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DESIGN AND EVALUATION OF LIPOSOMAL ENCAPSULATED ACYCLOVIR GEL FOR TOPICAL APPLICATION

Prepared & presented By-K. Mondal

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Content.2Serial no.Content1Introduction2Objectives of the study3Review of literature4Method5Result and discussion6References

1. INTRODUCTION1.1 LIPOSOMES-Its comes under novel drug delivery system(NDDS) Artificially prepared vesicles made of lipid bilayers.Phospholipids are the main component of liposomes e.g.-PC,PE,PSThis lipid vesicles entrapped aqueous solution. liposomes acts as drug carrier and releases the drug with a control rate

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1.2 TOPICAL LIPOSOMAL GELLiposomes are incorporated into the gelling substance.They are more effective and less toxic than conventional formulationsMainly use for local dermatologic diseases.Topical liposomal formulation has affinity to keratin and can penetrate deeper into skin hence gives better absorption.Liposomes can deliver sufficient amount of poor skin permeable drug to the site of infection.

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1.3 PERMEATION AND RELEASE OF DRUG TO THE SKIN FROM LIPOSOMAL FORMULATIONDifferent layers of human skin:-Consist of three layers Epiderdermis , dermis, hypodermis or subcutaneous adipose layer.

a) Epidermis composed of keratinocytes, with melanocytes and Langerhans cells . The epidermis can be further subdivided into:- 1. strata corneum 2. strata lucidum 3. strata granulosum 4. strata spinosum 5. strata basale Function- keeping water in the body.5

.2. Dermis layer The dermis layer is made of an irregular type of fibrous connective tissue consisting of collagen and elastin fibers. It consist of two layers- papillary layer and reticular layer This layer contains a number of structures including blood vessels, nerves, hair follicles, smooth muscle, glands and lymphatic tissue.

3. Hypodermis layer It consists of loose connective tissue and elastin Contain 95% 0f body fat Function - attach the skin to underlying bone and muscle.

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a fig.2 Different layers of skin 7

Permeation of liposome through skin Fig . 3 Permeation of liposome through skin Fig. 4 permeation of liposome through intercellular route8

Drug releases form liposome to the skin

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Fig. 6 Liposome comes in contact of the skin layer

Fig.7 liposome fuses with the skin barrier layer

Fig.8 liposome has dissolved and released the active agentsFig. 5 liposome dissolving in the dermis layer

1.4 MECHANISM OF VESICLE FORMATION Phospholipids [add excess amount of aqueous phase] Self-assemble and forms bilayer sheet [input of energy in the form of physical agitation, sonication, heat] Closed vesicle10

1.5 Material used for liposome preparation

Phospholipids e.g.-phosphatidyl choline(PC), phosphorylethanolamine(PE)Cholesterol Reducing the permeability of the membrane Stabilizing the membrane in the presence of biological fluid such as plasmaAntioxidant Antioxidant prolongs the auto-oxidation of a liposomal preparation thus increases the shelf life. e. g. - -tocopherolCharge inducing substance They increases the inter lamellar distance between successive bilayer s in MLV Positive charge inducer e.g. sterylamine Negative charge inducer e.g. phosphatidyl serine,phosphatidic acid,phosphatidyl glycerol

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1.6 METHODS OF PREPARATIONThere are many different strategies for the preparation of liposomes, which can be classified into 3 main groups. 1. Mechanical methods A. Film method B. Ultrasonication method. 2. Methods based on replacement of organic solvent. A. Reverse-phase evaporation. B: Ether vaporisation method. 3. Methods based on size transformation or fusion of preformed vesicles A: Freeze-thaw extrusion method. B: The dehydration-rehydration method.

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1.7 APPLICATION OF LIPOSOMESLiposomes act as carrier for drugs both in vivo and invitro In targeted drug delivery system liposome are used as anticancer drug such as methotrexate, actinomycin-DUsed in recombinant DNA technologyLiposome is used as carriers of radiopharmaceuticals for diagnostic imagingLiposomes in gene deliveryLiposomal encapsulation has been used as a step in enzyme purificationMost important use of liposome in cell biology is to manipulate the status of membrane lipid

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2. OBJECTIVES OF THE STUDY

To preparation the liposomes containing Acyclovir drug using different ratio of lipid and cholesterol using thin film hydration method according to the Response Surface Methodology (RSM).To carry out the in vitro drug release study & Skin Pearmeation Test of the prepared Acyclovir liposomal preparation to find out the pearmeability & rate of drug release form that preparation

Characterization of liposome with respect to :

Average size and size distribution Shape of liposome Encapsulation efficiency %drug content Skin retention study

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3. REVIEW OF LITERATURE A. DRUG PROFILE Name : AcyclovirMolecular formula: C8H11N5OStructure :

Molecular weight: 225.0 gmMelting point: 256.50CPhysico-chemical property: white, crystalline, odourless powder solubility in water is 1.3mg/ml ,solubility in alcohol is very lessMechanism of action : Acyclovir is converted to acyclovir monophosphate with help of thymidine kinase.Then it is converted to acyclovir triphosphate by cellular enzymes which inhibits viral DNA polymerase this results in chain termination. So viral DNA synthesis is stoped.

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.Pharmacokinetics : Acyclovir can be administered orally, intravenously or topically. protein binding: 9-30% Elimination half-life: 3 hours Excretion : glomerular filtration Adverse effect of acyclovir : Hallucinations Unusual bleeding Seizures Unusual decrease in urine production Precaution : Pregnancy - contra indicated orally and parentally Lactation May reduce lactation Uses : It treats cold sores around the mouth (caused by herpes simplex) shingles (caused by herpes zoster) chickenpox

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Literature review on acyclovir drug

N.Manjunatha et al prepared acyclovir liposomes by reverse phase evaporation method using various ratios of phosphatidyl choline with cholesterol and Cephalin (phosphatidyl ethanolamine) with cholesterol. Avinash kumar seth et al prepared twenty seven batches of acyclovir liposome by reverse phase evaporation (REV) method. prepared liposomal batches were evaluated for size,lamellarity,and percentage drug entrapment (PED). S.L.law at al prepared acyclovir-containing liposome system for ocular drug delivery . Corneal penetration of this preparation demonstrated that positively charged liposomes resulted in a penetration rate lower than those of negatively charged liposomes and free acyclovir in solutionBiswajit Mukherjee et al develop and compare acyclovir containing nano-vesicular liposomes and niosomes based on cholesterol, soya L--lecithin and non ionic surfactant, span 20. The effort was made to study in vitro whether acyclovir-loaded nano vesicles could sustain the release of the drug by increasing residence time and thus, acyclovir could reduce its dose-related systemic toxicity.

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B. LIPID PROFILE1. CholesterolFormula:- C27H75HOStructure:- Molecular weight:- 386.65Melting point:- 148.50CDensity:- 1.07gm/ml

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.2. soya lecithinMolecular formula:- C42H80NO8PChemical structure:-

Storage temp.:2-8C

Solubility:chloroform: 0.1g/mL, slightly hazy, slightly yellow to deep orange

Molecular weight:- 758.06

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LITERATURE REVIEW ON PHOSPHATIDYL CHOLINEMaria Glavas-Dodov et al prepared Liposomes composed of Soya lecithin and cholesterol, with lidocaine hydrochloride entrapped in the inner watercompartment, were prepared by the simple mechanical method vortexing the phospholipid dispersion in water. J.Jakirhussain et al formulated liposomes using phosphatidyl choline, cholesterol and those liposomes were modified using surfactants surfactants span 20 , span 40 , span 80 or tween 80 atvarious molar ratio and optimized for its stable vesicular formation. Harica chanda et al liposomes of Fluconazole were prepared by thin film hydration technique using soya lecithin, cholesterol and drug in different weight ratios.The studies demonstrated successful preparation of Fluconazole liposomes and effect of soya lecithin: cholesterol weight ratio on entrapment efficiency and on drug releaseDr. Rakesh P. Patel et al prepared ketoconazole liposome by thin film hydration technique using soya lecithin, cholesterol and drug in different weight ratios. The prepared liposomes were characterized for size, shape, entrapment efficiency, in-vitro drug release (by franz diffusion cell) and physical stability.

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C. LITERATURE REVIEW ON THIN FILM HYDRATION METHODS.S.Patel et al were prepared Tacrolimus loaded liposomal by thin film hydration method. The amount of drug loaded into vesicles ranged from 4.4 mg per 115mg to 8.2mg per 140mg of total lipid.

Vijaykumar A.R et al formulate diclofanac potassium liposome by thin film hydration method. Drug loaded ethosomes had been prepared using phospholipid and ethanol, were optimized and characterized for entrapment efficiency, vesicular size, shape, invitro skin permeation, skin retention, drugmembrane component interaction and stability.

Dhara B. Patel et al formulate liposomes containing metronidazole by simple thin film hydration technique using soya lecithin and cholesterol. Some preliminary trials and 32 factorial design were conducted to optimize the formulation.

El-Khordagui et al developed a liposomal dibucaine base (DB) local anesthetic delivery system by thin film hydration teachnique . DB-loaded multilamellar vesicles (MLVs)with different characteristics were obtained by varying lipid composition, drug loading, induced charge and pH of the hydration buffer.

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4. Materials and methodINGREDIENTSEXCIPIENTS TYPECOMPANY NAMEAcyclovirEAST INDIA PHARMACEUTICALSoya LecithinRM 637 (1x100 GMS)HI-MEDIACholesterolRM 637 (1x100 GMS)HI-MEDIA Tocophero AcetateCM 1870HI-MEDIACarbopol940ChloroformLRSD Fine-chem ltdMethanolLRSD Fine-chem ltd

22Serial no.InstrumentsCompany Name1Electrical balanceSartorius GD 103, USA2Digital pH MeterSartoriu PB-11, USA3UV-Visible SpectrophotometerShimadzu-1700, Japan4RotavapourCyberlab corporation5Diffusion cellTesting laboratory

List of ingredients usedList of instruments used

.4.2 PREPARATION pH 7.4 Weight 1.36gms of potassium dihydrogen phosphate and palce in a 1000ml volumetric flask.Then add 1000ml of distilled water and shake well until potassium di hydrogen phosphste dissolve completely.4.3 PREPARATION OF STANDERD STOCK SOLUTION 10mg of standard sample of acyclovir was dissolved in distilled water in 10 ml volumetric flask to get concentration 1mg/ml.Then take 1 ml from this solution and dissolve it into 10ml of distilled water.The concentration of acyclovir in this second stock solutin is 100m/ml. Series of dilution prepared by tranferring 0.2,0.4,0.6,0.8,1, 1.2,1.4, 1.6,1.8,and 2ml into a 10ml volumetric flask.Then make up the volume upto 10ml with distilled water.The final concentration of acyclovir in these solutions are 2,4,6,8,10,12,14,16,18,20g/ml.4.4 METHOD OF DETERMING CALIBRATION CURVE OF ACYCLOVIR Absorbance of the final diluted solution is measured in a UV spectrophotometer (Shimadzu 1700) at 252 nm against phosphate buffer pH7.4 as blank .The absorbance so obtained was tabulated and standard curve was plotted .

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4.5 METHOD OF PREPARING LIPOSOME

Method- Thin film hydration (by using rotary evaporator)Phospholipids- phosphatidyl choline and cholesterol Organic solvent- chloroform and methanol as solvents in 2:1 ratioProcedure- 1)Dissolve PC and cholesterol in the solvent mixture 2)transfer them in to the round bottom flask attached with rotary evaporator at 60 rpm and 400C. 3)A thin residual film will be obtain 4)add phosphate buffer solution pH 7.4 and shake for 30 minutes 5)A milky white suspension of liposome will be formed.

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Rotary evaporator

4.6 PREPARATION OF ACYCLOVIR LIPOSOME AS PER FACTORIAL DESIGN Independent variables are X1= PC X2=CHOL Independent variables are Y1= entrapment efficiency Y2=drug release(at 8hrs)

25variables+10-1X1 (mg)100140160X2(mg)406080

.Formulation code X1 X2F1-10F2+1+1F3-1-1F4+10F5+1-1F60-1F70+1F8-1+1F900

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.CharacterizationThe parameters to be checked are:-Microscopy- size distribution profile, lamellarity Drug polymar interaction study-Differential scanning calorimetry(DSC)-FTIRSurface morphology study-Scanning electron microscopy (SEM)Drug entrapment studies In vitro skin permeation studyDrug release studySkin retention study Storage stability

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5.RESULT AND DISCUSSION

5.1 RESULT

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5.2 VALIDATION OF THE METHODThe method was validated for linearity, accuracy, precision and interference by other material used in the present study. Accuracy and precision (reproducibility) of the method was studied by analyzing six individually weighted samples of acyclovir.

. 5.3 STANDARD CALIBRATION CURVE The max was found at 252 nm in PH7.2. The standard calibration curve for acyclovir with regression value of 0.9939 is shown in the following figure.The relation between drug concentration and absorbance is linear and the curve obeys Beer-lamberts law within the concentration range of 2 to 20 g/ml of acyclovir.

29Sl. No.Conc(g/ml)absorbane120.0549240.1042380.18254120.27365160.35476200.4819

.30 calibration curve for the estimation of acyclovir in pH7.45.4 DiscussionThe calibration curve of acyclovir in pH7.4 obeyed Beers law in the concentration range 2-20 g/ml. low RSD value ensured reproducibility of the method. The regression coefficient (R2) value was found 0.9939 with y value of 0.0229 at 0.005 intercept indicates the linearity of the method.

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. 5.5 CONCLUSION In present study acyclovir liposomal topical gel design has to be developed,using combination of phosphatidyl choline an cholesterol . So that the drug can release in a control rate from the liposomal preparation to the skin. Various combinations of the phosphatidyl choline and cholesterol are used in different proportions for the preparation of liposome of different batch, and their drug release profile is seen, to optimize the process to find the best formulation that shows good drug release pattern.

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6.REFERENCE

1 . Vyas. S.P.,Khar. R.K.,Targeted and Controlled Drug Delivery Novel Carrier System, 1st ed., CBS Publishers, New Delhi,5: 173-174, 2002. 2 .Mustafa M.A. Elsayed , Ossama Y. Abdallah ,Viviane F. Naggar, Nawal M. Khalafallah. Lipid vesicles for skin delivery of drugs: Reviewing three Decade. International Journal of Pharmaceutics. 2007 ; 332 : 116. 3 .Galvas-Dodov M,Simon O,Goracinova K .Formulation and characterization of topical liposomal gel bearing lidocaine Hcl. Bulletin of the Chemists and Technologists of Macedonia, 2005 ; 24(1): 5965 4 .Kirjavainen M, Monkkonene J, Saukkosaari M. Phospholipids affects stratum corneum lipid bilayer fluidity and drug partitioning into the bilayers. J. Control. Rel. 1999; 58: 207214 5.Kalia Y.N, Guy R.H., Modeling transderma drug release. Adv. Surg Delivery Rev. 48:2001; 159172 6.Tortora GJ,Sandra RG.principles of anatomy and physiology.10th edition. Hoboken John Wiley and Sons Inc; 2003:141-3 7.Sharma Vijay K, Mishra D N, Sharma A K,Srivastava B. Liposomes: Present Prospective and Future Challenges.internationa journal of crrent pharmaceutical review and research.2010;vol-1

8.Kumar A,Badde S,Kamble R,Varsa B.P. Development and characterization of lipomal drug delivary system for nimesulide.International journal of pharmacy and pharmaceutical science. 2010;2(4)

9. Manjunatha N,prakash naidu G,Vutrave B,Patel K,Samanta MK. Preparation and Evaluation of Liposomes of an Antiviral Drug. Indian Journal of Novel Drug delivery. 2009;1(1): 25-31.

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.10. Mukherjee A, Mukherjee B, Patra B, Layek B.Sustained release of acyclovir from nano-liposomes and nano-niosomes: An in vitro study. International Journal of Nanomedicine. 2007;2(2): 21322511. Seth AK, and MisraA. Preparation and Evaluation of Acyclovir Liposomes by Two Techniques:A Comparative Study. Iranian Journal of Pharmaceutical Research (2007); 6 (2): 75-81.12. Dr.Patel Rakesh P , Patel H and Ashok H. Baria. Formulation and Evaluation of Liposomes of Ketoconazole.International Journal of Drug Delivery Technology 2009; 1(1): 16-2313. Chanda H,Das P,Chakraborty R,ghos A.Developement and evaluation of liopsome of fluconazole.journal of pharmaceutical and biomedical science.2011;5(27).14. Glav-Dodov M,Simonoska M,Goracinova K.Formulation and characterisation of liposomal gel bearing lidocain Hcl. Bulletin of the Chemists and Technologists of Macedonia. 2005; 24( 1): 5965 15. Patel S, Salampure S, Vishwanath B, and Patel N M. Development and Evaluation of Liposomes for Topical Delivery of Tacrolimus J. Sci. Res.2010; 2 (3): 585-596 .16. Vijaykumar M.R., Sathalia A, ARUN K. Formulation and evaluation of diclofenac potassium Ethosome .International Journal of Pharmacy and Pharmaceutical Sciences2010; 2( 4):1945-1091

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.17. Dhara B. Patel, Jayvadan K. Patel . Liposomal Drug Delivery of Metronidazole for the Local Treatment of Vaginitis .International Journal of Pharmaceutical Sciences and Nanotechnology . 2009;2(1):422-42718. Nounou M.M, El-Khordagui, L.K., Khalafallah, N.A. and Khalil, S.A. Topical liposomal dibucaine delivery system:Development and Characterisation Continental J. Applied Sciences 2007; 2: 26 37.19. Dr.Patel R, Patel H and Ashok H. Baria. Formulation and Evaluation of Liposomes of Ketoconazole. International Journal of Drug Delivery Technology 2009; 1(1): 16-2320. S. S. Patel, M. S. Patel, S. Salampure, B. Vishwanath2, and N. M. Patel. Development and Evaluation of Liposomes for Topical Delivery of Tacrolimus.J. Sci. Res 2010; 2 (3): 585-596 21. Vijaykumr M.R., Hasan A, Formulation and evaluation of diclofenac potassium ethosome .Internationa journal of phaemacy and pharmaceutical science.2010;2(4):82-86.22 . Vijay K S, Mishra D N, Sharma A K,Srivastava B. Liposomes: Present Prospective and Future Challeng .IJCPR August October 2010; 1(2) 23. Vyas. S.P.,Khar. R.K.,Targeted and Controlled Drug Delivery Novel Carrier System, 1st ed., CBS Publishers, New Delhi,5:181 -191, 2002.

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