RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA,
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1. / Name of the Candidateand Address
/
MUDRABOYNA KAMALA DEVI
a. Permanent AddressD/O,M.Suri Babu
Door no:-10-125a,
District. Guntur,
Andhra Pradesh
Pin Code-522509
b. Postal Address
Al-Ameen College of Pharmacy
Bangalore – 560027,
Karnataka
2. /
Name of the Institute
/ Al-Ameen College ofPharmacy
3. /
Course of Study and Subject
/ Master of Pharmacy inPharmacology
4. /
Date of Admission to Course
/ 20115. / Title of the Topic:
EVALUATION OF CARDIO PROTECTIVE EFFECTS OF NANO-SIZED NOVEL CHITOSAN–FLUVASTATIN CONJUGATE AGAINST MYOCARDIAL INFARCTION IN HYPERLIPIDEMIC RATS
6. / BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study:
Globally, myocardial infarction is a major public health concern and the leading cause of mortality. Developing countries like India are also struggling to manage the impact of myocardial infarction along with the growing burden of obesity, Type II diabetes and hypertension1. In recent years, an increasing number of young Indians are succumbing to myocardial infarction due to unusual risk factors characterized by high triglycerides, low High Density Lipoproteins (HDL), glucose intolerance, insulin resistance, abdominal obesity and increased lipoprotein (a) levels2. The major abnormalities noticed following myocardial infarction are lipidaemia, peroxidation and loss of plasma membrane integrity3. A better understanding of the processes involved in myocardial infarction has stimulated the search for new drugs, which could limit the myocardial injury.
Fluvastatin (FS), one of the clinically used 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor to reduce cholesterol biosynthesis, exerts additional beneficial pleiotropic effects independent of its lipid-lowering action, such as scavenging of free radicals4, anti-inflammatory, antithrombotic and antioxidant actions5-7. Research has demonstrated that treatment of FS can prevent ischemic heart diseases through its antioxidant property8 ameliorate myocardial ischemic injury presumably by reducing the formation of free radicals9 and improve ventricular function and clinical symptoms in patients with cardiac failure10. Recent research demonstrated that FS has a significant effect on the protection of heart against isoproterenol-induced myocardial infarction through maintaining endogenous antioxidant enzyme activities11. FS is rapidly absorbed after oral administration, with time to reach peak concentrations (tmax) within 0.5–1 h but possess poor oral bioavailability (20%). The poor oral bioavailability is attributed for its low aqueous solubility, crystalline nature, and high hepatic first-pass metabolism12, 13. Poor bioavailability has necessitated the administration of higher than normally required oral doses which often leads to economic wastages, risk of toxicity, erratic and unpredictable responses. Therefore, it is needed to develop a novel approach that can resolve poor oral bioavailability.
Chitosan (CS) is a natural cationic polysaccharide consisting of (1-4)-2-amino-2-deoxy-D-glucopyranosyl units. It breaks down into harmless products (amino sugars), which are completely absorbed by the human body14. Due to its non-toxicity and high biocompatibility, CS has been formulated as dietary supplements, as carrier for oral peptide and protein drug delivery, as targeted drug delivery, and in the pharmaceutical and biomedical fields15-17. Several studies have shown that CS has cholesterol-lowering properties both in animals and humans18, 19. The results of the research conducted by Sivakumar et al indicated that the cardioprotective effect of CS might be ascribable to the hypolipidaemic property and/or antioxidant nature of CS20.
It has been reported that paclitaxel conjugate with low molecular weight CS exhibited favorable features for oral delivery including: (1) increased water solubility of paclitaxel, (2) prolonged retention of the conjugate in the GI tract, (3) ability to bypass the P-glycoprotein mediated efflux, and (4) ability to bypass cytochrome P450-mediated metabolism, all of which led to dramatically enhanced bioavailability and antitumor efficacy in vivo21. Further, nano-sized drug delivery system is a very promising way of improving drug bioavailability. Enhanced bioavailability may be observed for nanoparticles containing FS. Hence an attempt is being made to study a new nano-sized conjugate between chitosan and FS for oral delivery of FS. Synthesis, characterization, drug release profile of the nano-sized novel CS–FS conjugate will be reported. Further, the pharmacokinetics will be studied along with evaluation of cardioprotective activity of nano-sized novel CS–FS conjugate against myocardial infarction in hyperlipidaemic rats.
6.2 Review of Literature:
Hyperlipidaemia is one of the important factors associated with atherosclerosis, others being hypertension, smoking, diabetes mellitus, and other factors. To retard or prevent the formation of atherosclerosis comes hyperlipidaemia on one of the present therapeutic challenges22.Elevated plasma cholesterol levels have long been established as risk factors for CHD, and lowering cholesterol levels, particularly low – density lipoprotein cholesterol (LDL –C), has been the focus of the prevention of CHD and its sequelae for almost 25 years. However, the complex mechanisms by which these molecules act are only beginning to be appreciated. Evidences suggest that lipid – lowering modes of therapy also reduce inflammation, which may reduce the risk of cardiovascular events, even for individuals with LDL–C levels in the normal range (< 130 mg/dL) based on the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) guidelines23.
6.3 Objective of study: The objective of the proposed study is to investigate cardioprotective effects of different doses of nano-sized novel CS–FS conjugates against isoproterenol induced myocardial infarction in cholesterol induced hyperlipidemic rat models.
SPECIFIC OBJECTIVES:
a. Synthesis and characterization of nano-sized CS–FS conjugates, a novel drug formulation for the application in the treatment of isoproterenol induced cardiac dysfunction in cholesterol induced hyperlipidaemic rats.
b. To evaluate the drug release profile nano-sized CS–FS conjugates, a novel drug formulation for the application in the treatment of isoproterenol induced cardiac dysfunction in cholesterol induced hyperlipidaemic rats.
c. To evaluate the pharmacokinetics of the nano-sized CS–FS conjugates a novel drug formulation using rat model.
d. Pharmacological evaluation of various doses of nano-sized CS–FS conjugates, a novel drug formulation as compared to the FS and CS monotherapy on isoproterenol induced myocardial infarction in hyperlipidaemic rats.
7. / MATERIALS AND METHODS:
7.1 Source of Data:
Data will be obtained from CD-Rom, Internet facilities, Literatures and related articles from libraries of Al-Ameen College of Pharmacy, Indian Institute of Sciences, Government College of Pharmacy etc., and other Research Publications and Journals.
Web sites: www.sciencedirect.com
www.pubmed.com
www.google.com
www.ijp-online.com
www.elsevier.com
7.2 Method of Collection of Data: The data collected will be based on animal experimentation as per the parameters studied under each animal model, which are mentioned under the objectives of the study.
MATERIAL AND METHODS
Chemical and reagents
Fluvastatin (FS), Isoproterenol, Chitosan (CS) (chitoclear, degree of deacetylation 80-96%), 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), All other chemicals will be of analytical grade.
Synthesis and characterization of CS–FS conjugate
CS–FS conjugate will be prepared by using amide coupling reaction. A 10% (w/v) solution of FS in methanol (5 ml) will be activated by EDC (125 mM, 1 ml) treatment for 4 h at room temperature to afford an ester form of FS. Separately, 1% (w/v) aqueous CS solution will be prepared after hydrating CS with 1 N HCl (5 ml). The methanolic solution of FS will be then added drop wise to the aqueous acidic CS solution under continuous magnetic stirring. Throughout the experiment, pH will be maintained in the range of 5–6. After stirring for 24 h at room temperature, the excess reagent will be removed by washing with distilled water. The reaction mixture will be then purified using ultra -filtration, after which the CS–FS conjugate will be lyophilized. The conjugate will be then characterized by using FT-IR spectrometry and Differential Scanning Calorimetry (DSC) and the content of FS will be determined by UV method.
Preparation of CS–FS nano-conjugate
Nano-sizing of CS–FS conjugate will be achieved using (High pressure homogenization) HPH technique. Briefly, 100 mg of the synthesized conjugate will be dispersed in deionized water at a concentration of 0.1% (w/v). The suspension thus formed will be allowed to pass through a high-pressure homogenizer to obtain nano-conjugates. CS–FS nano-conjugates will be collected by lyophilization.
Characterization of CS–FS nano-conjugate
Nano-conjugate size and morphology Measurement of particle size will be carried out by Zetasizer (Malvern, zetasizer-nano s 90), Scanning Electron Microscope (SEM) will be carried out in order to study the morphology of prepared nano-conjugates.
Nano-conjugate crystallinity
The physical form of the lyophilized nano-conjugates will be determined by powder X-ray diffraction method.
In vitro release studies
In vitro release studies will be performed using pure FS and the formulations (CS–FS) conjugate and CS–FS nano-conjugate) equivalent to 100 mg of FS. Tests will be performed using and simulated gastric fluid (SGF, pH 1.2) and phosphate buffer (pH 7.4) at 37 ± 0.1 0C for up to 72 h at a rotation speed of 50 rpm. At designated time points, 4 ml samples will be withdrawn with replacement with equal volume of the fresh medium, filtered through 0.11 µm nylon syringe filter, appropriately diluted with methanol and assayed for drug concentration by UV method described by Saminathan et al24. Dissolution tests will be performed in triplicate and the percentage of drug dissolved at different time intervals will be estimated.
In vivo pharmacokinetic studies
For in vivo pharmacokinetics, two groups, each containing six female wister rats. After 12 h of fasting, the rats will be allowed to administer 0.5 ml aqueous dispersion of FS and CS–FS nano-conjugate (equivalent to 10 mg/ml FS) using oral feeding sonde. Blood samples (0.2 ml) will be withdrawn at pre-determined time intervals through the tail vein of rats in vacutainer tubes, vortexed to mix the contents and centrifuged at 5000 rpm for 20 min. The plasma will be separated and stored at -20 0C until drug analysis will be carried out using HPLC method described by Saminathan et al25. The animal protocol to carry out in vivo study was obtained from the Institutional Animal Ethics Committee, Al-Ameen College of Pharmacy, Bangalore, India.
Groups
Female Wistar rats were divided into 2 groups as follows:
Group 1: Fluvastatin 20 mg/kg, single dose, p.o.
Group 2: CS-FS nano conjugate 20 mg/kg, single dose, p.o.
Induction of myocardial injury
Isoproterenol (ISO) will be dissolved in normal saline and injected subcutaneously to rats (85 mg/kg) daily for 2 consecutive days to induce experimental myocardial infarction11. Hyperlipidaemia can be induced after excessive cholesterol feeding.
Experimental protocol
Female Wistar rats were divided into eight groups as follows:
Group 1: Normal vehicle treatment for 30 days, p.o.
Group 2: Hyperlipidaemic rats, (Cholesterol 500 mg/kg) for 30 days, p.o.26
Group 3: Hyperlipidaemic rats, ISO challenge, 85 mg/kg, s.c repeated at 24 h interval.
Group 4: Hyperlipidaemic rats treated with Fluvastatin 20 mg/kg, for 45 daysp.o.11 and ISO
challenge.
Group 5: Hyperlipidaemic rats treated with Chitosan 225 mg/kg, for 45 days, p.o.27 and ISO
challenge.
Group 6: Hyperlipidaemic rats treated with CS-FS nanoconjugate 20 mg/kg, for 45 days, p.o. and
ISO challenge.
Group 7: Hyperlipidaemic rats treated with CS-FS nanoconjugate 15 mg/kg, for 45 days, p.o. and
ISO challenge.
Group 8: Hyperlipidaemic rats treated with CS-FS nanoconjugate 10 mg/kg, for 45 days, p.o and
ISO challenge.
After the experimental period, the rats will be sacrificed by cervical decapitation. Blood will be withdrawn from retro-orbital vein 48 h after the first dose of ISO under anesthesia and serum will be separated by centrifugation for Cholesterol, triglycerides, High density lipoproteins (HDL), Low density lipoproteins (LDL), lactate dehydrogenase (LDH) and creatine kinase (CK) measurements.
The heart will be isolated from each animal under ketamine (70 mg/kg, i.p) and xylazine (10 mg/kg, i.p) anesthesia and homogenized to prepare heart tissue homogenate (HTH) using sucrose (0.25 M). The activity of LDH, CK-MB, superoxide dismutase (SOD) and catalase will be determined in HTH. Microscopic slides of myocardium will be prepared for histopathological studies. Volume fraction of interstitial space (VFITS) in myocardial tissue will be determined from hematoxylin and eosin (H & E) stained transverse sections by using the equation.
VFITS = 100 X Area of interstitial space
Total tissue area.
The myocardial damage will be determined by giving scores depending on the intensity as follows; no changes – score 00; mild – score 01 (focal myocytes damage or small multifocal degeneration with slight degree of inflammatory process); moderate – score 02 (extensive myofi brillar degeneration and/or diffuse inflammatory process); marked – score 03 (necrosis with diffuse inflammatory process).
7.3 Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly:
YES
Study requires investigation on animals. The effects of the drug will be studied on various parameters using rats as experimental animal model.
7.4 Has ethical clearance been obtained from your institute
Ethical Committee approval letter is awaited.
8. / List of References:
1. Tilak-Jain JA, Devasagayam TPA. Cardioprotective and other beneficial effects of some Indian medicinal plants. J Clin Biochem Nutr 2006; 38: 9-18.
2. Farvin KHS, Anandan R, Kumar SHS, Shiny KS, Mathew S, Sankar TV, Nair PGV. Cardioprotective effect of squalene on lipid profile in isoprenaline-induced myocardial infarction in rats. J Med Food 2006; 9: 531-536.
3. Farvin KHS, Anandan R, Kumar SHS, Shiny KS, Sankar TV, Thankappan TK. Effect of squalene on tissue defense system in isoproterenol-induced myocardial infarction in rats. Pharmacol Res 2004; 50: 231-236.
4. Yamamoto A, Hoshi K, Ichihara K. Fluvastatin, an inhibitor of 3-hydroxy-3- methylglutaryl-CoA reductase, scavenges free radicals and inhibits lipid peroxidation in rat liver microsomes. Eur J Pharmacol 1998; 361: 143–149
5. Fischetti F, Carretta R, Borotto G, Durigutto P, Bulla R, Meroni PL, Tedesco F. Fluvastatin treatment inhibits leucocyte adhesion and extravasation in models of complement-mediated acute inflammation. Clin Exp Immunol 2004; 135: 186–193.
6. Ferrara DE, Liu X, Espinola RG, Meroni PL, Abukhalaf I, Harris EN, Pierangeli SS. Inhibition of the thrombogenic and inflammatory properties of antiphospholipid antibodies by fluvastatin in an in vivo animal model. Arthritis Rheum 2003; 48: 3272–3279.
7. Bandoh T, Sato EF, Mitani H, Nakashima A, Hoshi K, Inoue M. Antioxidative potential of fluvastatin via the inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Biol Pharm Bull 2003; 26: 818–822.
8. Demyanets S, Kaun C, Pfaffenberger S, Hohensinner PJ, Rega G, Pammer J, Maurer G, Huber K, Wojta J. Hydroxymethylglutaryl-coenzyme A reductase inhibitors induce apoptosis in human cardiac myocytes in vitro. Biochem Pharmacol 2006; 71, 1324–1330.