AZAPROPAZONE PREVENTION OF POST-ISCHEMIC TISSUE DAMAGE

Abstract: Azapropazone can be used to prevent post-ischemic tis­ sue damage in mammals.

Description: This invention relates to the prevention of post-ischemic tissue damage. Ischemic and post-ischemic tissue damage results when blood flow into bodily tissues and/or organs is blocked and the tissue becomes hypoxic. Upon restoration of normal blood flow, i.e., reperfusion, large amounts of toxic oxygen free radicals (O - / 2) are produced which cause significant tissue and/or organ damage and impaired function. Ischemia-induced tissue damage is now believed to be a major and medically significant complication in a wide variety of cardiovascular, central nervous system, and intestinal disease processes. In addition, post-ischemic tissue damage is also a medically significant problem in organ transplantation and circulatory shock. (McCord, J.M.: "Oxygen-Derived Free Radicals in Post-Ischemic Tissue Injury," New England Journal of Medicine, 312:154-163, 1985.) IRCS Medical Sciences, 13, 565 - 566 (1985) discloses that various drugs, among them azapropazone, inhibit xanthine oxidase. The toxic oxygen radicals responsible for post-ischemic tissue injury originate from two biochemically different sources. Large amounts of radicals are produced in ischemic and reperfused tissue as a byproduct of the enzymatic conversion of hypoxanthine to xanthine by xanthine oxidase and also as metabolic products from activated blood neutrophils also known as activated polymorphonuclear leukocytes. There is substantial experimental evidence implicating both sources as major contributors to the pathology of ischemia-reperfusion tissue damage. (Hill, J.H., Ward, P.A.: "The Phlogistic Role of C3 Leukotactic Fragments in Myocardial Infarcts of Rats." Journal Exp. Medicine. 133:885-900, 1971; Rossen, R.D. et al: "Selective Accumulation of First Component of Complement and Leukocytes in Ischemic Canine Heart Muscle," Circ. Res. 57:119-129, 1985; Bednar, M., Smith, B., Pinto, A. and Mullane, K.M.: "Nafazatrom-Induced Salvage of Ischemic Myocardium in Anesthetized Dogs is Mediated Through Inhibition of Neutrophil Function," Circ. Res. 57:131-141, 1985.) Therapeutic approaches to treating post-ischemic tissue injury have concentrated initially on drugs that selectively inhibit xanthine oxidase (e.g., allopurinol) and, more recently, on drugs that inhibit O - / 2 production by activated polymorphonuclear leukocytes (e.g., aprotinin, nafazatrom, ibuprofen). Presently, there are no reports in the literature of any drug that inhibits post-ischemic tissue damage by inhibiting both xanthine oxidase and O - / 2 production by activated polymorphonuclear leukocytes. Azapropazone is a nonsteroidal anti-inflammatory drug efficacious in treating gouty arthritis by virtue of its ability to inhibit xanthine oxidase and increase the renal excretion of uric acid (Templeton, J.S.: "Azapropazone, In Anti-Rheumatic Drugs ," Vol. 3 (Edited by E. C. Huskisson), Praeger Publishers, New York, p. 97 (1983); U.S. Patents 3,349,088, 3,482,024, and 4,305,942). It has now been found that azapropazone also has the unique feature of inhibiting a variety of neutrophil functional responses including the generation of oxygen free radicals. Moreover, the concentrations of azapropazone found to inhibit neutrophil O - / 2 production as well as xanthine oxidase in vitro correlate well with the therapeutic plasma levels attained in man. Based upon azapropazone's ability to inhibit both sources of toxic O - / 2 radicals, this drug will have distinct and novel therapeutic advantage over the current therapies used in treating ischemic and post-ischemic tissue damage. The invention relates to the use of azapropazone for the manufacture of a medicament for preventing ischemic and post-ischemic tissue damage in a mammal. Azapropazone is the compound 3-dimethylamino-7-methyl-1,2-(n-propylmalonyl)-1,2-dihydrol,2,4-benzotriazine dihydrate. This compound has the formula: The compound is known by the WHO recommended International Nonproprietary Name (INN) "Azapropazone." Azapropazone is applied in an amount sufficient to reduce ischemic and reperfusion damage by inhibiting both xanthine oxidase and O - / 2 production by activated polymorphonuclear leukocytes. The exact amount will depend oil the nature of the ischemia and the rate of reperfusion following ischemia. Azapropazone will have therapeutic utility in a wide variety of disease states in which ischemia-reperfusion tissue damage is medically significant. Specific disease processes in which azapropazone would be efficacious are intestinal, myocardial, cerebral ischemia as well as in circulatory shock, frostbite and organ transplantation. Based upon its pharmacokinetic profile, azapropazone will be administered orally or as intravenous injections with doses ranging between 300-1800 mg/day in man. Azapropazone therapy will be short or long term depending upon the disease being treated. Azapropazone may also be administered prophylactically to patients judged to be at high risk of ischemic attack. Azapropazone can be manufactured as taught in U.S. 3,349,088 The active compound can be formulated as taught in U.S. 4,305,942, Particularly preferred is an injectable formulation for ease of administration to unconscious patients or in emergency situations. EXAMPLE 1 Azapropazone can be formulated for injection as follows: Azapropazone dihydrate (corresponding to 576.8 mg azapropazone sodium) 600.0 mg Manitol 30.0 mg Sodium metabisulfite 3.0 mg Sodium hydroxide to adjust pH 72.0 mg Water for injection 5.0 ml EXAMPLE 2 Azapropazone can be formulated as a 300 mg capsule as follows: Azapropazone dihydrate 300 mg Sodium lauryl sulfate, NF 4.2 mg Povidone U.S.P. 3.0 mg Silicon dioxide, NF 1.8 mg Calcium stearate 4.5 mg Form uniform aggregate with sufficient equal amounts of denatured alcohol and purified water, dry and mill the aggregates to the desired size. Insert the resulting powder into a hard opaque gelatin capsule containing approved colorants. EXAMPLE 3 The following experiments were performed to demonstrate azapropazone inhibition of O - / 2 production by polymorphonuclear leukocytes. Rat peritoneal neutrophils (PMN) were collected 4-6 hrs. after i.p. injection of 10% w/v sodium caseinate (Sigma) and washed several times with Hanks' buffer (pH 7.2) at 4°C to remove residual exudate fluids. The cells were stimulated to generate O - / 2 by exposing the cells to 100 ng/ml phorbol-12-myristate-13-acetate (PMA) in dimethylsulfoxide. PMN (5 x 10⁶ cells/ml) were pretreated with or without varying concentrations of azapropazone for 10 minutes at 37°C. Non-reduced cytochrome C (0.23mM) and PMA (100 ng/ml) were added to the cells and the mixture quickly transferred to quartz curvettes in a Beckman Du-6 spectrophotometer at 37°C. Superoxide anion (O - / 2) generation was assayed by measuring the O - / 2 dependent reduction of ferricytochrome C to ferrocytochrome C by monitoring absorbance at 550 nm over a 30 min. period. In order to calculate specific O - / 2 reduced cytochrome C, the change in Abs at 550 nm detected in the presence of 30 µg/ml superoxide dismutase (SOD) was also measured. Data were converted to nanomoles of O - / 2 reduced cytochrome C by first subtracting the SOD values and then dividing by the absorbance coefficient for reduced cytochrome C (21.2 mM⁻¹ cm⁻¹). Rates of O - / 2 production were calculated by linear regression analysis of the O - / 2 production vs. time curve at times between 0 and 15 minutes. All data are expressed as mean values of 3 or more separate experiments. It can be seen that azapropazone causes significant inhibition of both the rate of O - / 2 generation and maximal amounts of O - / 2 produced by PMN stimulated with PMA. Inhibition of Rat PMN O - / 2 Production BY Azapropazone Treatment nmoles Cyto C reduced/5.0x10⁶ PMN (minute) 0 5 10 15 20 30 + PMA 1.6± 5.4± 7.5± 7.8± 8.3± 8.1± 1.3 1.2 1.6 2.1 2.4 2.7 - PMA 0.3± 0.4± 0.5± 0.4± 0.1± 0.2± 0.6 0.4 2.4 0.8 1.1 1.8 PMA + 0.1 mM 0.3± 2.6± 3.8± 4.5± 5.6± 4.8± azapropazone 0.6 1.4 1.6 2.0 2.4 1.6 PMA + 0.01 mM 0.3± 4.7± 7.0± 8.3± 9.1± 9.7± azapropazone 0.2 2.7 3.8 4.4 4.4 5.9 PMA + 0.001 mM 0.2± 4.9± 6.7± 8.7± 8.7± 8.7± azapropazone 0.5** 3.2 4.4 5.6 5.6 6.2

Claims: Use of azapropazone for the manufacture of a medicament for preventing ischemic and post-ischemic tissue damage in a mammal. Use of Claim 1 wherein the medicament is an injectable.

Inventors: BOSWELL, GEORGE ALBERT, MACKIN, WILLIAM MICHAEL, THOOLEN, MARTIN JAN
Applicants: THE DU PONT MERCK PHARMACEUTICAL COMPANY
Application year: 1986
Main classification: A61K31
IPC codes: A61K31, C07D487