Specific Chymase Inhibitor Research Articles

Primacy of Chymase over Angiotensin Converting Enzyme in the Production of Angiotensin II in Rat Bone Marrow Tissue

BackgroundThe bone marrow is attracting attention as not only the nidus of circulating blood cells but the source of stem and progenitor cell‐based therapy. Renin‐angiotensin system (RAS) and angiotensin II (Ang II) receptor type 1 in bone marrow participate in hematopoiesis, a target in the evolution of inflammatory processes involved in cardiovascular disease pathogenesis. Extending the original demonstration of RAS components in the rat bone marrow (Strawn et al. Br J Haematol. 2004;126:120–6), this study investigated the existence of an Ang II‐forming pathway in which chymase rather than angiotensin‐converting enzyme (ACE) cleaves the peptide from the newly described substrate angiotensin‐(1‐12) [Ang‐(1‐12)] which functions as an endogenous tissue substrate for the direct formation of Ang II by chymase.MethodsBone marrow tissue was obtained from the tibia of twelve weeks‐old Sprague Dawley rats following systemic perfusion with phosphate buffered saline with or without the secondary addition of Zamboni's fixative. Bone marrow tissues, homogenized with 0.05 M Tris‐HCl and centrifuged at 28,000 g, were subjected to immunoblot and chymase enzymatic activity using high‐performance liquid chromatography (HPLC). Additional samples were prepared for quantitative RT‐PCR analysis normalized to GAPDH. All data were analyzed using GraphPad PRISM 7.0 with P<0.05 considered statistically significant.ResultsFluorescent immunohistochemistry showed the existence of immunoreactive (ir‐) Ang‐(1‐12) and ir‐chymase‐positive cells in rat's bone marrow. Immunoblots showed chymase protein expression in cell pellets but not in supernatants. Assessment of 125I‐Ang‐(1‐12) or 125I‐Ang I hydrolysis in cell pellets in the presence and absence of specific chymase or ACE inhibitors, revealed that production of Ang II by chymase was two‐thousand fold higher compared to ACE (4,531 ± 137 fmol/mg/min and 2.3 ± 0.3 fmol/mg/min, respectively, P<0.0001, n = 6). ANOVA and post‐hoc Tukey's test showed that gene expression of rat mast cell protease‐5 (rMCP‐5) was at least twice higher than rMCP‐1, rMCP‐2 and rMCP‐4.ConclusionThese studies reveal a major role of Ang‐(1‐12) and chymase as the critical pathway for Ang II‐formation in rat's bone marrow. The pleotropic actions of tissue chymase in the degradation of matricellular proteins, activation of matrix metalloproteinases, transforming growth factor‐β, and production of inflammatory cytokines posits this alternate pathway for Ang II‐production as most relevant in understanding and treating cardiovascular disease.Support or Funding InformationThis study was supported by the National Heart, Blood, Lung Institute of the NIH through HL‐051952 grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pivotal Role of Mouse Mast Cell Protease 4 in the Conversion and Pressor Properties of Big-Endothelin-1

The serine protease chymase has been reported to generate intracardiac angiotensin-II (Ang-II) from Ang-I as well as an intermediate precursor of endothelin-1 (ET-1), ET-1 (1-31) from Big-ET-1. Although humans possess only one chymase, several murine isoforms are documented, each with its own specific catalytic activity. Among these, mouse mast cell protease 4 (mMCP-4) is the isoform most similar to the human chymase for its activity. The aim of this study was to characterize the capacity of mMCP-4 to convert Big-ET-1 into its bioactive metabolite, ET-1, in vitro and in vivo in the mouse model. Basal mean arterial pressure did not differ between wild-type (WT) and mMCP-4(-/-) mice. Systemic administration of Big-ET-1 triggered pressor responses and increased blood levels of immunoreactive (IR) ET-1 (1-31) and ET-1 that were reduced by more than 50% in mMCP-4 knockout (-/-) mice compared with WT controls. Residual responses to Big-ET-1 in mMCP-4(-/-) mice were insensitive to the enkephalinase/neutral endopeptidase inhibitor thiorphan and the specific chymase inhibitor TY-51469 {2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonylphenyl]thiazole-4-carboxylic acid}. Soluble fractions from the lungs, left cardiac ventricle, aorta, and kidneys of WT but not mMCP-4(-/-) mice generated ET-1 (1-31) from exogenous Big-ET-1 in a TY-51469-sensitive fashion as detected by high-performance liquid chromatography/ matrix-assisted laser desorption/ionization-mass spectrometry. Finally, pulmonary endogenous levels of IR-ET-1 were reduced by more than 40% in tissues derived from mMCP-4(-/-) mice compared with WT mice. Our results show that mMCP-4 plays a pivotal role in the dynamic conversion of systemic Big-ET-1 to ET-1 in the mouse model.

Impact of Mast Cell Chymase on Renal Disease Progression

Chymase, a serine protease found in mast cell granules, is released into the interstitium following injury or inflammation. Chymase is the primary ACE-independent pathway of angiotensin II formation, and also functions to activate TGF-beta and other promoters of extracellular matrix degradation, thereby playing a role in tissue remodeling. In the diseased kidney, chymase-containing mast cells markedly increase and their density correlates with tubulointerstitial fibrosis severity. Studies in humans support the pathologic role of chymase in diabetic nephropathy, while animal studies form the basis for the importance of increased chymase-dependent angiotensin II formation in progressive hypertensive, diabetic and inflammatory nephropathies. Moreover, humans with kidney disease express chymase in diseased blood vessels in concordance with significantly elevated plasma chymase levels. Conversely, specific chymase inhibitors attenuate angiotensin II production and renal fibrosis in animal models, suggesting their potential therapeutic benefit in human nephropathy, where chymase-containing mast cells accumulate and contribute to progressive disease.

The specific chymase inhibitor TY-51469 suppresses the accumulation of neutrophils in the lung and reduces silica-induced pulmonary fibrosis in mice

ABSTRACTChymase is a chymotrypsin-like serine protease that is present in mast cells. Its activities include various effects associated with inflammatory responses. But little is known about the effects of chymase in pulmonary fibrosis. The mouse silicosis model was induced by intratracheal injection of 10 mg silica. The Ashcroft pathological score and the hydroxyproline content of lungs were measured to evaluate the effect of a chymase inhibitor, 2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonylphenyl] thiazole-4-carboxylic acid (TY-51469). The cellular composition and cytokine levels in bronchoalveolar lavage fluid (BALF) were also examined. Following TY-51469 treatment, the lung fibrosis score and hydroxyproline level were significantly reduced, and the number of neutrophils and the levels of macrophage inflammatory protein-2, monocyte chemoattractant protein-1, and transforming growth factor-β1 in BALF were reduced on day 21. The administration of TY-51469 at an early stage showed a greater reduction of fibrosis compared to administration at a later stage. The neutrophil number in BALF in mice treated with TY-51469 both at an early stage and late stage was significantly reduced. The level of mouse mast cell proteinase-4 mRNA increased with time in silica-induced fibrosing lung tissue. These results show that the chymase inhibitor TY51469 suppresses the migration of neutrophils, which results in the suppression of lung fibrosis.

Inhibition of chymase protects against diabetes-induced oxidative stress and renal dysfunction in hamsters

Accumulating evidence suggests that the intrarenal renin-angiotensin system may be involved in the progression of diabetic nephropathy. Chymase is a potent local angiotensin II-forming enzyme in several species, including humans and hamsters. However, the pathophysiological role of chymase is not fully understood. Here, we report a causal role of chymase in diabetic nephropathy and the therapeutic effectiveness of chymase inhibition. In the present study, renal chymase expression was markedly upregulated in streptozotocin-induced diabetic hamsters. Oral administration of a specific chymase inhibitor, TEI-F00806, completely ameliorated proteinuria, the overexpression of transforming growth factor-β and fibronectin in glomeruli, and renal mesangial expansion, by normalizing the increase in intrarenal angiotensin II levels in diabetic hamsters independently of blood pressure levels. In contrast, ramipril did not show such sufficient effects. These effects occurred in parallel with improvements in superoxide production and expression of NAD(P)H oxidase components [NAD(P)H oxidase 4 and p22(phox)] in glomeruli. This study showed for the first time that chymase inhibition may protect against elevated intrarenal angiotensin II levels, oxidative stress, and renal dysfunction in diabetes. These findings suggest that chymase offers a new therapeutic target for diabetic nephropathy.

Contribution of Chymase-Dependent Angiotensin II Formation to the Progression of Tubulointerstitial Fibrosis in Obstructed Kidneys in Hamsters

Recent studies indicate a role of chymase in the regulation of angiotensin II (AngII) formation in cardiovascular and renal tissues. We investigated a possible contribution of chymase to AngII formation and to renal fibrosis in unilateral ureteral obstruction (UUO). Eight-week-old Syrian hamsters were subjected to UUO and treated with vehicle, the specific chymase inhibitor (CI) 4-[1-(4-methyl-benzo[b]thiophen-3-ylmethyl)-1H-benzimidazol-2-ylsulfanyl]-butyric acid (50 mg/kg, twice a day, p.o.), or the selective AT1-receptor blocker olmesartan (10 mg/kg per day, p.o.) for 14 days. UUO-induced renal interstitial fibrosis was associated with increases in renal mRNA levels of α-smooth muscle actin (SMA), type I collagen, and transforming growth factor (TGF)-β. The UUO hamsters showed markedly higher AngII contents and increased AT1-receptor mRNA level in the obstructed kidney than sham-operated ones. In contrast, angiotensin-converting enzyme (ACE) protein expression was significantly lower in UUO hamsters. In UUO hamsters, treatment with CI or olmesartan significantly decreased AngII levels in renal tissue and mRNA levels of α-SMA, type I collagen, and TGF-β and ameliorated tubulointerstitial injury. On the other hand, neither CI nor olmesartan changed systolic blood pressure, renal ACE, and AT1-receptor protein levels. These data suggest that chymase-dependent intrarenal AngII formation contributes to the pathogenesis of interstitial fibrosis in obstructed kidneys of hamsters.

Abstract 5955: Role of Bradykinin and Chymase in Cardiorenal Function Under Long-Term ACE Inhibition in Heart Failure

Renal insufficiency is relatively common in patients with heart failure (HF) and patients with chronic kidney disease are known to have a significant increase in cardiovascular morbidity and mortality. Angiotensin (Ang) II plays an important role in water and sodium (Na) balance and renal injury. Although ACE inhibitors (ACEIs) suppress the formation of Ang II and are useful in the treatment of HF, the agents also prevent the breakdown of bradykinin (BK), which exerts its vasodilative action through BK type 2 (B2) receptors. On the other hand, over 50% of renal Ang II formation in HF is mediated by ACE independent chymase pathway. The roles of BK and chymase in the cardiorenal function under chronic treatment of ACEI has not been fully elucidated as yet. We therefore evaluated the cardiorenal effects of a specific B2 receptor antagonist, FR173657 (FR, 0.3mg/kg/day) or a specific chymase inhibitor, (ChyI, 100mg/kg/day) with an ACEI, enalapril (1mg/kg/day) compared to those of enalapril alone in dogs with tachycardia induced HF (22days). Cardiac contractile function markedly deteriorated and body fluid retention and impaired renal function accompanied with upregulation of renal ACE and chymase mRNA levels after the induction of severe HF. However, enalapril significantly improved the ejection fraction (vehicle 19% vs. enalapril 36%, p<0.01) and increased urine excretion (vehicle 0.36 vs. enalapril 0.55 ml/min, p<0.05) associated with increases in the glomerular filtration rate (GFR) and renal plasma flow (RPF) (vehicle 24 vs. enalapril 60 ml/min, and 74 vs. 169 ml/min, respectively p<0.05) in HF. There were no differences observed in blood pressure and LV ejection fraction among three treatment groups. FR did not statistically affect urinary Na excretion, GFR and RPF compared to the ACE inhibition alone, however, the chymase inhibition has more significantly increased water and Na excretion rate (0.70 vs. 0.55 ml/min and 56 vs. 26 μEq/min, respectively p<0.05) and GFR (70 vs. 59 ml/min, p<0.05) compared to the ACE inhibition alone. In conclusion, endogenous BK does not likely contributes to the renoprotective effects of ACEI, however, combining ACE and chymase inhibition has greater therapeutic potential in the amelioration of renal function in HF.

Abstract 1947: Chymase Inhibition Reduces the Incidence of Intraplaque Hemorrhage and Induces a Stable Atherosclerotic Plaque Phenotype in ApoE Deficient Mice

Mast cells are abundantly present in perivascular tissue during atherosclerotic plaque progression and were previously demonstrated to contribute significantly to plaque destabilization. In this study, we aimed to investigate to what extent inhibition of chymase, one of the mast cell proteases, could enhance plaque stability. We demonstrated earlier that the specific chymase inhibitor RO501 (1 μM) was able to quench mast cell activation in vitro , as illustrated by a decreased β-hexosaminidase (−41% and −80%, respectively; P<0.05) as well as chymase activity in the releasate of activated MC/9 and peritoneal mast cells (−71% and −65%, respectively; P<0.05). Next, we assessed whether chymase inhibition was also effective in vivo. Atherosclerotic carotid artery lesions were induced in ApoE −/− mice by perivascular collar placement and mast cells were activated by DNP challenge systemically during lesion development. RO501 (50 mg/kg/day) was administered as diet supplement, leading to serum concentrations of ~2 μM. While plaque size after 6 weeks of treatment did not differ, collagen content of the lesions was 2-fold enhanced in mice treated with RO501 compared to controls (1.4 ± 0.5% and 0.7 ± 0.2%, respectively). This was accompanied by a significant decrease in necrotic core size of the plaques (controls: 52 ± 3% versus RO501: 41 ± 4%, P<0.05). To determine the effects of chymase inhibition after acute mast cell activation in advanced plaques, perivascular mast cells were focally activated in the adventitia of advanced lesions in ApoE −/− mice, which were treated with RO501 as described above. At three days after focal mast cell challenge, the incidence of intraplaque hemorrhage (IPH) was inhibited from 23% in control mice to 4.5% in RO501 treated mice, while also the plaque erythrocyte area was reduced by >90% from 1.2 ± 0.6*10 3 to 0.1 ± 0.08*10 3 μm 2 (P<0.05). Also, we observed a reduction in apoptotic cells (RO501: 0.68 ± 0.20% vs. 1.01 ± 0.36% for IPH negative controls and 1.23 ± 0.42% for IPH + plaques). In conclusion, our data suggest that chymase inhibition at least partly prevents the detrimental effects of perivascular mast cells on plaque stability, identifying chymase inhibition as a new therapeutic approach in the prevention of acute coronary syndromes.

Upregulation of cardiac interstitial chymase after canine myocardial ischemia and reperfusion

The serine protease chymase is stored in mast cell secretory granules and is released upon mast cell activation into the cardiac interstitium where it activates matrix metalloproteinases, causes cardiomyocyte apoptosis, and generates angiotensin (Ang) II. Miyazaki et. al. (Life Sci. 71:437, 2002) reported beneficial effects on cardiac function and survival of chymase inhibition during the acute phase of myocardial infarction in hamsters. Here we determined if mast cell‐chymase is released into the cardiac interstitium after acute myocardial ischemia (MI) and reperfusion in dogs. To measure in vivo left ventricle (LV) interstitial fluid (ISF) chymase activity, we combined cardiac microdialysis with direct ISF infusion of the chymase‐specific substrate [Pro11, DAla12]Ang I after MI. The ISF chymase activity in the ischemic region was 3.5‐fold (n=6, p<0.001) increased after 60 min MI followed 100 min reperfusion and it was significantly suppressed by TEI‐F00806 (100 mg/kg/BID, pretreated 5 days), a novel, orally active and specific chymase inhibitor (CI). Importantly, there was also 5‐fold (n=6, p<0.001) increased ISF chymase activity in the non‐ischemic region after reperfusion and this increase was completely blocked by CI treatment. The current investigation indicates that LV ISF chymase in vivo can be regulated by factors that affect mast cell activation, in particular, MI and reperfusion. These findings demonstrated for the first time that cardiac interstitial chymase participates directly in the pathophysiologic state after acute MI in dog. Supported by TEIJIN Pharma Limited and SCCOR in Cardiac Dysfunction P50HL077100 (LJD).

Abstract 471: Systemic Chymase Inhibition Directs Atherosclerotic Plaques Towards a Stable Phenotype in ApoE Deficient Mice

Activated mast cells have been identified at the site of rupture in human coronary artery plaques and appear to contribute considerably to plaque progression and stability. We and others have previously demonstrated that the mast cell constituents chymase and tryptase promote apoptosis of plaque cells. In this study, we aimed to investigate whether inhibition of mast cell chymase by a specific chymase inhibitor indeed has a beneficial effect on plaque stability. Preincubation of 48/80 activated MC/9 murine mast cells or freshly isolated peritoneal mast cells with chymase inhibitor RO5010226 – 000 – 004 (RO501; 1 μM) inhibited mast cell activation, as illustrated by a decreased β-hexosaminidase activity in the releasate (−41% compared to control MC/9 cells, *P=0.04, and −80% compared to control peritoneal mast cells, *P=0.02) as well as chymase release and activity (−71% and −65%, *P=0.04, respectively). Next, we addressed whether chymase inhibition also was effective in vivo. Atherosclerotic carotid artery lesions were induced in ApoE −/− mice by perivascular collar placement; during lesion development mast cells were activated by a DNP challenge once weekly for 4 weeks. Concomitantly, a subset of mice received the chymase inhibitor (50 mg/kg/day, n=14) as diet supplement, leading to continuous serum concentrations of ~2 μM or control diet (n=12). After 6 weeks, the advanced plaques were analyzed for size and stability. While plaque size did not differ, collagen content of the lesions was 2-fold enhanced in mice treated with the chymase inhibitor compared to controls (RO501: 1.4 ± 0.5% versus controls: 0.7 ± 0.2%). This was accompanied by a significant decrease in necrotic core size of the plaques (RO501: 52 ± 3% versus controls: 41 ± 4%, *P=0.04) as well as by an increased plaque cellularity (RO501: 2.6 ± 0.1*10 3 versus controls: 2.3 ± 0.1*10 3 cells/mm 2 tissue). In agreement with these data we did observe increased peritoneal leukocyte numbers in the RO501 treated mice (RO501: 4.2 ± 1.1*10 6 cells versus 2.2 ± 0.3*10 6 cells in controls, *P=0.04). In conclusion, our data suggest that chymase inhibition indeed results in enhanced plaque stability, identifying chymase inhibition as a new therapeutic approach in the prevention of acute coronary syndromes.

Chymase Inhibitor Improves Renal Dysfunction Under Long-term Angiotensin-converting Enzyme Inhibition in Heart Failure

Impaired renal function is one of the predictors of mortality in heart failure (HF). Angiotensin-II (Ang-II) plays a deteriorational role in renal injury and half of renal Ang-II formation is mediated via chymase not ACE pathway. The interaction between the heart and the kidney is an important issue in the treatment of HF. Thus, we evaluated the concomitant therapeutic effects of an ACE inhibitor (ACEI, Enalapril) with a specific chymase inhibitor (ChyI, TEI) on cardiorenal function in dogs with tachycardia induced HF.

The Significance of Chymase in the Progression of Abdominal Aortic Aneurysms in Dogs

In this study, we investigated the effect of a specific chymase inhibitor, NK3201, in the progression of abdominal aortic aneurysm in a dog experimental model. Abdominal aortic aneurysms were induced in dogs by injecting elastase into the abdominal aorta. NK3201 (1 mg/kg per day, p.o.) or a placebo was started 3 days before elastase injection and continued for 8 weeks after the injection. On abdominal ultrasound, the aortic diameter was seen to gradually expand in the placebo-treated group, but not in the NK3201-treated group. Eight weeks after elastase injection, the ratio of the medial area to the total area in the placebo-treated group was significantly smaller than that in the normal group, but it was significantly larger than that in the NK3201-treated group. In addition to chymase activity, angiotensin II-forming and matrix metalloproteinase-9 activities were significantly higher in the placebo-treated group than in the normal group; in the NK3201-treated group, all of these activities were significantly decreased. On immunohistochemical analyses, there was a significantly greater number of chymase-positive cells in the placebo-treated group than in the normal group, but the number was significantly smaller in the NK3201-treated group than in the placebo-treated group. Thus, chymase inhibition may become a useful strategy for preventing abdominal aortic aneurysms.

Effect of chymase on intraocular pressure in rabbits

Chymase is a chymotrypsin-like serine protease that is stored exclusively in the secretory granules of mast cells and converts big endothelins to endothelin-1 (1–31). The aim of this study was to evaluate the effect of chymase on intraocular pressure in rabbits. Chymase injection (3 and 10 mU) resulted in a trend toward increased intraocular pressure and a significant increase in intraocular pressure at a dose of 10 mU compared with the control. A specific chymase inhibitor, Suc-Val-Pro-Phe P(OPh) 2, attenuated the ocular hypertension induced by chymase. Endothelin-1 (1–31) also caused ocular hypertension, which was inhibited by a selective endothelin ET A receptor antagonist, cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123). Moreover, chymase-induced ocular hypertension was inhibited by BQ-123. These results suggest that chymase influences the regulation of intraocular pressure, and it is likely that the formation of endothelin-1 (1–31) and subsequent activation of endothelin ET A receptors are involved in the development of ocular hypertension induced by chymase.

The beneficial effects of a novel specific chymase inhibitor, SUNC8257, on congestive heart failure-related atrial remodeling in dogs

The beneficial effects of a novel specific chymase inhibitor, SUNC8257, on congestive heart failure-related atrial remodeling in dogs

A specific chymase inhibitor, NK3201, suppresses bleomycin-induced pulmonary fibrosis in hamsters

We evaluated whether a chymase inhibitor, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)- N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-heptyl]acetamide (NK3201), suppressed bleomycin-induced pulmonary fibrosis. Hamsters were orally administered NK3201 (30 mg/kg per day) or placebo, beginning 5 days before intratracheal instillation of bleomycin (10 mg/kg). Four weeks after the instillation of bleomycin, pulmonary chymase activity in placebo-treated hamsters was significantly higher than in control hamsters, whereas the activity in NK3201-treated hamsters was significantly lower than in placebo-treated hamsters. The ratio of fibrotic area to total area in NK3201-treated hamsters was significantly decreased to 54.0% of the ratio in placebo-treated hamsters. Therefore, NK3201 may be useful in the prevention of pulmonary fibrosis.

Vascular chymase: pathophysiological role and therapeutic potential of inhibition.

Chymase is a chymotrypsin-like serine protease secreted from mast cells. Mammalian chymases are classified into two subgroups (alpha and beta) according to structure and substrate specificity; human chymase is an alpha-chymase. An important action of chymase is the ACE-independent conversion of Ang I to Ang II, but chymase also degrades the extracellular matrix, activates TGF-beta1 and IL-1beta, forms 31-amino acid endothelins and is involved in lipid metabolism. Under physiological conditions, the role of chymase in blood vessels is uncertain. In pathological situations, however, chymase may be important. In animal models of hypertension and atherosclerosis, chymase may be involved in lipid deposition and intimal and smooth muscle hyperplasia, at least in some vessels. In addition, chymase has pro-angiogenic properties. In human diseased blood vessels (e.g. atherosclerotic and aneurysmal aorta; remodeled pulmonary blood vessels), there are increases in chymase-containing mast cells and/or in chymase-dependent conversion of Ang I to Ang II. These findings have raised the possibility that inhibition of chymase may have a role in the therapy of vascular disease. The effects of chymase can theoretically be attenuated either by reducing availability of the enzyme, with a mast cell stabiliser, or alternatively with specific chymase inhibitors. The mast cell stabiliser, tranilast, was shown to be beneficial in animal models of atherosclerosis, where a prevention protocol was used, but was not effective in clinical trials where it was administered after angioplasty. Chymase inhibitors could have the advantage of being effective even if used after injury. Several orally active inhibitors, including SUN-C8257, BCEAB, NK3201 and TEI-E548, are now available. These have yet to be tested in humans, but promising results have been obtained in animal models of atherosclerosis and angiogenesis. It is concluded that orally active inhibitors of chymase may have a place in the treatment of vascular diseases where injury-induced mast cell degranulation contributes to the pathology.

A specific chymase inhibitor, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[[3,4-dioxo-1-phenyl-7-(2-pyridyloxy)]-2-heptyl]acetamide (NK3201), suppresses development of abdominal aortic aneurysm in hamsters.

In this study, we investigated the effect of a specific chymase inhibitor, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[[3,4-dioxo-1-phenyl-7-(2-pyridyloxy)]-2-heptyl]acetamide (NK3201), in the development of abdominal aortic aneurysm in a hamster experimental model. The abdominal aortic aneurysm was induced by application of elastase onto the abdominal aorta in hamster. Each hamster was administered NK3201 (30 mg/kg/day p.o.) or placebo beginning 4 days before application of elastase and continuing through the experiments. Sham-operated hamsters received saline application onto the abdominal aorta. Two weeks after application of elastase, the aortic diameter in the placebo-treated group was significantly increased to 1.6-fold compared with the value for the sham-operated group, whereas that in the NK3201-treated group was significantly reduced. The chymase activities in the sham-operated and the placebo-treated groups were 0.35 +/- 0.01 and 3.44 +/- 0.62 mU/mg protein, respectively, and this difference was significant. NK3201 significantly reduced the chymase activity in the placebo-treated group. Here, we demonstrated for the first time that a chymase inhibitor prevented the development of abdominal aortic aneurysm in a hamster experimental model.

An antiarrhythmic effect of a chymase inhibitor after myocardial infarction.

Chymase plays an important role in the regulation of local angiotensin (Ang) II formation in the cardiac tissue. We recently found that cardiac chymase was activated significantly and survival rate markedly improved by treatment with chymase inhibitors after myocardial infarction (MI) in hamsters. However, the mechanisms for this effect have not been established. Because lethal arrhythmias are generally believed to contribute to sudden cardiac death, we assessed whether inhibition of cardiac chymase would provide an antiarrhythmic effect during the 8-h ischemic period after 2-[4-(5-fluoro-3-methylbenzo-[b]thiophen-2-yl)sulfonamide-3-methanesulfonylphenyl]oxazole-4-carboxylicacid (TY51184) (a specific chymase inhibitor, 1 mg/kg i.v.) treatment by ligation of left anterior descending coronary artery (LAD) in dogs. Effects of candesartan (an Ang II type 1 receptor antagonist, 1 mg/kg i.v.) in this model were also assessed. Total Ang II-forming activity and chymase activity in the infarcted heart were increased significantly 8 h after LAD ligation. A time-dependent elevation of Ang II in plasma was also observed. A decrease in plasma Ang II levels after TY51184 treatment occurred concomitantly with suppression of cardiac chymase activity. LAD ligation resulted in a large number of ventricular arrhythmias (VAs). TY51184 and candesartan treatments largely suppressed the appearance of VAs, and the efficacy of the two agents was similar. These findings demonstrate that chymase inhibition can provide an antiarrhythmic effect after MI, and the reduction of Ang II by TY51184 may be mainly responsible for this beneficial effect. An antiarrhythmic effect of chymase inhibitors may contribute to reductions in the mortality rate during the acute phase after MI.

A Single Treatment With a Specific Chymase Inhibitor, TY-51184, Prevents Vascular Proliferation in Canine Grafted Veins

In this study, we evaluated whether a specific chymase inhibitor, TY-51184 (2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonylphenyl]oxazole-4-carboxylicacid), prevents the vascular proliferation in canine grafted veins. In the placebo- and chymase inhibitor-treated groups, the external jugular vein was infiltrated with saline and 10 μM TY-51184, respectively, and then it was grafted to the ipsilateral carotid artery. The non-surgical dogs were used as the control group. By 28 days after grafting, the chymase and ACE activities were significantly increased in the injured arteries. TY-51184 significantly reduced the chymase activity in the grafted veins, while it did not affect the ACE activity. The intimal areas in the placebo- and TY-51184-treated groups were 3.32 ± 0.16 and 1.96 ± 0.52 mm2, respectively, and this difference was significant. The ratios of intimal area to medial area in the placebo- and TY-51184-treated groups were 66.8 ± 3.5% and 34.9 ± 9.2%, respectively, and this difference was also significant. There was a significant relationship between vascular proliferation and chymase activity, but not ACE activity. In this study, we demonstrated that a single treatment with a specific chymase inhibitor, TY-51184, could prevent the vascular proliferation in canine grafted veins.

SF2809 compounds, novel chymase inhibitors from Dactylosporangium sp. 1. Taxonomy, fermentation, isolation and biological properties.

Six novel chymase inhibitors, SF2809-I, SF2809-II, SF2809-III, SF2809-IV, SF2809-V and SF2809-VI, were isolated from the fermentation broth of an actinomycete strain SF2809. The strain was identified as Dactylosporangium sp. by morphological, chemotaxonomical and phylogenetic studies. These six novel compounds inhibited recombinant human chymase in the range between IC50 of 0.014 and 7.3 microM. However, they showed little or no inhibitory activity against chymotrypsin or cathepsin G, even though these two and chymase belong to the chymotryptic serine protease family. This result indicates that these compounds work as specific chymase inhibitors.