Mefenamic Acid &#X2014; a Nonsteroid Antiinflammatory Agent

NEW DRUGS

MEFENAMIC ACID--A NONSTEROID ANTIINFLAMMATORY AGENT

F. P. Trinus, N. A. Mokhort, UDC 615.276 L. M. Yagupol'skii, A. G. Fadeicheva, V. S. Danilenko, T. K. Ryabukha, Yu. A. Fialkov, L. M. Kirichek, ~. S. Endel'man, and G. A. Get'man

The inflammatory process is, as known, a pathological symptom of many illnesses. There- fore, the regulation of the inflammatory reaction, including that by means of pharmacological compounds, is one of the important problems of modern medicine. In particular, medici- nal compounds are used in clinics, which inhibit the development of the inflammatory process. The suppression of inflammation makes it possible to shorten the time of the illness and to decrease the gravity of the disease and its consequences. A modern clinic has a vast store of antiphlogistic preparations, which we divide into steroid and nonsteroid antiimflammatory agents. Despite the considerable process achieved in curing inflammatory illnesses by antiinflammatory preparations, in view of the low effectiveness in several cases, and the side effects and complications caused by them, it was necessary to search for new antiinflammatory agents with different action mechanisms. In this connection, intense research has recently been carried out in the series of derivatives of anthranilic acid, of which mefenamic acid and fluofenamic acid have been introduced into the medical practice [1-3]. These compounds differ from known nonsteroid antiinflammatory agents in that they are only slightly toxic and rarely cause side effects, while they exhibit a marked antiinflammatory, antipyretic, and analgesic activity. In the USSR, mefenamic acid has been also introduced into the practical public health services. Mefenamic acid is N-(2,3-dimethylphenyl)anthranilic acid (synonyms: Ponstan, Parka- med): ~6"OOH

Research on the preparation has been carried out at the Kiev Sclentific-Research Insti- tute of Pharmacology and Toxicology and at the Institute of Organic Chemistry, Academy of Sciences of the Ukrainian SSR [4]. The compound is synthesized on an industrial scale by the following reaction:

~ CH 5 HNOt OH3 Fe v "OH~ [ OH% OH~ OH% ]

H2SO% OH%] --.- H7804 --,,,- [ '~ -OH~ OH 3

Kiev Scientific-Research Institute of Pharmacology and Toxicology. Institute of Or- ganic Chemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Khimiko- Farmatsevticheskif Zhurnal, Vol. ii, No. 12, pp. 123-129, December, 1977. Original article submitted August 2, 1977.

1706 0091-150X/77/II12-1706507.50 1978 Plenum Publishing Corporation ~,, ,~COOH .I%IOH= CH5 ~"~/~"01 = OOOH

OH% OH%

The starting material for the synthesis of the compound is o-xylene. This is nitrated and a mixture of o- and m-nitroxylenes obtained, which is reduced by iron in the presence of hydrochloric acid. The amines obtained are separated by crystallization of their sul- fates. The slightly soluble sulfate salt of o-xylidene precipitates from the solution. Thus, the isomers can be completely separated. By the action of sodium hydroxide, the salt is converted into a base. o-Xylidine which separates out is condensed with o-chlorobenzoic acid, according to Ullmann, in the presence of potassium carbonate and copper (as the catalyst) in boiling isoamyl alcohol. The N-(2,3- dimethylphenyl)anthranilic acid thus obtained is purified by reprecipitation with hydrochloric acid from dilute alkaline solutions followed by crystallization twice from ethyl or isopropyl alcohol. The compound is in the form of a white or light-gray powder: It is odorless and has a bitter taste. It is practically insoluble in water, soluble in acetone and chloroform, slightly soluble in alcohol, nonhygroscopic, stable on storage and during sterilization. Melting point 223-228@C. Mefenamic acid is produced at the Irbit Pharmaceutical Chemical Factory in accordance with VFS [All-Union Pharmaceutical Standard] 42-231-73, and is put on the market in tablets of 0.25 and 0.5 gby the Tyumen Pharmaceutical Chemical Factory according to VFS 42-305-77.

Identification Reactions The following tests have been proposed for the identification of the preparation: i) reaction for carboxylic group: a 0.l-g portion of the preparation is dissolved with heating at 40~ in 30 ml of 1% sodium hydroxide solution, and i0 ml of the solution thus obtained is acidified by hydrochloric acid; a white curd separates out; 2) reaction for the secondary aromatic amino group: to 1 ml of 0.01% aqueous solution of sodium nitrite, 3 drops of the obtained alkaline solution of the preparation are added, the solution is shaken in a test tube, and 2 ml of concentrated sulfuric acid are carefully added: a blue ring is formed; 3) fluorescence in ultraviolet light of a chloroformic solution of the preparation: a 0.1% solution of the preparation in chloroform fluoresces in ultraviolet light with a pale-blue color; 4) the characteristic (%max and e) of the UV spectrum of the alcoholic-hydrochloric acid solution of the preparation. In the latter case, we limit ourselves to the position of the absorption maximum only: a 0.002% solution of the preparation in a mixture of meth- anol and IN hydrochloric acid (99:1) has two maxima at 279 • 1 and 350 • 1 nm in the region from 220 to 400 nm.

Test for Purity To confirm the purity of the preparation, the method of thin layer chromatography on Silufol UV25~ was used. As reference spots, the possible admixtures of o-chlorobenzoic and salicylic acids were used. The compounds were applied in an ether solution, and a mixture of toluene, acetic acid, and dioxane in a ratio of 90:1:30 (by volume) serves as the eluent. The chromatogram was developed by UV irradiation. The Rf values obtained for mefenamic acid is 0.59, o-chlorobenzoic acid 0.38, salicylic acid 0.34. The method of thin layer chromatography worked out on artificial mixtures makes it possible to determine impurities up to 0.25%. Mefenamic contains scarcely any impurities.

1707 Quantitative Determination About 0.3 g of the preparation (accurately weighed sample) is placed in a i00 ml flask and dissolved in 30 ml of acetone. The solution is titrated with 0.i N sodium hydroxide to a rapidly disappearing blue coloration, using thymol blue as indicator. A blank experiment is carried out in parallel. A I ml portion of 0. i N sodium hydroxide corresponds to 0.02413 g of CIsHIsN02, of which not less than 99% should be present in the preparation, calculated on the dry compound.

EXPERIMENTAL

Pharmacological The pharmacological properties of mefenamlc acid and its sodium salt were studied on different types of animals using various models of the experimental pathology. In the early investigations itwas shown that mefenamic acid inhibits skin erythema during UV irradiation of guinea pigs, and suppresses the development of adjuvant arthritis in rats. It exhibits a marked antiinflammatory activity in experiments with kaolin, carra- geen, formalin, Viscarin, and serotonin induced edema of paws of the rats, and in several cases is more effective than Butadion and sodium salicylate [2, 5-9]. The antiexudative effectiveness of mefenamic acid is to a considerable extent caused by its ability to decrease the permeability of the vascular wall [3, I0, ii]. This effect is to a certain extent due to the ability of the preparation to stabilize protein ultrastructures of cellular and subcellular membranes, as the result of which the permeability of the latter is de- creased. This has been experimentally confirmed in studies on the influence of mefenamic acid on the thermal denaturation of bovine and human albumin, and during the clarification of its ability to stabilize membranes of erythrocytes, leucocytes,and lysosomes of neutrophilic leucocytes [12-16]. Butadlon has a similar activity, but in its intensity of the protein-stabilizing effect, it is inferior to mefenamic acid, while derivatives of salicylic acid, in general, did not show any activity in these experiments [I]. In the mechanism of the antiexudative action of nonsteroid antiinflammatory agents, in general, and of mefenamic acid, in particular, an important role is played by the ability of these compounds to act on other pathogenic units participating in the development of the exudative inflammation phase, such as histamine, serotonin, quinines, or prostaglandins systems [i, 17]. Like Butadion, mefenamic acid suppresses vascular and the antiinflammatory effects of serotonin, which may be due to its ability of blocking the serotonin receptors of the cardiovascular system, and also to the inhibition of 5-hydroxytryptophan decarboxylase enzyme [18]. According to available data, mefenamic acid prevents the development of inflammatory reaction to the introduction of exogenic histamine, and to a considerable extent, suppresses the synthesis of prostaglandins, which are the main causes of inflammation [19, 20]. Mefenamic acid disrupts the oxidative phosphorylation and inhibits the synthesis of mucopolysaccharides [21, 22]. A distinct property of mefenamic acid and its salts is their ability to inhibit proteases which actively participate in the development of the inflammatory reaction. In the experiments in vitro and in vivo it has been shown that sodium salicylate and Butadion in concentrations of 1-8 mg/ml do not suppress the enzymatic activity of trypsin, while mefenamic acid largely inhibits the proteolytic activity of free trypsin [i, 23]. A comparative study of the antiprotease activity of mefenamic acid and more than 50 other derivatives of N-phenylanthranilic acid with different functional groups in experiments in vitro has shown that mefenamic acid more than other compounds, prevents the hydrolysis by trypsin of its substrates BAPNA (l-N-benzoyl-DL-arginlne-p-nitroanilide) and casein. The EDso doses (in millimoles per ml) of the acid for the above substrates are 6.4"10 -2 and 5.6" 10-2 , respectively, and in their effectiveness, these correspond to the EDso doses of Trasylol, the classical inhibitor of proteases, amounting to 7 and 9 calllcrein inactivator units [24]. In experiments in vivo with a slow internal infusion of a 24% trypsin solution to rats, it was found that the preparation noticeably increases the overall dose of the enzyme, leading to the death of the animals (control 78.9 • 1.4 mg/kg, experiment 116.8 • 9.8 mg/kg), and in this respect is similar to Trasylol (128 • 8.1 mg/kg).

1708 The antiproteolytic activity of mefenamic acid was confirmed in experiments in vitro with another representative of serine proteinases, plasmin. The experimental data show that mefenamic acid inhibits the lysis by plasmin of fibrinous pellicula by 31.4 • 2.5% (this index for c-ACA [e-aminocaproic acid] and sodium salicylate is 30.6 • 2.1 and 10.4 • 0.4%, respectively). In contrast to the clearly pronounced antitrypsin and antiplasmin effect, mefenamic acid does not show any antibradykinin activity in tests for bradykinin induced hypotension and bradykinin induced spastic contraction of a fragment of the small intestines of guinea pigs, and during the administration in therapeutic doses, it does not decrease the bradykinin content in the undiluted venous blood of rats [25]. It is interesting to note that a preliminary incubation of dilminal (a callicrein preparation) with mefenamic acid does not prevent and does not inhibit the hydrolysis of a callicrein substrate BAEE (N-benzoyl-L-arginine ethyl ester). Comparison of these results with the data on the inhibition of callicrein and bradykinin induced edemas by mefenamic acid confirms the conclusion [I] that the antiinflammatory effect of mefenamic acid is ex- plained not by the direct antibradykinin action, but by its ability to inhibit the proteolytic enzymes, to stabilize protein structures of the cellular membranes and to disturb histamine and serotonin metabolism. Mefenamic acid not only has a pronounced influence on the exudative phase, but also suppresses proliferation processes in the inflammation source [2, 26]~ surpassing in its effectiveness Butadion and sodium salicylate. The I3o of mefenamic acid (the dose of the preparation inhibiting the formation of a granular tissue in the inflammation source by 30%) is 12 mg/kg, while the I3o of Butadion and sodium salicylate is 51 and 120 mg/kg, respectively [i]. Mefenamic acid did not prevent the formation of skin necroses during the application of acetic acid and urea to the latter. However, on subsequent treatment of the animals, in contrast to Butadion and sodium salicylate , it caused a more rapid normalization of their state, and more active cicatrization of the defects formed [i, 27]. The preparation has a less marked ulcerogenic effect than Butadion and the derivatives of salicylic acid [28, 29]. Mefenamic acid exhibits pronounced antipyretic and analgesic activity. These effects are more appreciable in the presence of acute inflammatory symptoms in the organism, i.e., in cases when the pain reaction and hyperthermia result from the development of the inflammatory process. In the mechanism of the antipyretic and analgesic action of the preparation, great importance is attached to its ability to inhibit the synthesis of prostaglandin E in tissues by suppressing the activity of prostaglandin synthetase enzyme [19]. In the intensity of the anesthetic action, mefenamic acid is equivalent to Butadion, and is appre- ciably better than the derivatives of salicylic acid, while in its antlpyretic activity, it is approximately equivalent to Butadion and sodium salicylate [30]. In conventional therapeutic doses, thepreparation did not have any negative effect on the cardiovascular system, or the gastroenteritic and urinal tracts of the experimental ani- mmls [31]. Mefenamic acid is rather slightly toxic: the LDbo is 150 • 3.17~mg/kg for mice during intraperitoneal, and 600 • 23.3 mg/kg during peroral administration. In the case of peroral administration of mefenamic acid to rabbits in doses of 400 to 2000 mg/kg, no death of the animals was observed, i.e., under the conditions in which it is used, its toxicity is insignificant [32]. In the group of chronic tests (the preparation was introduced through a stomach tube to rats, daily, for 30 days, in a dose equal to 0.i LDso, determined for these animals at a similar mode of administration), when administered to a total dose of 3 LDso, mefenamic acid was tolerated, did not exhibit any negative effects on the growth and development of young rats, did not affect the hematological indicators, and advantageously differed from the derivatives of pyrazolone, which as known, suppress leucopenia. In a special series of experiments, no tetratogenic and embryotoxic action of the preparation studied was revealed [32]. Mefenamic acid is administered orally. Its adsorbability from the gastroenteritic tract is good. After being absorbed in blood, it combined with serum albumins, and the aromatic part of the molecule becomes as it were "inserted" into the hydrophobic fold of the protein,

1709 while its carboxylic group reacts with the cationic portion of the protein molecule surface [33]. The preparation is irregularly distributed over organs and tissues. Thus, according to the data of A. V. Vinnikova, one hour after the peroral administration of mefenamic acid to rats in a dose of 54 mg/kg, it was detected in the blood plasma in an amount of 58.6 ~g/kg, in the liver 41.5 ~g/kg, kidneys 42.5 Bg/kg, heart 17.75 ~g/kg, and in lungs 12.5 Bg/kg. It was not determined in the brain. It is partially excreted from the organism in an unchanged state by kidneys [34], partially hydrolyzed at the amine group with the formaL tion of a diacid, and the biological half-removal period is about 15 hours [35].

Clinical In 1968, in accordance with the decision of the Pharmacological Committee of the Minis- try of the Public Health of the USSR, mefenamic was clinically tested in several clinics of the USSR. The following doses of the preparation were approved: a single dose of 0.25 to 0.5 g, a daily dose of 1.5 g. The course of the treatment, depending on the progress of the illness, lasted from 20 to 45 days. The preparation was administered orally in tablets. A considerable therapeutic effectiveness of mefenamic acid has been noted during the treatment of patients suffering from an active phase rheumatism, rheumatoid polyarthritis,and infectious-allergic myocarditis. Thus, according to the data of the faculty of the Department of Therapy (director Prof. N. B. Rudenko) of the Odessa Medical Institute, the preparation was found to be effective in two-thirds of patients suffering from rheumatism and rheumatoid polyarthritis, and according to the data of the hospital pediatric clinic (director Prof. V. M. Sidel'nikov) of the Kiev Medical Institute, in 17 out of 32 children with an active rheumatic phase. Mefenamic had a high index of therapeutic effectiveness during the treatment of patients with infectious-allergic myocarditis: A considerable improvement was observed in 19 out of 24 patients treated, i.e., almost 80%. Other authors also point to the effectiveness of mefenamic acid during rheumatoid polyarthritis and osteoarthroses [36, 37]. The preparation prescribed to patients with rheumatic polyarthritis, sciatica, and rheumatism in doses of 200 mg, in capsules three times daily, had a distinct analgesic and antiinflammatory action, caused normalization of ele- vated body temperature, leucocytosis and the erythrocyte sedimentation rate [38]. When the dose is increased to 500 mg three times daily, the therapeutic effectiveness increased con- siderably [39]. During a prolonged use of mefenamic acid, side effects are possible, but they are found much more rarely than during the use of salicylates, derivatives of pyrazolone and indo- methacin, as well as hormonal preparations. Out of 123 patients who received the preparation during its testing in the USSR clinics, side effects were noticed in three patients only, i.e., in 2.4% of the total number of persons treated. In two of them a stomach pain was observed, and in one an itching eruption on the skin. According to [40], the side effects during the treatment with mefenamic acid were observed in 19.3% of patients. Accord- ing to the data of [34, 41], a prolonged use of mefenamic acid can lead to loss of appetite, appearance of nausea, diarrhea, feeling of weakness, which rapidly disappear on the discon- tinuation of the treatment. The preparation has almost no irritating action on the gastroenteritic tract and blood-forming organs, by which it advantageously differs from glucocorti- colds and widely known other nonsteroid antiinflammatory preparations. On the basis of the experimental and clinical research, the Pharmacological Committee of the Ministry of Public Health of the USSR permitted the wide clinical use of mefenamic acid for treating active forms of rheumatism, infectious-allergic myocarditis, infectious nonspecific (rheumatoid) forms of polyarthritis, as well as an analgesic and antipyretic drug. The preparation is put on the market by the pharmaceutical industry, and supplied to the network of pharmacies.

LITERATURE CITED

I. F. P. Trinus, N. A. Mokhort, and B. M. Klebanov, Nonsteroid Antiinflammatory Agents [in Russian], Kiev (1975). C. V. Winder, J. Wax, L. Scotti, et al., J. Pharmacol. Exp. Ther., 138, 405-413 (1962). 3. Ch. A. Winter, Annu. Rev. Pharmacol., ~, 157-174 (1966). 4. L. M. Yagupol'skii, P. A. Yufa, Yu. A. Fialkov, et al., USSR Inventor's Certificate No. 331058, Otkrytiya, No. 9 (1972).

1710 5. N. S. Korkhova, "Pharmacology of anthranilic acid derivatives," Candidate's Disserta- tion, Dnepropetrovsk (1970). 6. N. A. Mokhort, in: Pharmacology and Toxicology [in Russian], No. 6, Kiev (1971), pp. ,108-111. 7-8.F.P. Trinus and N. A. Mokhort, Farmakol. Toksikol., No. 3, 306-311 (1971). 9. C. V. Winder, L. A. Lembke, and M. D. Stephens, Arthr. Rheum., 12, 472-482 (1969). i0. N. A. Mokhort and N. I. Sharikina,Fiziol. Zh., No. 3, 357-360 (1972). ii. F. P. Trinus and N. A. Mokhort, Farmakol. Toksikol., No. i, 60-63 (1972). 12. N. A. Mokhort and T. L. Sakun, Summaries of Lectures at the Third Congress of Pharma- cologists of the Ukrainian SSR [in Russian], Vinnitsa (1977), p. 122. 13. T. L. Sakun, "Phlogenic activity of neutrophilic leucocytes and nonsteroid antiinflammatory agents," Candidate's Dissertation, Kiev (1974). 14. F. P. Trinus, in: Pharmacology and Toxicology [in Russian], No. 7, Kiev (1972), pp. 55-62. 15. A. D. Inglot and E. Wolna, Biochem. Pharmacol., 17, 269-279 (1968). 16. Y. Mizushima and H. Suzuki, Arch. Int. Pharmacodyn., 157, 115-124 (1965). 17. B. B. Vargaftig, Agents and Actions, !, 357-365 (1973). 18. N. A. Mokhort, F. P. Trinus, and G. A. Get'man, Vrach. Delo, No. Ii, 109-112 (1971). 19. W. Losert, Arzneimittel Forsch., 25, 135-148 (1975). 20. J. R. Vane, in: International Congress on Pharmacology. 5th Pharmacology and the Future of Man. Proceedings. Vol. 5, Basel (1973), pp. 352-378. 21. B. M. Klebanov and F. P. Trinus, Farmakol. Toksikol., No. 5, 578-581 (1974). 22. F. P. Trinus and B. M. Klebanov, Farmakol. Toksikol., No. i, 112-117 (1972). 23. F. P. Trinus, K. M. Veremeenko, N. A. Mokhort, et al., Farmakol. Toksikol., No. 6, 715-718 (1969). 24. F. P. Trinus, V. S. Danilenko, and K. A. Chernoshtan, in: Physiologically Active Com- pounds [in Russian], No. 5, Kiev (1973), pp. 75-77. 25. V. S. Danilenko and K. A. Chernoshtan, in: Treatment of Rheumatism and Joints lll- nesses [in Russian], Kiev (1973), pp. 26-28. 26. N. A. Mokhort, Farmakol. Toksikol., No. 3, 297-200 (1971). 27. N. A. Mokhort, in: Pharmacology and Toxicology [in Russian], No. 12, Kiev (1977), pp. 77-80. 28. N. A. Mokhort and F. P. Trinus, Vrach. Delo, No. 6, 20-23 (1971). 29. U. Jahn and R. W. Adrian, Arzneimittel Forsch., 19, 136-152 (1969). 30. N. A. Mokhort, in: Mechanism of the Action of Drugs and Poisons. Su-mmries of Lec- tures at the Conference of Young Pharmacologists and Toxicologists [in Russian], Kiev (1967), pp. 63-65. 32. N. A. Mokhort and N. S. Korkhova, in: Pharmacology and Toxicology [in Russian], No. 4, Kiev (1968), pp. 87-89. 33. C. Chignell, Mol. Pharmacol., ~, 455-462 (1969). 34. E. Schupp, Therapiewoche, 18, 1531-1533 (1968). 35. C. V. Winder, D. H. Kaump, A. J. Glazko, et al., Ann. Phys. Med., Suppl., 7-49 (1967). 36. F. D. Chart, Drugs, 9, 321-325 (1975). 37. J. Villiauemy, Lyon Mediterrane4 Med., I0, 882-883; 875-876 (1974). 38. M. Barlattani and W. Carignola, Clin. Ter., 49, 577-588 (1969). 39. L. J. Cass and W. S. Frederik, J. Pharmacol. Exp. Ther., 139, 172-176 (1963). 40. H. Adamska - Dyniewska, J. Chojnacki, J. Chojnowska - Jezierska, et al., Wiad. Lek., 28, 269-274 (1975). 41. W. J. Cahill, R. D. Hill, J. Jessop, et al., Ann. Phys. Med., 8, 26-29 (1965).

*As in Russian original -- Consultants Bureau.

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