Inhibition of Tissue Damage by the Arachidonate Lipoxygenase Inhibitor BW755C (Leukocyte Accumulation/Prostaglandins/Leukotrienes/Anti-Inflammatory Drugs) GERALD A

Proc. Nati. Acad. Sci. USA Vol. 81, pp. 2890-2892, May 1984 Medical Sciences

Inhibition of tissue damage by the arachidonate lipoxygenase inhibitor BW755C (leukocyte accumulation/prostaglandins/leukotrienes/anti-inflammatory drugs) GERALD A. HIGGS, KENNETH G. MUGRIDGE, SALVADOR MONCADA, AND JOHN R. VANE Department of Prostaglandin Research, Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, United Kingdom. Contributed by J. R. Vane, January 30, 1984

ABSTRACT The effects of three anti-inflammatory zymes, but by stimulating the production of a phospholipase drugs, which interfere with arachidonic acid transformation inhibitor (15, 16) they reduce the availability of arachidonic by three different enzymes, have been compared by using a acid for conversion to both prostaglandins and leukotrienes. simple model of tissue damage and foreign body rejection. In This suppression of leukotriene as well as prostaglandin syn- groups of control rats, subcutaneously implanted polyester thesis may explain why steroids provide a more comprehen- sponges were rejected after a mean of 12 days. Indomethacin, sive anti-inflammatory activity than aspirin-like drugs. We which selectively inhibits prostaglandin synthesis, did not sig- have now compared the effects of BW755C with a selective nificantly change time to rejection but BW755C (3-amino-1- cyclo-oxygenase inhibitor, indomethacin, and with the ste- [m-(trifluoromethyl)phenylJ-2-pyrazoline), which is a dual in- roid dexamethasone in a simple model of tissue damage and hibitor of prostaglandin and leukotriene synthesis, prolonged foreign body rejection. time to rejection to a mean of 22 days. The anti-inflammatory steroid dexamethasone, which reduces arachidonic acid me- METHODS AND MATERIALS tabolism by stimulating the formation of a phospholipase in- Polyester sponges were soaked in 2% carrageenin (wt/vol in hibitor, prolonged time to sponge rejection as BW755C did. sterile saline) and implanted subcutaneously in male Wistar Treatment with BW755C or dexamethasone was also accom- rats (-200 g) (12). Drugs were administered orally twice dai- panied by a reduction in total leukocyte numbers in inflamma- ly in an aqueous vehicle. In some experiments animals were tory exudates collected at 1-14 days, whereas indomethacin observed on a daily basis until the sponges were rejected. increased leukocyte migration on days 1 and 2 and had no ef- Each animal was inspected twice a day and a subjective as- fect at later times. These results suggest that the inhibition of sessment of tissue damage was made on a randomized blind the leukotriene-forming lipoxygenase suppresses leukocyte ac- basis. Tissue damage was scored on a 0-5 scale according to tivation and that this leads to a reduced rate of tissue damage the following criteria: 0, no change from time of implanta- in experimental inflammation. tion; 1, first signs of hair loss; 2, hair loss and edema; 3, extensive hair loss, edema, and necrosis (blackening of the The inhibition of prostaglandin synthesis accounts for the skin); 4, skin split and sponge visible; 5, sponge rejected. anti-inflammatory and analgesic properties of aspirin-like Each animal was scored twice a day, giving a maximum pos- drugs (1). These drugs are much less effective in reducing sible score of 10 when the sponge was rejected. the accumulation of inflammatory leukocytes (2), and this In other experiments animals were killed 1-14 days after may explain why they do not alter the course of chronic ar- implantation, inflammatory exudates were collected from thritis or prevent tissue damage and incapacitation (3). the sponges for leukocyte counts, and samples of tissue were The pathogenesis of chronic inflammatory conditions is taken for histology. Smears of exudates were examined for closely associated with accumulation of phagocytic leuko- bacterial contamination and exudates were cultured on cytes. The appearance of first polymorphonuclear leuko- blood agar plates for 24 hr in aerobic and anaerobic condi- cytes (PMNs) and then mononuclear leukocytes in injured tions. tissues is characteristic of the chronic inflammatory re- BW755C was synthesized by F. C. Copp at the Wellcome sponse. Release of lysosomal enzymes from these cells, af- Research Laboratories. ter specific stimuli or cell death, contributes to tissue damage and necrosis (4). A putative mediator of leukocyte acti- RESULTS vation is leukotriene B4, which is a potent stimulator of Inflammatory exudates collected from the sponges of con- chemotaxis and degranulation (5, 6) and has been detected in trol rats 2 days after sponge implantation contained approxi- experimental inflammation (7) and human joint disease (8, mately 50 x 106 leukocytes per ml and these cells were pre- 9). dominantly (>95%) PMNs. The skin above the implant be- In common with the prostaglandins, leukotrienes are prod- came edematous and also heavily infiltrated with PMNs. ucts of arachidonic acid peroxidation (10). The cyclo-oxy- After 4 days, macrophages appeared in the lower dermis and genase that catalyzes prostaglandin formation is selectively by 6 days mononuclear leukocytes were present in exudates inhibited by aspirin-like drugs, but the lipoxygenase that ini- in approximately equal numbers to the PMNs. There was tiates leukotriene synthesis is resistant to these drugs (11). evidence of fibrin deposition around the sponge as early as The experimental compound BW755C (3-amino-1-[m-(tri- 24 hr after implantation and by 6 days there was extensive fluoromethyl)phenyl]-2-pyrazoline) (12) is a dual cyclo-oxy- deposition of fibrin and collagen in the area of the sponge. genase and lipoxygenase inhibitor that prevents prostaglan- Fibroblasts appeared in association with the collagen. In ex- din and leukotriene synthesis in vitro (13) and in vivo (14). udates collected after 6 days, mononuclear leukocytes were Anti-inflammatory steroids do not inhibit either of these en- predominant over PMNs and total leukocyte numbers increased to approximately 100 x 106 per ml. Externally, these The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: BW755C, 3-amino-1-[m-(trifluoromethyl)phenyl]-2- in accordance with 18 U.S.C. §1734 solely to indicate this fact. pyrazoline; PMNs, polymorphonuclear leukocytes.

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changes were associated with swelling, hair loss, and black- 10 Indomethacin ening of the skin. At 8 days, more than 90% of the epidermis above the sponge was undergoing necrosis, progressing to the formation of a dry scab by days 10-14, during which period the sponges were usually rejected. Normal hair growth quickly resumed at the implantation site. Indomethacin (0.5 mg/kg, twice per day) significantly in- Dexamethasone creased total leukocyte numbers in 1- and 2-day exudates, but this effect was not maintained after 4 days (Fig. 1). Also, 0 in indomethacin-treated animals the external signs of tissue u

damage were accelerated (Fig. 2). Sponges were rejected 7; from indomethacin-treated rats 1.5 days before the controls, but this difference was not significant (Fig. 3). BW755C (10 ._ mg/kg, twice per day) caused a 45-65% decrease in total leukocyte numbers in exudates collected 1-8 days after implantation (Fig. 1). Dexamethasone (0.1 mg/kg, twice per day) also reduced cell numbers by up to 80% on days 2-8 (Fig. 1). Both BW755C and dexamethasone significantly de- layed the onset of visible tissue damage (Fig. 2), and this was accompanied by a significant increase in rejection time (Fig. 3). Skin samples taken from animals treated with BW755C at day 14 showed signs of acute inflammation with edema and 6 8 leukocyte infiltration but epidermal necrosis was markedly Time, days reduced compared with control samples taken at 10 days. None of the drug treatments altered the relative proportion FIG. 2. Effects of drugs on tissue damage and necrosis around of PMNs and mononuclear leukocytes in sponge exudates. subcutaneous sponge implants. During a period of 14 days after im- Microscopic examination of exudates did not reveal sub- plantation, each animal was observed twice a day and a subjective stantial bacterial contamination, but cultures of exudates assessment of tissue damage was made on a randomized blind basis. Tissue damage was scored on a 0-5 scale as described in the text. produced a mixed population of organisms in which small Each animal was scored twice a day, giving a maximum possible Gram-negative rods were predominant. Baterial contamina- score of 10. Each point is the mean of daily scores from 8-62 ani- tion in exudates from animals treated with BW755C did not mals and the bars represent ± 1 SEM. Drugs were administered differ significantly from that in exudates from control ani- orally twice daily and control animals received vehicle alone. Dos- mals. In vitro, BW755C (1 mM) did not prevent the growth of ages were as follows: indomethacin, 0.5 mg/kg; dexamethasone, 0.1 mg/kg; and BW755C, 10 mg/kg. 200f Staphylococcus aureus or Escherichia coli. 180h Indomethacin, at the dose used in this study, acts as a selective inhibitor of prostaglandin synthesis, but higher doses Indomethacin also inhibit leukocyte migration (2). In such high amounts, 160k indomethacin (4-16 mg/kg) caused a dose-dependent reduc- - 140F - 22 r Ur_ 120k 0 18 F 10OF ------lo~

ci 80F 0o tX 14 60k BW755C C 40~ 0 10

20 .6wG Dexamethasone

0 1 2 3 4 5 6 7 8 Time, days FIG. 1. Effects of indomethacin (0.5 mg/kg, twice per day), 2 BW755C (10 mg/kg, twice per day), and dexamethasone (0.1 mg/kg, K twice per day) on total leukocyte numbers in 1- to 8-day inflamma- Control Indomethacin BW755C Dexamethasone tory exudates. Each point is the mean of total leukocyte counts from sponges of at least four animals; the bars represent ± 1 SEM and * FIG. 3. Effect of indomethacin, BW755C, and dexamethasone indicates P < 0.05 compared to control values obtained from similar on the rejection of subcutaneously implanted polyester sponges in groups of animals receiving vehicle alone. BW755C and dexametha- rats. Each histogram represents the mean from at least 10 animals sone caused similar effects on 8- to 14-day exudates but exudates and the bars represent ± 1 SEM. Drugs were administered orally could not be collected reliably from indomethacin-treated animals twice daily: indomethacin, 0.5 mg/kg; BW755C, 10 mg/kg; and after day 8 due to sponge rejection. dexamethasone, 0.1 mg/kg. Downloaded by guest on September 27, 2021 2892 Medical Sciences: Higgs et alPProc. NatL Acad ScL USA 81 (1984) tion in leukocyte numbers at 24 hr, but the drug at these 1. Higgs, G. A., Moncada, S. & Vane, J. R. (1983) in Anti-Rheu- doses was not well tolerated after repeated administration matic Drugs, ed. Huskisson, E. C. (Praeger, United King- for more than 3 days. BW755C was well tolerated in this dom), pp. 11-36. study, with no significant changes in weight gain from con- 2. Higgs, G. A., Eakins, K. E., Mugridge, K. G., Moncada, S. & trol animals. Effective doses of dexamethasone resulted in a Vane, J. R. (1980) Eur. J. Pharmacol. 66, 81-86. loss of weight compared with controls but there were no oth- 3. Woodbury, D. M. & Fingl, E. (1975) in The Pharmacological er clinical signs of toxicity. Basis of Therapeutics, eds. Goodman, L. S. & Gilman, A. (Macmillan, New York), 5th Ed., p. 325. DISCUSSION 4. Davis, P. & Allison, A. C. (1978) in Inflammation, Handbook ofExperimental Pharmacology, SO/1, eds. Vane, J. R. & Fer- These results support the theory that inhibition of leukocyte reira, S. H. (Springer, New York), pp. 267-294. migration reduces tissue inflammation, necrosis, and dam- 5. Ford-Hutchinson, A. W., Bray, M. A., Doig, M. W., Shipley, age. Corticosteroids and immunosuppressive agents such as M. E. & Smith, M. J. H. (1980) Nature (London) 286, 264- azathioprine and methotrexate are known to reduce leuko- 265. cyte migration into damaged tissues, and this could be an 6. Goetzl, E. J. & Pickett, W. C. (1980) J. Immunol. 125, 1789- important component of their anti-inflammatory activity (17, 1791. 18). BW755C also suppresses leukocyte migration, and this 7. Simmons, P. M., Salmon, J. A. & Moncada, S. (1983) Bio- may be directly linked to the inhibition of leukotriene B4 chem. Pharmacol. 32, 1353-1359. synthesis (14). The reduction of tissue damage and delay in 8. Klickstein, L. B., Shapleigh, C. & Goetzl, E. J. (1980) J. Clin. sponge rejection caused by BW755C cannot be explained by Invest. 66, 1166-1170. an anti-bacterial effect. 9. Rae, S. A., Davidson, E. M. & Smith, M. J. H. (1982) Lancet In some types of chronic degenerative disease such as ii, 1122-1123. rheumatoid arthritis the control of leukocyte accumulation 10. Samuelsson, B. (1983) Science 220, 568-575. could be an important factor in limiting tissue damage. Simi- 11. Higgs, G. A. & Flower, R. J. (1981) in SRS-A and Leuko- larly, inhibition of leukocyte accumulation has been shown trienes, ed. Piper, P. J. (Wiley, New York), pp. 197-207. to reduce tissue damage after myocardial infarction (19, 20). 12. Higgs, G. A., Flower, R. J. & Vane, J. R. (1979) Biochem. The results reported here indicate that conventional nonste- Pharmacol. 28, 1959-1961. roid anti-inflammatory drugs such as indomethacin do not 13. Randall, R. W., Eakins, K. E., Higgs, G. A., Salmon, J. A. & reduce leukocyte accumulation or leukocyte-mediated tissue Tateson, J. E, (1980) Agents Actions 10, 553-555. damage at anti-prostaglandin doses. 14. Salmon, J. A., Simmons, P. M. & Moncada, S. (1983) J. In common with other chemotactic factors, leukotriene B4 Pharm. Pharmacol. 35, 808-813. induces lysosomal enzyme release from leukocytes (6). Inhi- 15. Blackwell, G. J., Carnuccio, R., Di Rosa, M., Flower, R. J., bition of lipoxygenase could, therefore, result in a cytopro- Parente, L. & Persico, P. (1980) Nature (London) 287, 147- tective effect at the site of leukocyte activation. This may 149. explain why there is reduced necrosis in tissues from animals 16. Hirata, F., Schiffmann, E., Venkatasabramanian, K., Solo- treated with BW755C. The dual inhibition of arachidonate mon, D. & Axelrod, J. (1980) Proc. NatI. Acad. Sci. USA 77, cyclo-oxygenase and lipoxygenase may represent an im- 2533-2536. proved anti-inflammatory mechanism for certain chronic 17. Di Rosa, M. (1979) in Anti-Inflammatory Drugs, Handbook of diseases, giving a steroid-like therapeutic effect while avoid- Experimental Pharmacology, 50/If, eds. Vane, J R. & Fer- ing the toxicity normally associated with steroid treatment. reira, S. H. (Springer, New York), pp. 223-253. It is also possible that a drug which selectively inhibits lipid 18. Brune, K. & Whitehouse, M. W. (1979) in Anti-Inflammatory peroxidation could be a useful clinical tool in situations in Drugs, Handbook of Experimental Pharmacology, 50/II eds. tissue is a Vane, J. R. & Ferreira, S. H., (Springer, New York), pp. 531- which rejection problem. 597 We thank Dr. N. G. Read, Mr. N. C. Woods, and Mr. P. J. 19. Mullane, K. M. & Moncada, S. (1982) Prostaglandins 24, 255- Astbury for help with the histology and Dr. C. Adlam for performing 266. the bacteriological studies. 20. Jolly, S. R, & Lucchesi, B. R. (1983) Am. Heart J. 106, 8-13. Downloaded by guest on September 27, 2021