Therapeutic developments in matrix metalloproteinase inhibition

A family of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs) have the capacity to degrade all elements of the extracellular matrix (ECM) and are required for homeostatic maintenance of the ECM. However, interest in MMPs predominantly arises from the accumulating evidence implicating that dysregulated MMP expression plays a role in mediating or accompanying a diverse array of pathologies. These include tumour invasion and metastasis and inflammatory diseases characterised by excessive tissue destruction, such as arthritis, periodontal disease, atherosclerosis, plaque rupture, arterial aneurysms, postmyocardial infarction, ventricular remodelling and cardiac rupture. Several patents representing therapeutic drugs and strategies to treat the associated conditions have been claimed, some resulting in clinical drug trials. This review will: i) summarise the current status of our understanding of MMPs and how they participate in normal and functional ECM degradation; ii) review therapeutic efforts to favourably alter the balance between MMP proteolysis and ECM sythesis; and iii) critically evaluate recent studies that have importantly advanced our understanding of the complexities of MMP function and propose areas where future efforts to develop therapeutic strategies might be most beneficial and productive.     

四环素类似物的体外抗菌活性初探

目的检测四环素类似物的体外抗菌活性,以观察类似物的抗菌和趋骨活性的相关性。方法采用纸片琼脂扩散法及试管二倍稀释法。结果四环素类似物的抗菌活性较四环素大幅降低或完全消失。结论通过对四环素的结构简化,保留其趋骨活性而使抗菌作用减弱或消失。     

Study of the inactivation of copper, zinc superoxide dismutase by tetracycline analogues

Tetracycline analogues (oxytetracycline, doxycycline, and tetracycline) can inactivate copper, zinc superoxide dismutase (CuZnSOD). The interactions between tetracycline analogues and CuZnSOD were studied by using the UV–visible absorption and spectrofluorimetric method. Tetracycline analogues can cause fluorescence quenching of CuZnSOD by a static mechanism. The absorption spectrum and quenching constant all support this conclusion. The binding constants of tetracycline analogues with CuZnSOD were obtained at various temperatures. Based on the Förster nonradioactive energy transfer theory, we obtain the distance between the donors and acceptors. Based on the thermodynamic parameter we also determined that the main force acting between them is electrostatic gravitation. Tetracycline analogues can decrease the activity of CuZnSOD purified from garlic and the whole blood from rabbits injected with tetracycline. Such results provide a reference for clinical diagnosis using the activity level of CuZnSOD, which varies in patients with different diseases.     

Selective targeting of matrix metalloproteinase inhibition in post-infarction myocardial remodeling

BACKGROUND: A cause-effect relationship has been established between MMP activation and left ventricular (LV) remodeling following myocardial infarction. The goal of the present study was to examine a selective MMP inhibitor (sMMPi) strategy that effectively spared MMP-1, -3, and -7 with effect to regional and global left ventricular remodeling in a pig model of myocardial infarction. METHODS AND RESULTS: Pigs instrumented with coronary snares and radiopaque markers within the area at risk were randomized to myocardial infarction-only (n = 10) or sMMPi (PGE-530742, 1 mg/kg TID) begun 3 days prior to myocardial infarction. Ten weight-matched noninstrumented pigs served as reference controls. Left ventricular end-diastolic volume in the myocardial infarction-only group was increased from baseline (81 +/- 3 mL versus 55 +/- 4 mL, respectively, P < 0.05) but was attenuated with sMMPi (67 +/- 3 mL, P < 0.05). Fractional area of shortening of marker area was decreased in the myocardial infarction-only group (change from baseline -63 +/- 10%, P < 0.05) but this effect was attenuated with sMMPi (-28 +/- 14%, P < 0.05), indicative of less dyskinesis of the infarct region with sMMPi. Wall stress was reduced within both the septal and posterior wall regions with sMMPi. Myocardial MMP-2 activity was decreased in both remote and border areas of sMMPi-treated samples compared with myocardial infarction-only values, consistent with pharmacologic MMP inhibition. CONCLUSIONS: Selective MMP inhibition favorably affected regional myocardial geometry and decreased left ventricular dilation post-myocardial infarction. This study suggests that a strategy of selective MMP inhibition of a limited array of MMPs may be an achievable goal in preventing pathologic left ventricular remodeling post-myocardial infarction.     

Bioinformatical and in vitro approaches to essential oil-induced matrix metalloproteinase inhibition

Context: Essential oils carry diverse antimicrobial and anti-enzymatic properties.

Objective: Matrix metalloproteinase (MMP) inhibition characteristics of Salvia fruticosa Miller (Labiatae), Myrtus communis Linnaeus (Myrtaceae), Juniperus communis Linnaeus (Cupressaceae), and Lavandula stoechas Linnaeus (Labiatae) essential oils were evaluated.

Materials and methods: Chemical compositions of the essential oils were analyzed by gas chromatography–mass spectrometry (GC–MS). Bioinformatical database analysis was performed by STRING 9.0 and STITCH 2.0 databases, and ViaComplex software. Antibacterial activity of essential oils against periodontopathogens was tested by the disc diffusion assay and the agar dilution method. Cellular proliferation and cytotoxicity were determined by commercial kits. MMP-2 and MMP-9 activities were measured by zymography.

Results: Bioinformatical database analyses, under a score of 0.4 (medium) and a prior correction of 0.0, gave rise to a model of protein (MMPs and tissue inhibitors of metalloproteinases) vs. chemical (essential oil components) interaction network; where MMPs and essential oil components interconnected through interaction with hydroxyl radicals, molecular oxygen, and hydrogen peroxide. Components from L. stoechas potentially displayed a higher grade of interaction with MMP-2 and -9. Although antibacterial and growth inhibitory effects of essential oils on the tested periodontopathogens were limited, all of them inhibited MMP-2 in vitro at concentrations of 1 and 5 µL/mL. Moreover, same concentrations of M. communis and L. stoechas also inhibited MMP-9. MMP-inhibiting concentrations of essential oils were not cytotoxic against keratinocytes.

Discussion and conclusion: We propose essential oils of being useful therapeutic agents as MMP inhibitors through a mechanism possibly based on their antioxidant potential.     

Ramiprilate inhibits functional matrix metalloproteinase activity in Crohn's disease fistulas

Increased expression of matrix metalloproteinase (MMP)-2, -3 and -9 has been demonstrated in Crohn's disease fistulas, but it is unknown whether these enzymes are biologically active and represent a therapeutic target. Therefore, we investigated the proteolytic activity of MMPs in fistula tissue and examined the effect of inhibitors, including clinically available drugs that beside their main action also suppress MMPs. Fistula specimens were obtained by surgical excision from 22 patients with Crohn's disease and from 10 patients with fistulas resulting from other causes. Colonic endoscopic biopsies from six controls were also included. Total functional MMP activity was measured by a high-pressure liquid chromatography (HPLC)-based, fluorogenic MMP-substrate cleavage assay, and the specific activity of MMP-2, -3 and -9 by the MMP Biotrak Activity Assay. The MMP inhibitors comprised ethylene-diamine-tetraacetic acid (EDTA), the synthetic broad-spectrum inhibitor, GM6001, the angiotensin-converting enzyme (ACE) inhibitor, ramiprilate, and the tetracycline, doxycycline. In Crohn's disease fistulas, about 50% of the total protease activity was attributable to MMP activity. The average total MMP activity was significantly higher (about 3.5-times) in Crohn's fistulas (471 FU/μg protein, range 49-2661) compared with non-Crohn's fistulas [134 FU/μg protein, range 0-495, (p < 0.05)] and normal colon [153 FU/μg protein, range 77-243, (p < 0.01)]. MMP-3 activity was increased in Crohn's fistulas (1.4 ng/ml, range 0-9.83) compared with non-Crohn's fistulas, [0.32 ng/ml, range 0-2.66, (p < 0.02)]. The same applied to MMP-9 activity [0.64 ng/ml, range 0-5.66 and 0.17 ng/ml, range 0-1.1, respectively (p < 0.04)]. Ramiprilate significantly decreased the average total MMP activity level by 42% and suppressed the specific MMP-3 activity by 72%, which is comparable to the effect of GM6001 (87%). Moreover, MMP-9 activity was completely blunted by ramiprilate. Doxycycline had no effect on MMP activity. Increased functional MMP activity, notably MMP-3 and -9, is present in Crohn's fistulas and may be inhibited by ramiprilate, a widely available ACE inhibitor.     

Oxaprozin: A new hope in the modulation of matrix metalloproteinase 9 activity

Oxaprozin (4,5‐diphenyl‐2‐oxazolepropionic acid) is a non‐steroidal, analgesic and antipyretic propionic acid derivative, whose activity in treating inflammatory disorders is well known. The aim of this study was to investigate the ability of oxaprozin to modulate the activity of matrix metalloproteinase 9 (MMP‐9), a zinc‐dependent endopeptidase involved in a wide range of physiological and pathological events associated with extracellular matrix (ECM) remodelling. The interaction between oxaprozin and MMP‐9 was firstly investigated in silico by molecular docking and analysis with LIGPLOT software. Subsequently, the potential inhibitory activity of oxaprozin against MMP‐9 and the possible mechanism of the ligand–enzyme interaction were investigated in vitro. Taking into account the in silico findings, MMP‐9 can be considered a potential target of oxaprozin, which seems to be able to chelate the catalytic zinc ion through the nitrogen of the oxazole ring and the carboxylate moiety. Moreover, one of the phenyl rings interact with the S1′ inhibitor‐binding pocket through hydrophobic interaction. Gelatin zymography and enzymatic inhibition assay confirmed the potential role of oxaprozin as a competitive inhibitor of MMP‐9. These observations sound particularly interesting if we consider the pathological role of MMP‐9, especially evident in inflammatory conditions and cancer. This work may represent a starting point to improve the understanding of the role of oxaprozin, as well as its structural analogues, in modulating the MMP‐9 function.     

四环素类化合物对膀胱癌EJ细胞增殖和金属酶活性的影响

目的：金属蛋白酶是降解细胞外基质的重要酶．最近有研究表明四环索族化合物有抑制金属蛋白酶活性和某些肿瘤细胞增殖的作用。但其对层粘连蛋白和Ⅳ型胶原蛋白的降解以及对人EJ细胞增殖作用影响尚不清楚。方法：用EJ细胞体外培养加入不同浓度的四环素和米诺环索．用MTT法测定其对EJ细胞的增殖作用的影响。EJ细胞体外培养后．加入不同浓度的四环索和米诺环索培养24h后再加入外源性的Ⅳ型胶原和层粘连蛋白,继续培养24h．用放射免疫法测定培养液中的Ⅳ型胶原和层粘连蛋白含量。结果：四环索和米诺环索对层粘连蛋白的降解无明显影响．但两者均能阻止Ⅳ型胶原的降解．且相互间无显著性差异。四环素和米诺环对EJ细胞的增殖均无影响。结论：四环素和米诺环索均能抑制Ⅳ型胶原的降解．保护细胞外基质,这可能是由于两者络合Ca^2 或Zn^2 影响了Ⅳ型胶原酶的结构和活性。本实验发现四环素和米诺环索对层粘连蛋白的降解无明显影响．可能由于层粘连蛋白的降解有多种酶参与．如血浆纤维蛋白溶解酶等。米诺环素和四环索对EJ细胞增殖无明显影响．其原因可能是两者透过EJ细胞膜困难．不能影响细胞内线粒体内蛋白质合成。     

Selective inhibition of matrix metalloproteinase isozymes and in vivo protection against emphysema by substituted gamma-keto carboxylic acids

The synthesis and matrix metalloproteinase (MMP) inhibitory activity of a series of gamma-keto carboxylic acids are described. Among nine MMP isozymes tested, compound 1j displays selective inhibition of MMP-2, -9, and -12 with IC(50) values between 0.20 and 1.51 microuM, and in male golden Syrian hamsters, it shows protection against PPE-induced emphysema.     

Anti-invasive activity of diallyl disulfide through tightening of tight junctions and inhibition of matrix metalloproteinase activities in LNCaP prostate cancer cells

Diallyl disulfide (DADS) is a major component of an oil-soluble allyl sulfide garlic (Allium sativum) derivative, which has been shown to exert a potential for anti-cancer activity. However, the biochemical mechanisms underlying DADS-induced anti-invasiveness and anti-metastasis have not been thoroughly studied. In this study, we investigated the effect of DADS on the correlation between tightening of tight junctions (TJs) and anti-invasive activity in human prostate carcinoma LNCaP cells. Inhibitory effects of DADS on cell motility and invasiveness were found to be associated with increased tightness of the TJ, which was demonstrated by an increase in transepithelial electrical resistance (TER). Additionally, immunoblotting results indicated that DADS repressed the levels of the claudin proteins, which are major components of TJs that play a key role in control and selectivity of paracellular transport. Furthermore, the activities of matrix metalloproteinase (MMP)-2 and -9 in LNCaP cells were dose-dependently inhibited by treatment with DADS, and this was also correlated with a decrease in expression of their mRNA and proteins. Although further studies are needed, the present study indicates that TJs and MMPs are critical targets of DADS-induced anti-invasiveness in human prostate cancer LNCaP cells.     

Influence of aldose reductase inhibition on the microvascular reactivity in experimental diabetes

• 1.1. To verify if tolrestat, an aldose reductase inhibitor, corrects the impaired responses of microvessels to histamine and bradykinin in alloxan-diabetic rats, the mesenteric microcirculation was studied in vivo in anaesthetised animals.
• 2.2. The impaired responses were corrected by tolrestat 5 mg/kg/day for 7 days p.o. Similar responses to acetylcholine and sodium nitroprusside were obtained in preparations of diabetic and control rats and were not altered by tolrestat treatment.
• 3.3. As in diabetes, galactosemia induced impaired responses to histamine and bradykinin; these altered responses were corrected by tolrestat treatment.
• 4.4. These data allow us to suggest that the polyol pathway activity might be involved in the altered responses of microvessels observed in diabetic rats. It is possible that polyol activation may play an important role in the development of vascular dysfunction in diabetes mellitus.

     

Resveratrol as a novel agent for treatment of multiple myeloma with matrix metalloproteinase inhibitory activity

AIM: To examine the in vitro antitumor activity of resveratrol against multiple myeloma (MM) cell lines (RPMI 8226, U266, and KM3), and the mechanisms involved. METHODS: The growth inhibition of resveratrol was determined by 3-(4, 5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The effect of resveratrol on the apoptosis was investigated by combined annexin V-propidium iodide staining. The effect of resveratrol on the invasion through Matrigel matrix was detected by transwell invasion analyses. The activity of matrix metalloproteinase (MMP)-2 and -9 proteins were determined by gelatin zymography analysis. The expression of MMP-2, MMP-9, Bcl-2, Bcl-x(L), XIAP and Bax protein were detected using Western blotting analysis. RESULTS: Resveratrol inhibited proliferation of MM cells in a dose- and time-dependent manner. Incubation of MM cells with resveratrol resulted in apoptotic cell death. Resveratrol down-regulated the expression of the antiapoptotic proteins Bcl-2, Bcl-x(L) and XIAP and up-regulated the expression of the proapoptotic protein Bax. Furthermore, resveratrol inhibited invasion of RPMI 8226, U266, and KM3 cells with IC50 values of 64+/-8 micromol/L, 93+/-11 micromol/L, and 153+/-11 micromol/L, respectively.Resveratrol inhibited the constitutive expression of MMP-2 and -9 proteins of MM cells and suppressed its gelatinolytic activity. CONCLUSION: Resveratrol inhibits the proliferation of MM cells by inducing apoptotic cell death. Resveratrol also inhibits MM cell invasion. The inhibition of invasion may be associated with the attenuation of the enzymatic activities of MMP-2 and -9.     

Gadolinium metallofullerenol nanoparticles inhibit cancer metastasis through matrix metalloproteinase inhibition: imprisoning instead of poisoning cancer cells

The purpose of this work is to study the antimetastasis activity of gadolinium metallofullerenol nanoparticles (f-NPs) in malignant and invasive human breast cancer models. We demonstrated that f-NPs inhibited the production of matrix metalloproteinase (MMP) enzymes and further interfered with the invasiveness of cancer cells in tissue culture condition. In the tissue invasion animal model, the invasive primary tumor treated with f-NPs showed significantly less metastasis to the ectopic site along with the decreased MMP expression. In the same animal model, we observed the formation of a fibrous cage that may serve as a physical barrier capable of cancer tissue encapsulation that cuts the communication between cancer- and tumor-associated macrophages, which produce MMP enzymes. In another animal model, the blood transfer model, f-NPs potently suppressed the establishment of tumor foci in lung. Based on these data, we conclude that f-NPs have antimetastasis effects and speculate that utilization of f-NPs may provide a new strategy for the treatment of tumor metastasis. FROM THE CLINICAL EDITOR: In this study utilizing metallofullerenol nanoparticles, the authors demonstrate antimetastasis effects and speculate that utilization of these nanoparticles may provide a new strategy in metastatic tumor therapy.     

Effect of flavones on rat brain and lung matrix metalloproteinase activity measured by film in-situ zymography

We have evaluated the inhibitory activity of flavone, nobiletin, and heptamethoxyflavone on matrix metalloproteinase (MMP) activity in the rat. MMP in 9000-g supernatant fraction of lung homogenate was activated by p-aminophenyl mercuric acetate (APMA), and gelatinolytic activity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. This activity should be related to MMP-2 and/or MMP-9 and was confirmed by gelatin zymography. Fluorescent-conjugated collagen used as a substrate for collagenolytic activity wasinvestigated by SDS-PAGE also. The film in-situ zymography method was applied to rat brain and lung tissue in the same manner. Flavone and nobiletin inhibited the APMA-stimulated gelatinolytic activity and also the collagenolytic activity by more than 75%. The film in-situ zymography method indicated that these compounds might be potent inhibitors of MMP, suggesting the specific inhibition of localized MMP in brain hippocampus and/or lung terminal bronchioles, which may contribute to the prevention of some types of brain disease or cancer invasion and metastasis.     

柔肝化纤颗粒对四氯化碳诱导的肝纤维化大鼠基质金属蛋白酶1及其抑制因子的影响

目的 探讨柔肝化纤颗粒对四氯化碳诱导的肝纤维化大鼠基质金属蛋白酶1（MMP-1）和金属蛋白酶组织抑制因子1（TIMP-1）的影响.方法 建立四氯化碳诱导的肝纤维化大鼠模型,健康清洁SPF级大鼠85只,雌雄各半,随机数字表法分为正常对照组（15只）、病理模型组（16只）、柔肝化纤颗粒组（16只）、大黄座虫丸组（16只）及秋水仙碱对照组（16只）.正常对照组不做其他处理,常规饲养;肝纤维化造模成功后,正常对照组、病理模型组4周后灌胃0.9％氯化钠注射液,柔肝化纤颗粒组、大黄廑虫丸组、秋水仙碱对照组造模成功4周后分别给予柔肝化纤颗粒、大黄=虫丸（0.2 g/kg）、秋水仙碱（100 μg/kg）灌胃,各组灌胃液体量为10 ml/kg体重,每日1次.各组于用药治疗后5、10周,免疫组织化学染色及计算机图像分析技术检测Ⅳ胶原.利用半定量反转录-聚合酶链反应检测MMP-1和TIMP-1 mRNA的表达.结果 用药治疗后5、10周,病理模型组肝组织炎症活动度计分、肝纤维化程度计分均明显高于正常对照组[用药后5周：（4.93±2.56）分比（1.08 ±0.29）分,（15.57±6.12）分比0分,用药后10周：（5.03±2.66）分比（1.10±0.22）分,（16.27 ±6.21）分比0分],差异均有统计学意义（均P＜0.05）;经柔肝化纤颗粒和秋水仙碱、大黄廑虫丸治疗后,大鼠肝组织炎症活动度、肝纤维化程度计分及Ⅳ胶原蛋白水平均明显降低（P＜0.05）.用药治疗后10周,病理模型组MMP-1 mRNA和TIMP-1 mRNA表达分别为（1.30±0.15）和（20.62 ±4.56）,均明显高于正常对照组[分别为（0.94±0.44）、（10.52±3.20）],差异均有统计学意义（均P ＜0.05）;柔肝化纤颗粒组MMP-1 mRNA表达为（1.58±0.34）,明显高于病理模型组、大黄廑虫丸组（1.39±0.35）和秋水仙碱对照组（1.41±0.41）;柔肝化纤颗粒组TIMP-1 mRNA为（13.6±3.3）,明显低于病理模型组、大黄廑虫丸组（18.3±4.4）和秋水仙碱对照组（17.3±4.47）（P＜0.05）;柔肝化纤颗粒组TIMP-1/MMP-1 mRNA为（8.9±2.0）,明显低于病理模型组、大黄廑虫丸组（11.1±1.9）和秋水仙碱对照组（10.4±2.4）（P＜0.05）.结论 柔肝化纤颗粒能通过调节TIMP-1/MMP-1比例来促进细胞外基质降解,从而发挥对肝纤维化的防治作用.     

Induction of mutagenic activity of tetracycline by synergistic action with nitrite

Synergistic mutagenicity of tetracycline(TC) and nitrite was investigated by the bacterial mutation test in the Salmonella/microsome system by using the reaction products obtained under neutral condition as well as under acidic condition (in simulated gastric juice). Results from tests using Salmonella typhimurium TA 100 disclosed a significant increase in the appearance of histidine (+) (his+) revertants by the reaction product between TC and nitrite in the presence of the rat liver-microsomal enzyme system (S9 mix), while the mutagenic potency of the reaction product of the two compounds in simulated gastric juice was extremely weak. In the process of the reaction of TC with and without nitrite in the presence of S9 mix, formaldehyde was detected, indicating the demethylation of TC by demethylase in S9. To explain the induction mechanism of the synergistic mutagenicity of TC and nitrite, it was suggested that the alkylating reaction of nitroso compound formed by nitrosation of the 4-demethylated intermediate of TC by the aid of microsomal metabolism is more important than the well known nitrosation mechanism under acidic condition such as in gastric juice in rats and in simulated gastric juice.     

Icariin attenuates cardiac remodelling through down-regulating myocardial apoptosis and matrix metalloproteinase activity in rats with congestive heart failure

Objectives In this study, the anti‐heart failure effect of icariin, a natural flavonol glycoside, and the underlying mechanisms were investigated. Methods Heart failure was induced by isoproterenol in male Sprague–Dawley rats. Matrix metalloproteinase activity was determined by gelatin zymography assay. The mRNA expression was determined by real‐time PCR. The protein expression was determined by Western bolt. Mitochondria structure was examined by transmission electron microscopy. Key findings Isoproterenol administration resulted in a severe heart failure, as shown by the increased levels of left ventricular weight index, heart rate, left ventricular end diastolic pressure, maximal rate of left ventricular pressure decline (dp/dt_min), decreased levels of left ventricular systolic pressure and maximal rate of left ventricular pressure rise (dp/dt_max). Against these, icariin dose‐dependently reversed the changes of these cardiac morphometric and haemodynamic parameters. In addition, icariin significantly inhibited serum levels of tumour necrosis factor‐α, noradrenaline, angiotensin II and brain natriuretic peptide in rats with congestive hear failure and improved the histological changes, including cardiocyte hypertrophy, cardiocyte degeneration, inflammatory infiltration and cardiac desmoplasia. Furthermore, the expression and activity of matrix metalloproteinase (MMP)‐2 and MMP‐9, which regulate collagen production, were also blocked by icariin. Moreover, myocardial apoptosis was remarkably attenuated by icariin through regulating Bcl‐2/Bax axle. Conclusions Icariin ameliorates left ventricular dysfunction and cardiac remodelling through down‐regulating matrix metalloproteinase‐2 and 9 activity and myocardial apoptosis in rats with congestive heart failure.