CH 5450

Aim:To investigate the ability of proteinase inhibitors to modulate nucleated cell infiltration into the peritoneum of mice induced by tryptase and chymase.Methods:Human lung tryptase and skin chymase were purified by a similar procedure involving high salt extraction, heparin agarose affinity chromatography followed by S-200 Sephacryl gel filtration chromatography. The actions of proteinase inhibitors on tryptase and chymase induced nucleated cell accumulation were examined with a mouse peritoneum model.Results:A selective chymase inhibitor Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPPF) was able to inhibit approximately 90 % neutrophil, 73 % eosinophil, 87 % lymphocyte and 60 % macrophage accumulation induced by chymase at 16 h following injection. Soy bean trypsin inhibitor (SBTI), chymostatin, and alpha1-antitrypsin showed slightly less potency than ZIGPPF in inhibition of the actions of chymase. While all tryptase inhibitors tested were able to inhibit neutrophil, eosinophil, and macrophage accumulation provoked by tryptase at 16 h following injection, only leupeptin, APC366, and aprotinin were capable of inhibiting tryptase induced lymphocyte accumulation. The inhibitors of tryptase tested were also able to inhibit tryptase induced neutrophil and eosinophil accumulation at 6 h following injection. When being injected alone, all inhibitors of chymase and tryptase at the concentrations tested by themselves had no significant effect on the accumulation of nucleated cells in the peritoneum of mice at both 6 h and 16 h.Conclusion:Proteinase inhibitors significantly inhibited tryptase and chymase-induced nucleated cell accumulation in vivo, and therefore they are likely to be developed as a novel class of anti-inflammatory drugs.

1. Functional recordings of smooth muscle tension and biochemical experiments on membrane fractions were performed to characterize angiotensin II (AII) formation in human isolated bladder smooth muscle. 2. A novel human chymase inhibitor CH 5450 (Z-Ile-Glu-Pro-Phe-CO2Me) and a recently developed human chymase substrate Pro11-,D-Ala12)-angiotensin I, claimed to be resistant to angiotensin converting enzyme (ACE) and carboxypeptidase, were used. 3. Angiotensin I (AI) (0.3 microM) induced a contractile response amounting to 58 +/- 5% (n=12) of the initial K+ (124 mM)-induced contractions. This response was reduced to 36 +/- 3% (n=8) by the ACE-inhibitor enalaprilat (10 microM), while pretreatment with soybean trypsin inhibitor (STI 200 microg ml(-1)) or CH 5450 (10 microM) had no effect. However, the combination of enalaprilat and STI reduced the AI-induced contractions to 19 +/- 5% (n=6), and the combination of enalaprilat and CH 5450 caused an almost complete inhibition of the AI-induced contractions to 1+/-1% (n=6). 4. The substrate (Pro11-,D-Ala12)-AI (3 microM) produced contractions which amounted to 57 +/- 4% (n=13) of the initial K+ (124 mM) contractions. These contractions were not affected by enalaprilat (10 microM). On the other hand, STI (200 microg ml(-1)) and CH 5450 (10 microM) added separately, depressed the (Pro11-,D-Ala12)-AI-induced contractions to 34 +/- 5% (n=6) and 24 +/- 4% (n=6), respectively. The combination of enalaprilat and STI or enalaprilat and CH 5450 did not produce any further inhibition. 5. Experiments with detrusor membrane fractions incubated with AI (50 microM) were performed. In the presence of enalaprilat (100 microM), carboxypeptidase inhibitor CPI (10 microg ml(-1)) and aprotinin (15 microM), CH 5450 (10 nM-1 microM) caused a concentration-dependent inhibition of AII formation. 6. The results confirm that AII is a potent contractile agent in the human isolated detrusor muscle. They also indicate that the serine protease responsible for AII formation in the human bladder in vitro is human chymase or an enzyme similar to human chymase.

There has long been evidence that inhibitors of chymotryptic proteinases can inhibit the degranulation of rodent mast cells, but their actions on human mast cells and the contribution of mast cell chymase itself have received little attention. We investigated the ability of the selective chymase inhibitor Z-Ile-Glu-Pro-Phe-CO(2)Me and other proteinase inhibitors to inhibit chymase and cathepsin G activity, and we examined their potential to modulate the responsiveness of mast cells dispersed from human skin, lung, and tonsil tissues. IgE-dependent histamine release from skin mast cells was inhibited by up to about 80% after preincubation with Z-Ile-Glu-Pro-Phe- CO(2)Me (up to 0.1 microM), 70% with chymostatin (17 microM), and 60% with soybean trypsin inhibitor (0.5 microM). The mast cell-stabilizing properties of chymase inhibitors appeared to be greater for skin mast cells than for those from lung, whereas tonsil mast cells were relatively unresponsive. There were marked differences in the time course of responses to inhibitors, and the effect was dependent on the stimulus, with calcium ionophore-induced histamine release being unaffected. Incubation of dispersed skin, lung, or tonsil cells for up to 45 min with purified chymase failed to induce histamine release, although preincubation of cells with chymase was able to suppress IgE-dependent activation. Chymase could thus contribute to mast cell degranulation and after secretion could provide a feedback mechanism to limit this process. Nevertheless, inhibitors of chymase can be potent mast cell stabilizers, particularly in the skin.