RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

M. PHARM SYNOPSIS

YEAR OF ADMISSION: AUG-2012

TITLE OF THE SYNOPSIS

DEVELOPMENT AND EVALUATION OF ISRADIPINE SOLID LIPID NANOPARTICLES FOR ENHANCED ANTI-HYPERTENSIVE ACTIVITY

BY SATISH M HAVANOOR M. PHARM., PART-I DEPARTMENT OF PHARMACEUTICS

UNDER THE GUIDANCE OF

Dr. K. MANJUNATH M.Pharm., Ph.D. Professor DEPARTMENT OF PHARMACEUTICS

INSTITUTION SREE SIDDAGANGA COLLEGE OF PHARMACY B. H. ROAD, TUMKUR-572 102, KARNATAKA

2 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. NAME OF THE SATISH M HAVANOOR CANDIDATE I M. PHARM AND ADDRESS DEPARTMENT OF PHARMACEUTICS SREE SIDDAGANGA COLLEGE OF PHARMACY B.H. ROAD. TUMKUR-572 102. KARNATAKA.

2. NAME OF THE SREE SIDDAGANGA COLLEGE OF PHARMACY INSTITUTION B. H. ROAD, TUMKUR- 572 102 KARNATAKA

3. COURSE OF STUDY AND MASTER OF PHARMACY IN PHARMACEUTICS SUBJECT

4. DATE OF ADMISSION OF AUG 2012 COURSE

5. TITLE OF THE TOPIC

“DEVELOPMENT AND EVALUATION OF ISRADIPINE SOLID LIPID NANOPARTICLES FOR ENHANCED ANTI-HYPERTENSIVE ACTIVITY”

3 6. BRIEF REVIEW OF THE INTENDED WORK

6.1 – NEED FOR STUDY

High blood pressure, also called hypertension, refers to blood pushing against the walls of the arteries with chronically elevated force. Blood pressure that rises above normal levels and remains high can lead to serious health problems including heart attack, heart failure, stroke and kidney failure as well as other health problems. Isradipine is indicated in the management of hypertension. It is usually prescribed for the treatment of high blood pressure in order to reduce the risk of stroke and heart attack. Isradipine is 90-95% absorbed and is subject to extensive first-pass metabolism, resulting in a bioavailability of about 15-24%. The elimination of isradipine is biphasic with an early half-life of 1.5- 2 hours, and a terminal half-life of about 8 hours1. Apparent volume of distribution is 3 L/kg. Isradipine is completely metabolized prior to excretion, and no unchanged drug is detected in the urine. Isradipine is a dihydropyridine calcium channel blocker. It binds to calcium channels with high affinity and specificity and inhibits calcium flux into cardiac and smooth muscle.

In the recent years, vast research trials have been made by throughout the globe for development of solid lipid nanoparticles (SLNs) as drug carriers. These are in submicron size range and are made up of biocompatible and biodegradable materials capable of incorporating lipophilic and hydrophilic drugs. A wide variety of materials such as polymers and lipids were utilized as carrier matrix materials. Appropriately, suitable methods such as high pressure homogenization, solvent diffusion, solvent evaporation, ultrasonication methods were used to develop them with the help of stabilizers and suitable solvents2. The significance of these systems was proved by pharmacokinetic and pharmacodynamic activities. Positively charged nitrendipine SLNs of different triglycerides enhanced the bioavailability of nitrendipine from 3 to 5 folds on intraduodenal administration to rats3. DHA-doxorubicin-loaded SLN promoted a significant improvement of the in vitro antitumor activity when compared to the free doxorubicin and docosahexaenoic acid (DHA)4.

4 SLNs offer selective advantages over conventional dosage form in terms of high stability systems, high specificity, high drug carrying capacity and ability for controlled release, possibility to use in various routes of administration and possibilities of reducing toxicities. Anti-hypertensive drugs such as lercanidipine5, ramipril6, nimodipine7, nitrendipine8 and valsartan9 have been prepared as solid lipid nanoparticles for sustained release and improvement of bioavailability.

High blood pressure cannot be treated once and ignored. Treatment of hypertension requires long term therapy. Single or combinations of anti-hypertension drugs are used in the treatment. Isradipine needs to be administered chronically in divided doses (b.i.d.) in the range of 5-20 mg daily for treatment to be effective. Conventional treatment with isradipine tablets may not maintain the constant blood levels. Sustain release of drug from nanoparticles avoid the fluctuations of drug concentration in blood. Till today, there are no attempts were made to formulate isradipine into SLN. Hence, there is need to develop stable, safe and effective isradipine SLNs. The objectives of the present study are to formulate isradipine SLNs using suitable solid lipids like trimyristin, glyceryl monostearate etc. and to evaluate the anti-hypertensive action using Wistar rats.

6.2 – REVIEW OF LITERATURE

Various researchers developed anti-hypertensive drugs into solid lipid nanoparticles in order to utilize the benefits such as slow release, improvement bioavailability and also stability. Literature survey is made for different class of anti-hypertensive drugs formulated as SLNs by different methodologies and summary of it is given below.

Kiran et al.5 developed lercanidipine loaded solid lipid nanoparticles by modified solvent injection method and characterized the prepared SLN for shape, surface morphology, particle size, and drug entrapment, etc. Lercanidipine is a lipophilic drug which is used in hypertension, was formulated into SLNs using tristearin. SLNs were spherical in shape and possessed particles of size range 110.1 to 148.5 nm, with good entrapment efficiency (85.1%), zeta potential values indicated stability of the formulations. FTIR

5 and DSC studies revealed absence of interaction between the drug and used excipients. Around 50% of drug released from SLNs formulation within 24 hours and the best fitting model was ascertained as Peppas and Matrix equation.

Ekambaram and Sathali6 prepared solid lipid nanoparticles of ramipril using lipids (glyceryl monostearate and glyceryl monooleate) along with stabilizers (tween 80, poloxamer 188, and span 20) in order to overcome the side effects and to increase the bioavailability of poor water soluble drug ramipril. The prepared formulations were evaluated for entrapment efficiency, drug content, in-vitro drug release, particle size analysis, scanning electron spectroscopy, Fourier transform-infrared studies and stability. The hot homogenization and ultrasound dispersion method was used to incorporate ramipril and achieved high drug entrapment efficiency. A formulation containing glyceryl monooleate, stabilized with span 20 showed prolonged drug release, smaller particle size, and narrow particle size distribution, when compared to other formulations with different surfactants and lipids. The adopted method was useful for the successful incorporation of the poor water-soluble drug ramipril with high entrapment efficiency 72 to 86%. The entrapment efficiency of various SLNs stabilized with different nonionic surfactants, and decreased in the order of poloxamer 188 > tween 80 > span 20.

Nimodipine-loaded solid lipid nanoparticles (NMD-SLNs) were prepared by chalikwar et al.7 with palmitic acid (PA), poloxamer 188 and soya lecithin as a lipid, surfactant and co-surfactant, respectively using high pressure homogenizer. A 23 factorial design was employed; three factors such as lipid, surfactant and co-surfactant concentration were used. Optimized formulation had showed particle size of 116 ± 21 nm, zeta potential of −10 ± (−4.8) mV, entrapment efficiency of 93.6 ± 9.7% and cumulative drug release of 87.5 ± 2.5% in 10 hours. Pharmacokinetic study was conducted in male Albino Wistar rats showed 2.08-fold increase in relative bioavailability than that of NMD solution, when administered orally. Differential scanning calorimetry study revealed absence of any chemical interaction between NMD and PA while SEM study confirmed the non spherical shape of optimized SLNs. Accelerated stability studies showed that there was no significant change in the mean particle size and PDI after stability period of three months. Due to enhanced bioavailability, they considered NMD-SLNs are promising

6 vehicles for oral delivery.

Nitrendipine loaded solid lipid nanoparticles were developed using triglyceride (tripalmitin), monoglyceride (glyceryl monostearate) and wax (cetyl palmitate) to improve the oral bioavailability of nitrendipine. Poloxamer 188 was used as surfactant. Hot homogenization of melted lipids and aqueous phase followed by ultrasonication at temperature above the melting point of lipid was used to prepare SLN dispersions. In vitro release studies were performed in phosphate buffer of pH 6.8 using Franz diffusion cell. Pharmacokinetics of nitrendipine loaded solid lipid nanoparticles after intraduodenal administration to conscious male Wistar rats was studied. Bioavailability of nitrendipine was increased three to four fold after intraduodenal administration compared to that of nitrendipine suspension. The obtained results were indicative of solid lipid nanoparticles as carriers for improving the bioavailability of lipophilic drugs such as nitrendipine by minimizing first pass metabolism. The method was consistently produced smaller size nanoparticles in the range of 110–138 nm with narrow size distribution and good entrapment efficiency.8

Sawant et al.9 developed and characterized valsartan loaded solid lipid nanoparticles (VSLNs) to enhance the solubility, avoid the first pass hepatic metabolism, enhance the lymphatic absorption leading to improve bioavailability. VSLNs were prepared using glyceryl behenate (compritol 888 ATO) as the lipid and poloxamer 407 (pluronic F 127) as the surfactant by the solvent injection method. Dialysis diffusion bag method was used for in vitro drug release studies using phosphate buffer of pH 6.8. Ex Vivo drug release studies for VSLNs and valsartan suspension were performed in stomach and intestine of animal models. From in vitro and Ex Vivo drug release studies, they concluded that release of valsartan from SLNs follows Peppas-Korsemeyer kinetic model with a non-Fickian diffusion mechanism followed by combination of lipid swelling, erosion and diffusion through the hydrated lipid matrix. Stability studies revealed negligible increase in mean particle size and reduction in percentage assay.

6.3 – OBJECTIVE OF STUDY:

Following are the objectives of the present study

7  Development of isradipine solid lipid nanoparticles using solid lipids.  Characterization of isradipine solid lipid nanoparticles including in vitro drug release evaluation.  Comparison of anti-hypertensive activity of prepared isradipine solid lipid nanoparticles with isradipine conventional suspension using Wistar rats.

7. MATERIALS AND METHODS

Materials Drug : Isradipine Lipids such as trimyristin, glyceryl monostearate etc. Suitable stabilizers such as egg lecithin or soy lecithin etc. Co-surfactants such as poloxomer 188, tween 80 etc.

Methods Homogenization followed by ultrasonication.

7.1 - Source of Data

 Journals such as, 1. International Journal of Biopharmaceuticals

2. Colloids and surfaces B:Biointerfaces

3. Journal of Young Pharmacists

4. International Journal of Pharmaceuticals

5. International Journal of Pharmaceutical Science and Nanotechnology

 Web sites such as 1) Science direct (www.sciencedirect.com) 2) Pubmed (www.ncbi.nlm.nih.gov/pubmed)

7.2 - Method of collection of data

a) Calibration graph of isradipine shall be done by UV Spectrophotometry at 239

8 nm.

b) Formulation of isradipine SLNs using different solid lipids.

c) Characterization of prepared isradipine SLNs for particle size, zeta potential, entrapment efficiency, assay and other parameters.

d) In vitro drug release studies of isradipine SLNs using Franz diffusion cell.

e) Induction of hypertension in rats by subcutaneous administration of dexamethasone for 12 days.

f) Comparison of anti-hypertensive effect of isradipine SLNs with conventional suspension of isradipine on dexamethasone induced hypertensive Wistar rats for the following parameters.

1. Body weight measurements 2. Electrocardiographic changes. (by Bio-Pac instrument). 3. Blood pressure i) Systolic blood pressure (SBP) ii) Diastolic blood pressure (DBP) iii) Mean arterial blood pressure (MABP) iv) Heart rate (HR) 4. Thymus weight 5. Hematocrit value g) Statistical analysis of data obtained from the results.

7.3 - Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so, Please describe briefly.

9 “YES” The present study requires investigation to be done on the Wistar rats for the comparison of antihypertensive activity of developed isradipine SLN and conventional isradipine suspension. The male Wistar rats (150-180 gm) will be selected and randomized into following 7 groups for conducting ant-hypertensive activity. Each group consists of six animals.

Groups Treatment

I Normal control (Saline)

II Disease control (Dexamethasone)

III Blank treatment of Formulation 1 (Dexamethasone + SLNs without drug) IV Blank treatment of Formulation 2 (Dexamethasone + SLNs without drug) V Conventional treatment (Dexamethasone +Isradipine suspension) VI SLN treatment Formulation 1 (Dexamethasone + Isradipine SLNs) VII SLN treatment Formulation 2 (Dexamethasone + Isradipine SLNs)

7.4 - Has ethical clearance been obtained from your institution in case of 7.3?

“ YES ”

The study has been referred to the ethical committee of the institution and committee has approved the same.

10 8.

REFERENCES

1. http://dailymed.isradipine

2. Manjunath K, Reddy JS, Venkateswarlu V. Solid lipid nanoparticles as drug delivery systems. Method Find. Exp. Clin. 2005;27(2):127-44.

3. Manjunath K, Venkateswarulu V. Pharmacokinetics, tissue distribution and bioavailability of nitrendipine solid lipid nanoparticles after intravenous and intraduodenal administration. J Drug Target 2006;14(9):632-45.

4. Mussi SV, Silva RC, Oliveira MC, Lucci CM, Azevedo RB, Ferreira LAM. New approach to improve encapsulation and antitumor activity of doxorubicin loaded in solid lipid nanoparticles. Eur. J. Pharm. Sci. 2013;48:282–90.

5. Kiran VKP, Nair R, Raju PY, Chakrapani M, Dhanalakshmi P. Preparation and characterization of lercanidipine loaded solid lipid nanoparticles. Int. J. Biopharm. 2012;3(2):82-8.

6. Ekambaram P, Sathali AA, Formulation and evaluation of solid lipid nanoparticles of ramipril. J. Young Pharmacists 2011;3:216-20

7. Chalikwar SS, Belgamwar VS, Talele VR, Surana SJ, Patil MU. Formulation and evaluation of nimodipine-loaded solid lipid nanoparticles delivered via lymphatic transport system. Colloids and Surfaces B: Biointerfaces, 2012;97:109-16.

8. Kumara VV, Chandrasekar D, Ramakrishna S, Kishan V, Rao YM, Diwan VP. Development and evaluation of nitrendipine loaded solid lipid nanoparticles: Influence of wax and glyceride lipids on plasma pharmacokinetics. Int. J. Pharm. 2007;335:167–75.

9. Parmar B, Mandal S, Petkar KC, Patel LD, Sawant KK. Valsartan loaded solid

11 lipid nanoparticles: Development, characterization, and In-vitro and Ex-vivo evaluation. Int. J. Pharm. Sci. Nanotech. 2011;4(3):1483-90.

9. SIGNATURE OF THE CANDIDATE

10 REMARKS OF THE GUIDE Recommended

11 NAME AND DESIGNATION OF

11.1 Guide Dr. K. MANJUNATH, M. Pharm., Ph. D. Professor Department of Pharmaceutics

11.2 Signature

11.3 Co-Guide ( If any)

11.4 Signature

11.5 Head of the Department Dr. SURESH V KULAKARNI, M.Pharm.,Ph. D. Professor & Head Department of Pharmaceutics

12 11.6 Signature

12 12.1 Remarks of the Chairman Forwarded to the University for approval and Principal

12.2 Signature

( Dr. S. BADAMI ) Principal Sree Siddaganga College of Pharmacy B. H. Road, Tumkur-572 102.

13