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SVMP Inhibitor Isolation and Characterisation

Patricia B.J. et al., 2003 – Isolation, characterisation of SVMP inhibitor.
Patricia et al. Investigated the PO41 as a SVMP inhibitor which was isolated from Philander opossum serum. The study was carried out using proper grade chemicals, the wistar rats were used as the test animal.
For the isolation of the PO41 a DEAE-sephacel column (1.0 x 10.5) was used. 10mM sodium acetate (pH 3.7, flow rate 0.16ml/min) was used for column equilibriation. Isocratic elution with same buffer was done following the linear NaCl gradient. ABF-Po was pooled and further processed to get desired product. For the characterisation of it the polyacylamide gel electrophoresis, isoelecric focusing, amino acid analysis and molecular mass determination was done.
The results of serum fractionation of P. opossum on a DEAE-Sephacel column, showed two peaks. ABF-Po eluted on the second peak (0.25M NaCl). The ABF-Po here obtained was futher chromatographed on Phenyl-Superose which gave five peaks. Authors mentioned that peak V showed antihaemorrhagic activity.

FIG: Purification of P.opossum serum:
(A) DEAE-Sephacel chromatography. (B) Phenyl-Superose chromatography of ABF-Po obtained from DEAE-Sephacel. (Jurgilas et al. 2003)
Reducing SDS PAGE carried out in this study showed the following results.

Lane 1- Molecular mass markers;
Lane 2- Philander opossum serum (20 mg);
Lane 3- ABF-Po (10 mg)
Lane 4- PO41 (6 mg)
Fig: SDS PAGE(12.5%) under reducing conditions (Jurgilas et al. 2003)
Authors used the method developed by Kondo et al.1960 for estimating the haemorrhagic activity of B. jararaca venom and jararhagin. One minimum haemorrhagic dose (MHD) was injected intradermally into the shaved back skin. Inhibition was tested by simultaneous injection of Bjv / jararhagin with PO41. A one-fold molar excess of PO41 subunit was used. measurement of Haemorrhagic spots was done after 24 h.
The ability of PO41 to inhibit the fibrinogenolytic activity of SVMP was shown by doing the reducing SDS-PAGE. The PO41 and SVMPs were used in 1:1 molar ratio. The authors said when the 15µg of jararhagin was injected along with PO41 subunit, no haemorrhagic activity can be seen.
These results proves that PO41 is a potent SVMP inhibitor. It is very capable of neutralizing the haemorragic effect of metalloproteases isolated from B. Jararaca venom. Its further use as as drug can undertaken for broad action against other variable SVMPs.
Elda et al 2008
Metalloprotease are enzymes which split peptide bonds in proteins and are involved in many biological and pathological processes. These metalloproteases are in a delicate balance with protease inhibitors to control biological functions. Matrix metalloproteases (MMPs) or matrixins are a family of zinc endopeptidases that function in the degradation of extracellular matrix proteins and play a key role in tissue degradation during embryogenesis, tissue growth, and wound healing in mammals.(28) They also belong to the metalloproteinase class which is one of the four major classes of proteases (serine, cysteine, aspartyl. And metalloproteases).
Both native and synthetic inhibitors have been considered for therapeutic approaches to cancer but the large size of the molecules was a problem in tissue penetration.(28) Synthetic matrix metalloprotease inhibitors (MMPIs) have been developed since the early 1980s. Most of these inhibitors were peptide derivatives of shorter sequences which would neutralize the effects of metalloproteases.(29–31) The British Biotech inhibitor batimastat (BB-94) was shown to reduce metastasis of melanoma, mammary carcinoma, and colorectal tumor cells in experimental metastasis assays.(29)
Marimastat is another type of synthetic MMPIs that is being used in comparative phase III trials.(32) However most of these protease inhibitors used to treat pathological processes are problematic due to poor specificity and low bioavailability.(28)
The fact that mammalian and venom metalloproteases are similar and both possess natural protease inhibitors that regulate them makes them quite intriguing. In pathological processes such as cancer, one critical step in the initiation of tumor metastasis is known to be the unregulated degradation of basement membranes by MMPs.(33) Studies with native tissue inhibitors of metalloproteases (TIMPs) have shown that they can prevent the growth and spread of experimental tumors.(29–31) Similarly, protease inhibitors from resistant animals neutralize the hemorrhagic effects of snake venom metalloproteases however, they have not always demonstrated to be completely effective or impede the damage of the lesion.(36–44)
The possibility of inhibiting toxin activity has been explored using peptidic inhibitors designed to interact specifically through non-covalent associations with their lateral chains. Among these inhibitors, coupled to the peptidic sequence, there are diverse ligands (carboxylic, sulfhydryl, chloromethyl, pyro-glutamates. or hydroxamates), which block the proteolytic activity of the enzyme located in the catalytic center where the Zn ion is present. The inhibitory potential of these synthetic peptids depends on the sequence of chosen peptides.(1,45)
Romero F.V. et al. (2012)
Local tissue damage induced by viperid snake venoms are caused predominantly by the action of hemorrhagic zink depedent metalloproteases (SVMP). Romero F.V. et al. (2012) have identified a snake venom metalloprotease inhibitor targeted against the Bothrops asper metalloproteinase P1(BaP1). First they screened the Prestwick Chemical Library (PCL). It was consisting of 880 off-patent small molecules of known pharmacology. They selected 16 compounds for their dose and found out their responses. Following active compounds were identified after triplicate assay.
Biological activity of the PCL-derived hits as Inhibitors of BaP1.

But these compound were not containing any ZBG (Zinc binding group) and the future improvements of it could be questioned. These results lead the authors to think of new ways for BaP1 inhibition. After this the authors focused their attention towards the production of Bap1 peptide with a ZBG present at the carboxy terminus. The authors collected information after the compound screening. According to information they decided that their design should have amphipathic nitrogen-heterocyclic side chain amino acids (His, Trp?Arg was also included as an amphiphatic side chain) or aromatic side chains (Phe,Tyr) . At position P2? (contact site to the S2?pocket), Leu, was selected. Then authors went for solid phase synthesis of the peptide, either by coupling or not to hydroxamate. The N-Fmoc chemistry was used for this synthesis.
Authors carried out BaP1 proteolytic activity assay and used Batimastat (IC50 =1.2µM) as a positive control. Biological activities of these sytnetic peptides were assayed. And following results were obtained:
IC50 (µM)
C terminus
>1 mM
TABLE 2: defferent synthetic tripeptides and their biological activity (Romero F.V et al (2012))
FLP and FLP sequence were found very effective. the authors studied the interaction between enzymes active site and tripeptides using computer simulated docking. Different binding modes were seen. These two effective inhibitor tripeptides were further studied for Zn2 binding. The binding was studied by a competitive Zn2 binding assay based in 2-(4-benzylpiperazin-1-yl)-N-[(2-hydroxy-3-prop-2-enyl-phenyl)-methylideneamino]acetamide (PAC-1). PAC-1 is a strong affinity Zn2 compound. According to the authors the results of both the assays suggested that the two peptides have specificity for BaP1. Computer simulated docking of Phe-Leu-Phe-NHOH to BaP1 showed that the tripeptide interacts with the active site. In the active site, it specifically bind to Zn2 ion. From this work it can be concluded that the production of synthetic peptides targated against the ZBG of mettaloproteases offer promising future as potential drug candidates.
Jurgilas, B. et al., 2003. PO41 , a snake venom metalloproteinase inhibitor isolated from Philander opossum serum. , 42, pp.621–628.

Unprotected Left Main Coronary Artery Disease Treatment

Comparison of percutaneous coronary intervention with drug-eluting stents and coronary artery bypass grafting for treatment of unprotected left main coronary artery disease in Chinese patients
Objective: To compare drug-eluting stents (DES) and coronary artery bypass grafting (CABG) in the treatment of unprotected left main coronary artery (ULMCA) disease. Methods: A total of 227 patients received revascularization because of continuous ULMCA disease in Cangzhou Center Hospital, of which 106 patients were implanted DES and 121 patients underwent CABG. The death and the major adverse cardiovascular and cerebrovascular events (MACCE) in hospital and in 1 year out of hospital were recorded to evaluate the treatment effects. Results: Compared with CABG group, the incidence of MACCE in hospital was less in DES group (p0.05). Conclusion: Percutaneous coronary intervention (PCI) with DES was a feasible and safe treatment for ULMCA disease. And it seemed to have favorable early clinical outcomes compared with CABG in our center.
Key words: unprotected left main coronary artery; percutaneous coronary intervention; coronary artery bypass grafting; drug-eluting stents; prognosis
Abbreviations and acronyms
ULMCA, unprotected left main coronary artery;
PCI, percutaneous coronary intervention;
CABG, coronary artery bypass grafting;
MACCE, the major adverse cardiac cerebrovascular event(s);
CAG, coronary angiography;
DES, drug-eluting stent(s);
BMS, bare metal stent(s);
LMCA, left main coronary artery;
LAD, left anterior descending coronary artery;
LCX, left circumflex coronary artery;
RCA, right coronary artery;
TVR, target vessel revascularization;
PTCA, percutaneous transluminal coronary angioplasty.
Unprotected left main coronary artery (ULMCA) disease occurs in 3% to 5% of patients undergoing coronary angiography. About 75-90% patients with ULMCA disease combine with multivessel disease. Coronary artery bypass grafting (CABG) is regarded as an accepted gold standard treatment for ULMCA disease according to current guidelines(1, 2). Early studies using bare-metal stents (BMS) have shown high restenosis rates and mortality rates, so that BMS was only used in patients chosen carefully(3, 4). In recent years, with the rapid development of percutaneous coronary intervention (PCI), especially the introduction of drug-eluting stents (DES), there was a renewed interest for the percutaneous treatment of ULMCA disease. Several studies have shown that PCI with DES was a feasible treatment of ULMCA disease with favorable midterm outcomes(5, 6). It seems that both PCI and CABG had similar long-term mortality; however, PCI with DES was associated with higher rates of repeated revascularization than CABG(7-9). In this study, we evaluated the in-hospital and long-term outcomes of PCI with DES and CABG for the treatment of ULMCA disease.
Patients and groups
A total of 227 patients received revascularization because of continuous ULMCA disease in Cangzhou Center Hospital during the time period of January 2009 to January 2013. Coronary heart disease (CHD) patients with evident angina or myocardial ischemia symptoms having more than 50% diameter stenosis in a quantitative coronary angiogram were diagnosed with ULMCA. Patients with patent graft to the left anterior descending artery or left circumflex artery were excluded. The patients excluded in the study met the following criteria: 1) ULMCA was induced by diseases except coronary artery atherosclerotic lesions; 2) myocardial infarction (MI) patients with ST elevation induced by the acute occlusion of left main; 3) patients with serious liver and renal insufficiency; 4) patients with diseases of the blood system; 5) patients combined with infection, tumors and diseases of the immune system; 6) patients had acute cerebral stroke events such as cerebral hemorrhage and cerebral infarction; 7) patients combined with serious infectious diseases; 8) patients with surgical contraindications.
Patients were evaluated by both interventional cardiologists and cardiac surgeons and the decision to perform PCI or CABG was made on the basis of: 1) hemodynamic conditions; 2) lesion characteristics; 3) vessel size; 4) the presence of comorbidities; 5) quality of arterial and/or venous conduits for grafting; and 6) patient and/or referring physician preferences.
Procedural Characteristics
PCI was performed using the standard percutaneous transfemoral approach. All patients treated with PCI were premedicated with 300 mg aspirin and 300mg clopidogrel 12 to 24 hours before the operation. Different stenting techniques for left main coronary artery (LMCA) bifurcation lesions were decided by the operators according to the actual angiographic findings. Simple stenting techniques were preferred over complicated techniques. Before stent implantation, the lesion regions were expanded by balloon and expansion with balloon was also performed after stent implantation. Final kissing balloon techniques were used if necessary. The poststenting antiplatelet regimen included lifetime aspirin if without contraindications and clopidogrel for at least 6 to 9 months, and the patients were recommended to take reexamination using coronary angiogram in 6 months after the operation.
Coronary artery bypass grafting was routinely performed. For those patients taking aspirin and clopidogrel, surgery was delayed to 5 days after discontinuing aspirin and clopidogrel. The postoperative antiplatelet regimen included lifetime aspirin if without contraindications and clopidogrel for at 1 year, and the patients were recommended to take reexamination using coronary angiogram if there were clinical symptoms like chest pain.
Follow up
The major adverse cardiovascular and cerebrovascular events (MACCE) of patients in DES and CABG group were recorded during hospitalization and at 1 year after leaving hospital. The death (cardiac death and stroke death), nonfatal MI, cerebral stroke and target vessel revascularization (TVR) were included. The deaths caused by unknown reasons were recorded as cardiac death. Patients with ischemic symptoms, electrocardiogram (ECG) changes and myocardial enzyme 3 times higher than upper limit of normal were recorded as nonfatal MI. Cerebral stroke included cerebral infarction, intracerebral parenchyma hemorrhage (IPH), transient ischemic attack and so on. TVR was defined as any repeated revascularization in the left anterior descending artery or left circumflex artery as well as in the target segment.
Statistical analysis
SPSS 13.0 was used for statistical analysis. The continuous variables following normal distribution were presented as mean ± SD (±s) and were compared using t test. The continuous variables of non-normal distribution were presented as median and interquartile range and compared using rank sum test. A multivariate logistic regression analysis was performed to take potential baseline differences between groups. Survival analysis was conducted using Kaplan–Meier method. p<0.05 indicated a significant differences.
Clinical characteristics of patients
As shown in Table 1, there were no significant differences between DES and CABG groups in clinical characteristics.
Coronary arteriography of patients
LMCA stenosis combined with bifurcation lesion and the diseased regions of LMCA were recorded. And there were no significant differences between DES and CABG groups (Table 2).
In-hospital clinical outcomes of patients in DES and CABG groups
In-hospital clinical outcomes of patients were shown in Table 3 and Fig. 3. During hospitalization there were no deaths and was one patient with nonfatal perioperative microvessel infarction in DES group. In CABG group there were 4 deaths (3 patients with cardiac death and 1 patient cerebral infarction), 1 patient with nonfatal stroke, 1 patient with nonfatal MI and 1 patient with TVR. The rate of MACCE during hospitalization in DES group was significantly smaller than CABG group (0.94% vs 5.78%, p<0.05).
Long-term clinical outcomes of patients in DES and CABG groups
As show in Table 4 and Fig. 4, there were no significant differences of one-year clinical outcomes between DES and CABG groups. In DES group, there were no deaths but 4 patients with TVR after 1 year. There were 2 deaths (were all cardiac death) in CABG group, 1 patient with nonfatal MI and 1 patient with TVR.
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