Mary Chou

Effect of Matrix Metalloproteinase Inhibition on Progression of Atherosclerosis and Aneurysm in LDL Receptor‐Deficient Mice Overexpressing MMP‐3, MMP‐12, and MMP‐13 and on Restenosis in Rats after Balloon Injury

ABSTRACT: The broad‐spectrum MMP inhibitor CGS 27023A was tested to determine its potential as a therapy for atherosclerosis, aneurysm, and restenosis. LDL receptor‐deficient (LDLr −/−) mice fed a high‐fat, cholic acid‐enriched diet for 16 weeks developed advanced aortic atherosclerosis with destruction of elastic lamina and ectasia in the media underlying complex plaques. Lesion formation correlated with a 4.6‐ to 21.7‐fold increase in MMP‐3, ‐12, and ‐13 expression. Treatment with CGS 27023A (p.o., b.i.d. at 50 mg/kg) had no effect on the extent of aortic atherosclerosis (36 ± 4% versus 30 ± 2% in controls), but both aortic medial elastin destruction and ectasia grade were significantly reduced (38% and 36%, respectively, p < 0.05). In the rat ballooned‐carotid‐artery model, CGS 27023A (12.5 mg/kg/day via osmotic minipump) reduced smooth muscle cell migration at 4 days by 83% (p < 0.001). Intimal lesions were reduced by 85% at 7 days (p < 0.001), but intimal smooth muscle proliferation was unaffected, and inhibitory efficacy was lost with time. At 12 days, intimal lesion reduction was less potent (52%, p < 0.01). At 3 and 6 weeks, reductions of 11% and 4%, respectively, were not significant. This demonstrates that it is essential to include late time points when the ballooned‐carotid‐artery model is employed to ensure that lesion size does not “catch up” when a compound solely inhibits smooth muscle cell migration. In summary, MMP inhibitor therapy delayed but did not prevent intimal lesions, thereby demonstrating little promise to prevent restenosis. In contrast, MMP inhibitor therapy may prove useful to retard progression of aneurysm.

Sulfonamide-based hydroxamic acids as potent inhibitors of mouse macrophage metalloelastase

The structural requirements of sulfonamide-based hydroxamic acid 1 for inhibition of macrophage metalloelastase (MME) were investigated. A short aliphatic group at the R2 position together with an aromatic group at the R3 position significantly improved the inhibitory activity. Compounds 32, 34, and 40 were the most potent inhibitors of MME with IC50 values between 5 and 6 nM.

Enhanced Expression of Matrix Metalloproteinase-3, -12, and -13 mRNAs in the Aortas of Apolipoprotein E-deficient Mice with Advanced Atherosclerosis

Increased expression of matrix metalloproteinases (MMPs) has been reported at sites of advanced atherosclerosis and aneurysm in humans. 1,2 An animal model that has been reported to develop advanced atherosclerosis and medial elastin degradation with progression to ectasia and aneurysm is the apolipoprotein E–deficient (apoE -/) mouse. 3–5 Using immunohistochemistry, a variety of MMPs have been demonstrated at sites of atherosclerosis and elastin degradation in apoE -/mice on a high-fat diet. 5 However, little information is available on MMP expression in apoE -/mice on a normal diet or in mice that are not as susceptible to atherosclerosis on either normal or high-fat diet. The present study compares mRNA expression of MMP-3, -9, -12, and -13 in mice that are highly susceptible (apoE -/), moderately susceptible (C57BL/6J and B6129F1/J), or resistant (C3H/HeJ and Balb/cJ) to diet-induced atherosclerosis. 6

Enhanced expression of renal endothelin-converting enzyme-1 and endothelin-A-receptor mRNA in rats with interstitial fibrosis following ureter ligation.

Endothelin-1 (ET-1) has been implicated in many chronic renal glomerular diseases. The purpose of this study was to investigate whether the levels of mRNA expression of endothelin-converting enzyme-1 (ECE-1) and endothelin-A- and -B- (ET(A) and ET(B)) receptors are altered during the progression of interstitial fibrosis following ureter ligation. Rats were subjected to left ureter ligation or a sham operation and euthanized 5 days afterward. Kidneys were fixed in Carnoys fixative, embedded in paraffin, and sectioned for assessment of interstitial fibrosis by staining for collagen III using immunofluorescence techniques. The area occupied by collagen staining was quantified by image analysis. Kidneys from obstructed rats showed a 54% increase in the area occupied by collagen III staining compared to the contralateral kidney, and an 89% increase compared to sham-operated kidneys. The mRNA levels of ECE-1, as well as ET(A)- and ET(B)-receptors in the kidney were analyzed by Northern blots. It was found that the ECE-1 and ET(A)-receptor mRNA levels in kidneys subjected to ureter ligation increased by 92% and 71%, respectively, when compared with those obtained from the contralateral kidneys. In contrast, mRNA levels of ET(B)-receptors were not significantly different between the two groups. These results suggest that ET-1, through interaction with the ET(A)-receptors, may play a role in the progression of interstitial fibrosis following ureter ligation.

Design and synthesis of a potent and selective endothelin-converting enzyme inhibitor, CGS 35066.

CGS 26303 has previously been shown to inhibit human endothelin converting enzyme-1 (ECE-1) with an IC50 of 410 nM and to be efficacious in several animal disease models. However, it is a more potent inhibitor of neutral endopeptidase 24.11 (NEP) with an IC50 of 1 nM. The aim of this study was to optimize CGS 26303 for greater potency and selectivity towards ECE-1 inhibition. The in vivo activity of the compounds was assessed by inhibition of the big endothelin-1 (ET-1)-induced pressor response in anesthetized rats at 90 min after treatment with a dose of 10 mg/kg, i.v. Under these conditions, CGS 26303 inhibited the pressor response to big ET-1 by 50%. Replacement of the biphenyl and tetrazol groups in CGS 26303 with a dibenzofuran and carboxylic acid, respectively, yielded CGS 35066, a potent ECE-1 inhibitor having an IC50 of 22 nM. In contrast, these substitutions markedly weakened the NEP inhibitory activity of the compound to an IC50 of 2.3 microM. CGS 35066 also exhibited a potent and sustained ECE-1 inhibitory activity in vivo, blocking the pressor response to big ET-1 by 84%. Its orally active prodrug, CGS 35339, was obtained by introducing two phenyl groups at the phosphonic acid substituent in CGS 35066. Therefore, CGS 35066 and CGS 35339 represent novel compounds for assessing the pathogenic role of ET-1 overproduction in various disease states.

Blockade of Integrin VLA-4 Prevents Inflammation and Matrix Metalloproteinase Expression in a Murine Model of Accelerated Collagen-Induced Arthritis

DBA/1LacJ mice were immunized with type II collagen and boosted with bacterial lipopolysaccharide (LPS) 17 days later to induce accelerated arthritis. Clinical signs of inflammation were observed as early as Day 20. Matrix metalloproteinases MMP-2, -3, -9, and -13, but not MMP-12, mRNA levels were increased on Day 24. Administration of anti-VLA-4 antibody (mAb; 8 mg/kg/day for 3 days) at the time of LPS treatment strikingly inhibited arthritis-induced paw inflammation and histological scores, but not the increase in MMP expression. A higher dose of mAb (20 mg/kg/day for 4 days) inhibited pathology and normalized the levels of MMP mRNAs. In conclusion, the pathophysiology of this accelerated model of arthritis is VLA-4-dependent, and VLA-4-mediated events have a role in inflammation-induced MMP expression. Inhibition of arthritis-induced increases in MMP expression is not necessary to reduce pathology. This model is well suited for identifying agents that block integrin VLA-4 in vivo.

CGS 34043: a non-peptidic, potent and long-acting dual inhibitor of endothelin converting enzyme-1 and neutral endopeptidase 24.11.

Endothelin-1 (ET- 1) is a potent vasoconstrictor. Its biosynthesis is catalyzed by endothelin converting enzyme (ECE). In contrast, atrial natriuretic peptide (ANP) is a potent vasorelaxant and diuretic, and it is mainly degraded by neutral endopeptidase 24.11 (NEP). Therefore, compounds that can suppress the production of ET-1 by inhibiting ECE while simultaneously potentiating the levels of ANP by inhibiting NEP may be novel agents for the treatment of cardiovascular and renal dysfunction. CGS 34043 is one such compound, which inhibited the activities of ECE-1a and NEP with IC50 values of 5.8 and 110 nM, respectively. In vivo, it inhibited the pressor response induced by big ET-1, the precursor of ET-1, dose-dependently in rats, and the inhibition was sustained for at least 2 hr. In addition, CGS 34043 increased plasma ANP by 150% up to 4 hr after an intravenous dose of 10 mg/kg in conscious rats infused with ANP. However, this compound had no effect on the angiotensin I-induced pressor response. These results demonstrate that CGS 34043 is a potent and long-lasting dual inhibitor of ECE-1 and NEP. Consequently, it may be beneficial for the treatment of diseases in which an overproduction of ET-1 and/or enhanced degradation of ANP plays a pathogenic role. The activity of CGS 34753, an orally active prodrug of CGS 34043, is also described.

Attenuation of puromycin aminonucleoside-induced glomerular lesions in rats by CGS 26303, a dual neutral endopeptidase/endothelin-converting enzyme inhibitor.

Endothelin-1 (ET-1) has been implicated in the pathogenesis of various renal diseases. The purpose of this study was to investigate the effect of CGS 26303, an endothelin-converting enzyme (ECE) inhibitor, on puromycin aminonucleoside (PA)-induced nephrosis in rats. The animals (three groups; n = 8 per group) received 50 mg/kg PA or NaCl, intravenously. CGS 26303 (5 mg/kg/day, s.c. via osmotic minipumps) or vehicle was administered to the PA-treated animals for 4 weeks, starting within 5 min after PA injection. Uninephrectomy was performed 2 weeks after PA to accelerate the renal damage. Rats received no treatment between 4 and 8 weeks. At 8 weeks rats were euthanized and kidneys removed for histology and analysis for mRNA levels of endothelin-A- and -B- (ET(A) and ET(B)) receptors and ECE-1. Glomeruli (100 glomeruli/section; 800/group) were graded as normal (N), mild lesion (ML = few periodic acid-Schiff positive [PAS+] droplets and small adhesions to Bowmans capsule), and moderate to severe lesion (SL = many PAS+ droplets, adhesions to and thickening of Bowmans capsule, mesangial expansion, and cystic dilations of glomerular capillaries). In the PA + vehicle group N, ML and SL were 39.5%, 11.9% and 48.6%, respectively, while the respective values were 68.3%, 9.4%, and 22.3% in PA + CGS 26303-treated rats. However, the renal mRNA levels of ECE-1 and ET(A)- and ET(B)-receptors were not significantly different among the three groups. These results confirm the efficacy of ECE inhibition in this disease model. On the other hand, the mRNA data suggest that either there was no change in the expression of the genes examined or their levels had already returned to normal by the end of the experiment.