Section I: Pharmacology, Toxicology and Pharmacokinetics of Ketoprofen SOME PHARMACOLOGICAL AND TOXICOLOGICAL STUDIES ON KETOPROFEN

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1 Section I: Pharmacology, Toxicology and Pharmacokinetics of SOME PHARMACOLOGICAL AND TOXICOLOGICAL STUDIES ON KETOPROFEN BY L. JULOU, J. C. GUYONNET, R. DUCROT AND J. PASQUET SUMMARY Evidence for the effectiveness of ketoprofen in animal models of inflammation and its antipyretic effect is reviewed. Its toxicity in animals is discussed and the low tendency to produce ulcers is stressed. 2-(3-BENZOYLPHENYL)PROPIONIC ACID or ketoprofen was selected in our laboratories during a systematic study, which started in 1966, of anti-inflammatory activity amongst the numerous benzoylphenyl alkyl acids. In our first publication which appeared in 1971 (Julou et al.) we explained all the points of interest which this product seemed to show experimentally: its very potent antiinflammatory activity and its low toxicity, especially as regards the gastro-intestinal tract. Clinical studies carried out firstly in France and then in Britain and finally on a world-wide basis confirmed the therapeutic effect of ketoprofen. In this paper we have summarized the essential pharmacological and toxicological facts based on the study of ketoprofen in comparison with indomethacin and acetylsalicylic acid. The primary modes of action of the aryl alkyl and aryl carboxylic acids still remain to be worked out but it seems probable, as Shen pointed out in 1972, that these products have a variety of actions on the complex process of inflammation. PHARMACOLOGICAL STUDY The techniques which we have used for the study of the anti-inflammatory, analgesic and antipyretic activities of ketoprofen and of reference products have been described previously (Julou et al., 1971, 1975). As can be seen in Table I, ketoprofen exerts a TABLE I ANTI-INFLAMMATORY ACTIVITY OF KETOPROFEN, INDOMETHACIN AND ACETYLSALICYLIC ACID Product Indomethacin Acetylsalicylic acid Carrageenin abscess (rat) ED 5 * mg/kg p.o. (a) 1.(.25-4) 1.3 (.5-3.2) 16 (74-326) U.V. erythema (guinea-pig) ED 6 * mg/kg p.o. (a) 6 ( ) 1.2 ( ) 9 (5-2) Adjuvant arthritis (rat) active threshold dose, P=.5 mg/kg p.o./ day/18 days (b) (a) ED 5 and fiducial limits (P=.5) calculated according to Litchfield and Wilcoxon (1949). (b) Day 1 is the day when the adjuvant was administered. * Effective dose, mean. 5

2 A SYMPOSIUM ON KETOPROFEN potent anti-inflammatory activity in carrageenin-induced abscess in the rat and ultraviolet erythema in the guinea-pig. This is at least equal to indomethacin and very much more potent than acetylsalicylic acid, 15 to 15 times more, depending on the test used. In the adjuvant arthritis test in the rat, an attractive model (Spector and Willoughby, 1968; Walz et al., 1974), ketoprofen shows a significant activity from a daily dose of 2.5 mg/kg by mouth; the minimal effective dose of indomethacin is also around 2.5 mg/kg by mouth. A study of a higher dosage of indomethacin could not be carried out because of the toxic effects it produced (1% mortality after 1 days of treatment of 5 mg/kg orally). On the other hand, ketoprofen at a daily dose of 1 mg/kg orally was well tolerated by polyarthritic rats and the inhibitory effect on the arthritis was very marked, around 7 %. The activity of acetylsalicylic acid only became apparent at a very high dosage of around 2 mg/kg orally. The analgesic effects of ketoprofen and of the two reference products were tested by two classical techniques used for 'antipyretic analgesics': pressure test in the rat paw (Randall and Selitto's test) and intraperitoneal injection of phenylbenzoquinone in the mouse. The results which are given in Table II show that there is no significant difference TABLE II ANALGESIC ACTIVITY OF KETOPROFEN, INDOMETHACIN AND ACETYLSALICYCLIC ACID Randall and Selitto's Test Pain induced by phenyl- Product (rat) benzoquinone in the mouse ED 5 mg/kg p.o. (a) EDgo mg/kg p.o. (a) 2.4 (.8-7.2) 2.3 ( ) Indomethacin 3.5(.8-14.) 2.2( ) Acetylsalicyclic acid 7 ( ) 16 (97-264) (a) ED 5 and fiducial limits (P=.5) calculated according to Litchfield and Wilcoxon (1949). between the activity of ketoprofen and indomethacin but that the active dosage of acetylsalicylic acid is 2 to 7 times higher than that of the other two products. Fig. 1 shows that ketoprofen, administered orally to the rat, acts against hyperthermia induced by the subcutaneous injection of brewers' yeast, an antipyretic effect about four times greater than that of indomethacin and about 1 times greater than that of acetylsalicylic acid. The antipyretic activity of ketoprofen (given subcutaneously) has also been studied in the rabbit in comparison with indomethacin. The hyperthermia was provoked by an intravenous injection of Proteus vulgaris and in this model ketoprofen is about 3 times more active than indomethacin (Fig. 2). It is interesting to note that ketoprofen is well absorbed when given orally, percutaneously (as a water-miscible 1 % cream) and by the rectal route (as suppositories of.1 to.3 or.9% of the active product in an excipient composed of semisynthetic glycerides), as is shown by a comparison of ED 5 of the product by these different routes and when given subcutaneously (see Table III) in the carrageenin abscess test in the rat. This good absorption has been confirmed in pharmacokinetic studies carried out both in experimental animals (Julou et al., 1975; Populaire et al., 1973) and in man (Delbarre et al., 1975; Populaire et al, 1973). It should especially be noted that the very good local tolerance of the injectable solution when given by intramuscular injection allows this route to be used without difficulty in man.

3 L. JULOU, J. C GUYONNET, R. DUCROT AND J. PASQUET RECTAL TEMPERATURE C CONTROLS: normal rats f~] CONTROLS: hyperthermic ^ rats J lndomethacin Dosage Acetylsalicylic acid (mg/kgp.a) FIG. 1. Antipyretic activity of ketoprofen in the rat compared with indomethacin and acetylsalicylic acid. HYPERTHERMIA Controls lndomethacin Dosage (mg/kgs.c.) FIG. 2. Antipyretic activity of ketoprofen in the rabbit compared with indomethacin.

4 A SYMPOSIUM ON KETOPROFEN TABLE III EFFECTIVENESS OF DIFFERENT ROUTES OF ADMINISTRATION OF KETOPROFEN (CARRAGEENIN ABSCESS IN RAT) Route ED 5 mg/kg (a) Oral 1.4(.4^.) Sub-cutaneous.4(.1-1.4) Percutaneous 1 Rectal 4.8 ( ) (a) ED 5 and fiducial limits CP=.5) calculated according to Litchfield and Wilcoxon (1949). Toxicity Sub-acute Mouse Rat Mouse Species TABLE IV TOXICITY OF KETOPROFEN 36 (257-54) 16( ) 18 ( ) LD 5 o* mg/kg p.o. (a) Indomethacin Acetylsalicylic acid 2 (12-34) 15 (8-135) 38 (29-5) 155(12-2) 5.7 ( ) about 68 Rat 21 (15-29) 6 about 78 (a) The animals were observed for 8 days after the single (acute toxicity) or the last (subacute toxicity) administration of the compounds. *LDg (mean lethal dose expressed in mg/kg p.o/day in the subacute toxicity studies) and fiducial limits (P=.5) calculated according to Litchfield and Wilcoxon (1949). TOXICOLOGICAL STUDY, when given orally to the mouse and rat (see Table IV) in acute and subacute (5-day) toxicity tests, is about 2 and 4 times respectively less toxic than indomethacin. In these two species, the acute toxicity of acetylsalicylic acid is respectively 3 and 1 times less than that of ketoprofen. The long-term toxicity studies of ketoprofen, given orally, were carried out in the rat, dog and monkey, for 1, 3 and 18 months in the rat, 1 and 3 months in the dog and 12 months in the monkey. As is the case with indomethacin and in general with potent steroidal and non-steroidal antiinflammatory agents, ketoprofen may give rise to gastro-intestinal lesions (the gastrointestinal mucosa represents the most sensitive target for this type of compound). In one-month studies in the rat and dog, ketoprofen was compared with indomethacin and the results obtained regarding the gastro-intestinal toxicity and mortality are given in Table V. This table shows that the maximum dose tolerated by the gastrointestinal tract is about three times higher for ketoprofen when compared with indomethacin. Also, at the two highest dosages studied, the ulcerogenic activity may cause mortality which was much greater with indomethacin than with ketoprofen. In the rat, after a 3 or 18 month treatment, ketoprofen was well tolerated by the gastro-intestinal tract at daily doses, respectively, of 6 and 4.5 mg/kg orally. In the dog, a species especially sensitive to the ulcerogenic properties of the antiinflammatory agents, after a 3-month treatment at an oral daily dose of 3 mg/kg,

5 L. JULOU, J. C. GUYONNET, R. DUCROT AND J. PASQUET ONE-MONTH ORAL TOXICITY TABLE V STUDY OF KETOPROFEN AND INDOMETHACIN Daily dose Mortality Gastric and intestinal ulcers (a) Rat Dog Indomethacin /2 17/2 2 1/2 2/2 + -f (1 Dog) + (1 Dog) (a) The severity of the ulcerogenic effect has been recorded as: =no ulceration; + =minimal ulceration; + + =numerous well marked ulcers. ketoprofen produced only minimal changes in the digestive tract (a few small ulcerations or erosions most often in the process of healing). Finally, in the monkey after 12-month treatment, ketoprofen was well tolerated by the gastro-intestinal tract at an oral daily dose of 9 mg/kg. At an oral daily dose of 27 mg/kg, one of the 12 monkeys treated had a small lesion on the pyloric antrum which suggested a healed ulcer. Teratogenicity studies of ketoprofen were carried out in the mouse, rat and rabbit, the drug being given during the period of organogenesis. There was no evidence of a teratogenic effect in the rabbit and mouse at oral daily doses of 3, 6 and 12 mg/kg, and in the rat at 3, 6 and 9 mg/kg. Slight embryo-toxicity was seen in the rabbit at the highest dose, an effect most probably linked to the impaired general condition of the does at this sub-toxic dose level. DISCUSSION AND CONCLUSIONS The experimental studies show that ketoprofen, like indomethacin, is a potent nonsteroidal anti-inflammatory agent. Both compounds have a similar anti-inflammatory and analgesic activity but ketoprofen is 3 to 4 times more active as an antipyretic agent. The active dose of acetylsalicyclic acid is 15 to 15 times higher than that of the other two products. In the mouse and rat, the acute toxicity of ketoprofen, given orally, is 4 to 8 times less than that of indomethacin and 3 to 1 times greater than that of acetylsalicyclic acid. In the case of daily oral administration maintained over several weeks in the rat and dog, the comparison of the toxicity of ketoprofen and indomethacin, and especially the gastro-intestinal tolerance, is also clearly in favour of the former product. With regard to the mechanism of the anti-inflammatory action of ketoprofen, numerous studies carried out during the past 12 years on the modes of action of the anti-inflammatory agents, and especially with the aryl alkyl acids (see Shen, 1972) have opened up a number of lines of research the action on various enzymes and mediators involved in the development of the inflammatory process, the inhibition of prostaglandins synthesis, modifications in the function of leucocytes and platelets, a stabilizing effect on the lysosome membranes etc. It should, however, be noted that the results obtained from these studies, especially those which were done in vitro, are often controversial, and that the resultant hypotheses on the mechanisms of action of these products are often tenuous and should therefore be viewed with caution (Paulus and Whitehouse, 1973). + +

6 1 A SYMPOSIUM ON KETOPROFEN In 1974 at the end of a critical study of the various actions of the anti-inflammatory drugs at the cellular and molecular levels, which could have some bearing on the mode of action of these products, Hichens (1974) concluded that the most interesting hypotheses to consider in the present state of our knowledge were the inhibition of prostaglandins synthesis and the stabilizing action on the lysosome membranes, this latter effect may be a consequence of the former. REFERENCES DELBARRE, F., ROUCAYROL, J. C, AMOR, B., INGRAND, J., BOURAT, G., FOURNEL, J. and COUR- JARET, J. (1975) "Pharmacokinetics Study of ( R.P.) in Man, using the Tritiated Compound". Scand. J. Rheumatol. In the press. HICHENS, M. (1974) "Molecular and Cellular Pharmacology of the Anti-inflammatory Drugs: Some in vitro Properties Related to their Possible Modes of Action". In: Anti-inflammatory Agents. Ed.: R. A. Scherrer and M. W. Whitehouse. 2, pp New York and London: Academic Press. (Vol. 13 of the collection Medicinal Chemistry A Series of Monographs Ed.: G. de Stevens). JULOU, L. GUYONNET, J. C., DUCROT, R., FOURNEL, J. and PASQUET, J. (1975) "2-(3-Benzoylphenyl) propionic acid or ( R.P.) "Main Pharmacological Properties: Outline of Toxicological and Pharmacokinetic Data". Scand. J. Rheumatol. In the press. GARRET, C, BARDONE, M. C, MAIGNAN, G. and PASQUET, J. (1971) "Etude des proprietes pharmacologique d'un nouvel anti-inflammatoire, l'acide (benzoyl-3 phenyl)-2 propionique ( R.P.)". /. Pharmacol. (Paris) 2, LITCHFIELD, J. T. and WILCOXON, F. (1949) "A Simplified Method of Evaluating Dose - Effect Experiments. /. Pharmacol. Exp. Ther. 96, PAULUS, H. E. and WHITEHOUSE, M. W. (1973) "Nonsteroid Anti-inflammatory Agents". Ann. Rev. Pharmacol. 13, POPULAIRE, P., TERLAIN, B., PASCAL, S., DECOUVELAERE, B., RENARD, A. and THOMAS, J. P. (1973) "Comportement biologique: taux seriques, excretion et biotransformation de l'acide (benzoyl-3 phenyl)-2 propionique ou ketoprofene chez l'animal et chez l'homme". Ann. Pharm. Fr. 31, SHEN, T. Y. (1972) "Perspectives in Nonsteroidal Anti-Inflammatory Agents". Angew. Chem. Int. Ed. 11, SPECTOR, W. G. and WILLOUGHBY, D. A. (1968) The Pharmacology of Inflammation. London: English Universities Press. WALZ, D. T., DI MARTINO, M. J. and SUTTON, B. M. (1974) "Design and Laboratory Evaluation of Gold Compounds as Anti-inflammatory Agents". In: Anti-inflammatory Agents Ed.: R. A. Scherrer and M. Whitehouse, New York and London: Academic Press. 1, (Vol. 13 of the collection Medicinal Chemistry A Series of MonographsEd.: G. de Stevens.