketoprofen in adult and young cats. The percentage of the lesion area was calculated as 00% of total small intestine area. Ketoprofen (.0 mg/kg body weight, s.c.) was administered for days; once a day after a morning meal. The animals were sacrificed hr after the final dose of ketoprofen, and gastrointestinal mucosal lesions were examined. Lesion rate (%) = (lesion area in small intestine / total of small intestine area ) 00. Data 0 show the mean value and S.E. of cats. In young cats, few small intestinal lesions were produced by ketoprofen. Fig.. Time-dependent changes of plasma concentration of R (-) ketoprofen in both adult and young cats. Ketoprofen (.0 mg/kg) was injected s.c. at 0 min. Data show the mean value and S.E. of cats. There was no significant difference on plasma concentration of ketoprofen between adult and young cats.

19 Fig.. Time-dependent changes of plasma concentration of S (+) ketoprofen in both adult and young cats. Ketoprofen (.0 mg/kg) was injected s.c. at 0 min. Data show the mean value and S.E. of cats. There was no significant difference on plasma concentration of ketoprofen between adult and young cats.

20 Effect of ketoprofen on hyperthermia induced by LPS in young cats. LPS (0. µg/kg body weight) was injected i.v. at 0 hr. Ketoprofen was administered s.c. at 0. hr before LPS injection. The control group was received 0. ml/kg physiological saline solution (PSS, s.c. and i.v.). The LPS group was injected PSS before LPS injection. Those groups are referred to as CONT, LPS and KP. Data show the mean value and S.E. of cats. At 0.,, and hr after LPS injection, body temperature was significant higher than CONT. At and hr after LPS injection, ketoprofen significantly suppressed hyperthermia induced by LPS. Body temperature in the KP group was not significantly different from the CONT group. * P<0.0; ** P<0.0, significantly different from body temperature at -0. hr value. # P<0.0; ##P<0.0, significantly different from the CONT group. P<0.0, significantly different from the LPS group.

21 Effect of ketoprofen on hyperthermia induced by LPS in adult cats. LPS (0. µg/kg body weight) was injected i.v. at 0 hr. Ketoprofen was administered s.c. at 0. hr before LPS injection. The control group was received 0. ml/kg physiological saline solution (PSS, s.c. and i.v.). The LPS group was injected PSS before LPS injection. Those groups are referred to as CONT, LPS and KP. Data show the mean value and S.E. of cats. At,, and hr after LPS injection, body temperature was significant higher than CONT. At, and hr after LPS injection, ketoprofen significantly suppressed hyperthermia induced by LPS. Body temperature in the KP group was not significantly different from the CONT group. * P<0.0, significantly different from body temperature at -0. hr value. # P<0.0; ##P<0.0, significantly different from the CONT group. P<0.0; P<0.0, significantly different from the LPS group.

22 Gastrointestinal lesions induced by ketoprofen in adult and young cats. Ketoprofen (.0 mg/kg body weight s.c.) was administered for days; once a day after a morning meal. The animals were sacrificed hr after the final dose of ketoprofen, and gastrointestinal mucosal lesions were examined. Data show the mean value and S.E. of cats. In young cats, very few gastrointestinal lesions were produced by ketoprofen. * P<0.0; ** P<0.0, significantly different from adult cats.

23 Percentages of small intestinal lesion area induced by ketoprofen in adult and young cats. The percentage of the lesion area was calculated as 00% of total small intestine area. ketoprofen (.0 mg/kg body weight, s.c.) was administered for days; once a day after a morning meal. The animals were sacrificed hr after the final dose of ketoprofen, and gastrointestinal mucosal lesions were examined. Lesion rate (%) = (lesion area in small intestine / total of small intestine area ) 00. Data show the mean value and S.E. of cats. In young cats, few small intestinal lesions were produced by ketoprofen.

24 Time-dependent changes of plasma concentration of R (-) ketoprofen in both adult and young cats. Ketoprofen (.0 mg/kg) was injected s.c. at 0 min. Data show the mean value and S.E. of cats. There was not significant difference on plasma concentration of ketoprofen between adult and young cats.

25 Time-dependent changes of plasma concentration of S (+) ketoprofen in both adult and young cats. Ketoprofen (.0 mg/kg) was injected s.c. at 0 min. Data show the mean value and S.E. of cats. There was not significant difference on plasma concentration of ketoprofen between adult and young cats.