Development and Evaluation of Gel Containing Ethosomes Entrapped with Nsaids

DEVELOPMENT AND EVALUATION OF GEL CONTAINING ETHOSOMES ENTRAPPED WITH NSAIDS

M.Pharm dissertation protocol submitted to

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore – 560041

Mr.SYED IRFAN BASHAB.Pharm

Under the Guidance of

Mrs.A. GEETHA LAKSHMIM.Pharm, (Ph.D)

Asst.Professor

Department of Industrial Pharmacy

Acharya & B.M.Reddy College of Pharmacy

Soldevanahalli,Chikkabanavara (Post),

Hesarghatta main road, Bangalore – 560 090

2010– 2012

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE.

ANNNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1 / Name and address of candidate / Mr. Syed Irfan Basha.
H/N-40-36;472-A1,
Jani complex,
Near LIC office,
Kurnool,518001.
Andhra pradesh.
2 / Name of institution / ACHARYA & B.M. REDDY COLLEGE OF
PHARMACY.
Soldevanahalli, Hesarghatta Main Road,
Chikkabanavara Post,
Bangalore-560090.
3 / Course of study and subject / M. Pharm
(Industrial Pharmacy)
4 / Date of admission /

08-09-2010

5 / Title of the project / DEVELOPMENT AND EVALUATION OF GEL CONTAINING ETHOSOMES ENTRAPPED WITH NSAIDS
6
6.1 / BRIEF RESUME OF INTENDED WORK
NEED FOR THE STUDY:
ARTHRITIS:
Arthritis can be defined as the inflammation of joints which may lead to the damage of joints. The arthritis can be classified into various types like Rheumatoid arthritis, osteoarthritis, gout, psoriatic arthritis. Rheumatoid arthritis is an autoimmune disorder that causes the immune system to attack the joints where it causes inflammation (arthritis) and destruction. It can also damage some organs such as the lungs and skin.Osteoarthritis also known as degenerative arthritis or degenerative joint disease. It is a clinical syndrome in which low grade inflammation results in pain in the joints caused by abnormal; wearing of the cartilage that covers and act as a cushion inside joints and destruction or decrease of synovial fluid that lubricates those joints. Rheumatoid arthritis and osteoarthritis is most common chronic condition and have a substantial impact on quality of life and health care resource use. Fifty percent of patients with rheumatoid arthritis are unable to work within 10 years of the onset of disease. Pharmacotherapy for rheumatoid arthritis and osteoarthritis is aimed to relieving pain,suppressing inflammation and maintaining movement with analgesics and non-steroidal antiinflammatory drugs. Anti inflammatory drugs are heterogeneous group with differing chemical and physical properties.Various NSAIDS used in the treatment of RA are:Etodolac,Ibuprofen,Naproxen etc.The NSAIDS act as an non selective inhibitors of the enzyme cyclooxygenase. Cyclooxygenase catalyzes the formation of prostaglandins and thromboxane from archidonic acid. Prostaglandins act as a messenger molecule in process of inflammation1.
Many clinicians believe that higher dose of NSAIDS are more efficient than lower dose for treatment of osteoarthritis and rheumatoid arthritis, but are associated with higher rates of adverse events.The side effects include development of high blood pressure, worsening kidney function, fluid retention, gastro intestinal bleeding etc.2
The NSAIDS is mainly used for the treatment of rheumatoid arthritis and osteoarthritis. Transdermal application as an alternative route of administration has demonstrated better to sort-out
these problems, and it was speculated that topical administration of NSAIDS may increase the patient compliance and a possibility for continuous transdermal absorption of drug in controlled manner.Apart from this, number of novel drug delivery systems have been reported for various routes of administration,to achieve local and systemic drug delivery in controlled manner,whereas lipidic
vesicular drug delivery systems such as liposomes,niosomes,transfersomes, and pharmacosomes were
also developed for topical/transdermal administration of drug molecules.The vesicular approach i.e. liposomes in transdermal drug delivery system has been studied for many purposes but the unstable nature limits their use at clinical and industrial levels. In order to increase the stability of liposomes concept of proliposomes has been proposed, but because of poor skin permeability,breaking of vesicles,these system are not much successful transdermal drug delivery.Recently an innovative flexible vesicular system,ethosomes has been developed for topical/transdermal delivery of a drug. This system has wonderful property to permeate intact through the human skin due to its high elasticity properties,which have an immense consequence for design of carrier system to be, applied topically both for local and systemic delivery of hydrophilic and lipophilic drugs.3
ETHOSOMES:
Ethosomes are soft malleable vesicles composed mainly of phospholipids, water and ethanol.These soft vesicles represent novel vesicular carries for enhanced delivery through skin.The size of ethosomes vesicles can be modulated for tens of microns to nanometres.This carries presents interesting feature correlated with its ability to permeate intact through the human skin due to its high deformability.Due to the ethanol high concentration it help in enhancing the permeation of the ethosomes.Ethosomes are also non invasive drug delivery carriers that enable drug to reach the deep skin layers and systemic circulation.The high concentration of ethanol makes the ethosomes unique,as ethanol is known for the disturbance of skin lipid bilayer. Therefore when integrated into a vesicle membrane it gives that vesicle the ability to penetrate the stratum corneum.4
6.2 / REVIEW OF LITERATURE:-

Meenakshi chauhan et al.,reviewed the importance of gel containing ethosomes vesicles for transdermal delivery of aceclofenac to evaluate the potential of ethosomes to increase the transdermal transport of aceclofenac. The size of the vesicles was found to have increased with increasing lecithin concentration (2‐6%). Also it was observed that the size of the vesicles decreased significantly with increasing ethanol concentratio. EF‐2 ethosomes with 3% lecithin and 20% ethanol were found to have shown highest aceclofenac release (92.72 ± 1.04%). Ethosomes containing aceclofenac were converted to gel using three different carbopol concentrations. The gel containing aceclofenac encapsulated .And found to have excellent drug release.5

Priyanka rathore et al., reviewed the importance of comparative studies on skin permeation of miconazole using different novel carries. Miconazole nitrate is a widely used antifungal agent but its use in topical formulation is not efficacious because deep ereated fungal infections are difficult to treat with conventional topical formulation. The purpose of this study was to prepare miconazole incorporated different novel carriers such as liposomes, ethosomes and to compare their in vitroskin permeation studies with plane ointment using skin model. In vitrorelease studies of miconazole from the liposomal and ethosomal suspension incorporated ointment to an aqueous receptor phase through pig skin was analyzed spectrophotometrically at a wavelength of 272nm. Invitroskin permeation study results showed that the steady state fluxes of drug was higher in case of ethosomal suspension incorporated ointment as compared to liposomal ointment.6

Dayan Net al., studied carriers for skin delivery of trihexyphenidyl HCl: ethosomes vs. liposomes. The purpose of this work was to characterize a novel ethosomal carrier containing trihexyphenidyl HCl (THP) the delivery of THP from ethosomes versus classic liposomes. When compared with standard liposomes, ethosomes had a higher entrapment capacity and a greater ability to deliver entrapped fluorescent probe to the deeper layers of skin. It was observed that the THP was87, 51 and 4.5 times higher than from liposomes, phosphate buffer and hydroethanolic solution, respectively. The quantity of THP remaining in the skin at the end of the 18-h experiment was statistically significantly greater from the ethosomal system than from liposomes or a control hydroethanolic solution.7

Fang Y et al., studied thecomparison of 5-aminolevulinic acid-encapsulated liposome versusethosome for skin delivery for photodynamic therapytopical photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) is an alternative therapy for many non-melanoma skin cancers. The objective of the present work was to characterize ethosomes containing ALA and to enhance the skin production of protoporphyrin , compared to traditional liposomes. Results showed that the average particle sizes of the ethosomes were less than those of liposomes. It was also observed that the entrapment efficiencyof ALA in the ethosome formulations was 8–66% depending on the surfactant added. The study also showed that the particle size of the ethosomes was still approximately <200 nm after 32 days of storage. The results indicated that the penetration ability of ethosomes was greater than that of liposomes. The enhancements of all the formulations were ranging from 11 to 15-fold in contrast to that of control (ALA in an aqueous solution) in terms of protoprophyrin intensity.8

Touitou E et al.,studied the intracellular delivery mediated by an ethosomal carrierinvestigated the efficiency of ethosomes,phospholipid vesicular carriers containing ethanol. Different flourescent probes were used and penetration of these fluorescent probes into fibroblasts and nude mice skin was examined by CLSM and FACS. CLSM micrographs showed that ethosomes facilitated the penetration of all probes into the cells, as evident from the highintensity fluorescence. The intracellular presence of each of the three probes testedwas evident after 3 min of incubation. Moreover, with ethosomal , fluorescence was also seen in the fibroblast nucleus. Enhanced delivery of molecules from the ethosomal carrier was also observed in permeation experiments with the hydrophilic calcein and lypophilic to whole nude mouse skin. Calcein penetrated the skin to a depth of 160,80 and 60 mm from ethosomes, hydroethanolic solution and liposomes,respectively. Maximum fluorescence intensities measured for RR delivered from ethosomes, hydroethanolic solution and liposomes were 150, 40 and 20 AU,respectively.9

A.N Misraet al.,studied the Preparation and characterisation of Fluconazole encapsulated ethosomes, incorporated in suitable dermatological base and its comparative clinical efficacy in the treatment of candidiasis patients against liposomal gel and hydroethanolic solution. Drug encapsulated ethosomes and liposomes were prepared and optimised by “Hot” method technology and lipid film hydration technology. Vesicle size and drug entrapment efficiency of optimized ethosomes and liposomes were found to be 144±6.8nm and 82.68% and 216±9.2nm and 68.22% respectively. DSC of the study shown high fluidity of ethosomes and liposoms and the drug release was twice from ethosomes than liposomes and is three times higher than the hydroethanolic solution. The study demonstrated an enhanced activity compared to liposomal formulation and hydroethanolic solution of the drug.10

Keith glaser et al.,reviewed the importance of etodolac selective inhibits human prostaglandin G/H synthase 2 (PGHS-2)versus human PGHS-1.The isozymes of prostaglandin synthase are shown to be differentially inhibit invitro by current marketed nonsteriodal anti-inlamatory drugs using microsomal rhPGHS-1 and rhPGHS-2. Comparision of selective ratios demonstrated a 10-fold selectivity of etodolac for rhPGHS-2,whereas the other NSAIDS evaluated demonstrated no preference or a slight preference for inhibition of rhPGHS-1. In human whole blood assay where etodolac also demonstrated a 10-fold selectivity for inhibition of PGHS-2. These explains the clinically observed gastrointestinal safety of etodolac versus other marketed NSAIDS.11

P.Anitha et al., reviewed the importance of ethosomes as a noninvasive vesicular carrier for transdermal drug delivery. Ethosomes are noninvasive delivery carriers that enable drugs to reach the deep skin layers and the systemic circulation. These are soft, malleable vesicles tailored for enhanced delivery of active agents. Ethosomes are soft lipid vesicles containing phospholipids, alcohol in relatively high concentration and water. The size range of ethosomes may vary from tens of nanometers to microns (μ). Hot and cold methods are used for formulation of ethosomes. Evaluation parameters include size, shape, drug content, zeta potential, stability studies, skin permeation studies etc. These carriers open new challenges and opportunities for the development of novel improved therapies.12

Kumar R.et al.,reviewed the importance of novel visicular carries in transdermal drug delivery.Several methods have been tried to increase the permeation rate of drugs. one simple and convient approach is application of drug in formulation with elastic vesicles or skin enhancers.Elastic vesicles were more efficient at delivering a low and high moleclar weight drug to skin in terms of quantity and depth. Their effectiveness strongly depends on the physicochemial properties. The mechanism is due to ethanol effect where intercellular of ethanol into intercellular lipids increasing lipid fluidity and decrease density of lipid multilayer. Resulting in release of drug in deep layer of skin.13

Abdul Hasan Sathali A et al., reviewed the importance of formulation and evaluation of diclofina potassium ethosomes. 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, drug‐membrane component interaction and stability. The ethosomal formulation having 4%w/v of phospholipid and 40%v/v of ethanol showing the greatest entrapment efficiency (72.91±0.64%) with small particle size (251±23nm) was selected for further skinpermeation studies. Scanning electron microscopy confirmed the three dimensional nature of ethosomes. Dynamic light scattering technique proved that the ethosomes has smaller vesicular size than the liposomes prepared without alcohol. FT‐IR studies revealed no interaction between the drug and membrane components. The ethosomal vesicles were incorporated in carbopol gel base and its anti‐inflammatory efficiency was compared with the marketed diclofenac gel. The pharmacodynamic studies showed the enhanced anti‐inflammatory activity of ethosomal gel than the marketed gel formulation.14

Vaibhav Det al., reviewed the transdermal potential of novel ethanolic liposomes (ethosomes) bearing Melatonin (MT) was investigated. MT loaded ethosomes were prepared and characterized for vesicular shape and surface morphology, vesicular size, entrapment efficiency, stability, in vitro skin permeation and in vivo skin tolerability. % Entrapment efficiency of MT in ethosomal carrier was found to be 70.71±1.4. Stability profile of prepared system assessed for 120 days revealed very low aggregation and growth in vesicular size (7.6±1.2%). MT loaded ethosomal carriers also provided an enhanced transdermal flux of 59.2±1.22μg/cm2/h and decreased lag time of 0.9h across human cadaver skin. Fourier Transform-Infrared (FT-IR) data generated to assess the fluidity of skin lipids, after application of formulation, revealed a greater mobility of skin lipids on application of ethosomes as compared to that of ethanol or plain liposomes. Further, a better skin tolerability of ethosomal suspension on rabbit skin suggested that ethosomes may offer a suitable approach for transdermal delivery of melatonin.15

6.3 / OBJECTIVES OF THE STUDY:
The objectives of the present study are following:-
To formulate ethosomes containing NSAIDS.
To Characterize the prepared formulation using various methods which includes vesicular shape and surface morphology, vesicular size, size distribution, storage-physical stability of ethosomes, entrapment efficience.
Invitro drug diffusion study of ethosomes containing NSAIDS.
To carry out invivo studies with the optimized formulation.
To carry out the stabilities studies on the selected formulation as per ICH guidelines.
7.0
7.1
7.2 / MATERIALS AND METHODS:
SOURCE OF DATA
1)Review of literature from:
a. Journals – such as

Indian Journal of Pharmaceutical Sciences

International Journal of Pharmaceutics
International Journal of Pharmacy and Pharmaceutical Science.
European Journal of pharmacology.

2)
3) J-Gate@Helinet.
4) Reference books:

Drug Discovery and Evaluation by H. Gerhard Vogel. Second Edition

MATERIALS
DRUG: NSAIDS.
POLYMERS: Biodegradable/non biodegradable polymers.
EXCIPIENTS: excipients as required,Carbopol,Ethanol,Propylene glycol,Lecithin etc.
METHOD OF COLLECTION OF DATA:
Selection of the drug and other excipients.
Authentication of the drug and excipients.
Characterisation of drug and excipients for intended formulations and to carry out the compatability studies for selected drugs and polymers by FTIR, DSC etc.
Formulation of ethosomes by Cold or Hot method.
Characterization of ethosomal formulations:
a) Vesicular shape and surface
morphology

Scanning electron microscopy (SEM)

b) Vesicular size and size distribution

Dynamic light scattering (DLS)

1)Storage: Physical stability of ethosomes will be determined by the ability of vesicles to retain the drug (i.e., drug-retentive behavior) and will be assessed by keeping the suspensions at different temperatures, i.e., 4, 20, 30, 40º ±2ºC for different periods of time (1,20,40,60,80 and 120days). The vesicular suspensions will be kept in sealed vials (10mlcapacity) after flushing with nitrogen. The stability of ethosomes will be assessed quantitativelyby monitoring size and morphology of the vesicles over time using DLS and TEM.
2) Entrapment efficiency will be determined as prepared ethosomal vesicles will be separated from the free (unentrapped) drug by a Sephadex G-50 minicolumn centrifugation technique. The method will be repeated at least three times with fresh syringe packed with gel each time until the fraction collected is free from unentrapped drug. The vesicles will be lysed by TritonX-100 (0.5%w/w) and entrapped drug will be estimated using HPLC.
3)The anti inflammatory activity of gel will be tested by inducing inflammation using archidonic acid on the ear of the mice.
7.3

7.4 / DOES THE STUDY REQUIRE ANY INVESTIGATION TO BE CONDUCTED ON PATIENT OR OTHER HUMANS OR ANIMALS?
“ YES”
Has ethical clearance been obtained from your institution in case of 7.3?
“Obtained”
8 / LIST OF REFERENCES:-
1.Rheumatoid arthritis[home page on internet]. 2011[updated 2011 may 20; cited 2001 aug21].Available from :http//wikipedia.org/wiki/Rheumatoid_arthritis.
2.Emry P, Elliot W, Tanveer E, Douglas J, Ehrich MD, Watson et al., Dose-Effect Relationships of Nonsteroidal Anti-Inflammatory Drugs. Clin Thera,2002;24(8):1225-291.
3.Barupal AK, Vandana G, Suman R, preparation and characterization of ethosomes for topical delivery of aceclofenac. IJPS,2011;72(5):582-86.
4. Maurya SD. Enhanced transdermal permeation of indinavir sulfate through Stratum Corneum Via.Novel permeation enhancers:Ethosomes.Der Pharmacia Lettre,2020;2(5):208-20.
5.Atul KG, Lalit MN, Meenakshi C.Gel containing ethosomal vesicles for transdermal delivery of aceclofenac.Int J Pharmacy Pharma Sci, 2010; 2(2):102-8.
6.Anjali S, Priyanka R, Meenakshi S, and Satish N. Comparative studies on skin permeation of miconazole using different novel carriers. IJPSR, 2010;1 (9):61-6.
7. Dayan N, Touitou E. Carriers for skin delivery of trihexyphenidyl HCl: ethosomes vs. liposomes .Biomaterials,2000;21(3):1879-85.
8.Ping Fang Y, Hung Tsai Y, Chu Wu P, Bin Huang Y.Comparison of 5-aminolevulinic acid-encapsulated liposome versusethosome for skin delivery for photodynamic therapy.Int J Pharma,2008;356(1-2) :144–52.
9. Touitoua E, Godin B,Dayana N,Weissa C, Piliponsky A, Schaffer F. Intracellular delivery mediated by an ethosomal carrier. Biomaterials, 2001; 22: 3053–59.
10. Misra AN,Bhalaria MK, Sachin N.Ethosomes: A novel delivery system for anti fungal in the treatment of topical fungal diseases. Indian J Exp Biol,2009;vol47;368-75.
11.Glaser K, Mei LS, Kim O, Mary B, David H, Richard C et al., Etodolac selectively inhibits human prostaglandin G/H synthase 2(PGHS-2) versus human PGHS-1 .Euro JPharmaco,1995;281: 107-11.
12.Anitha P, Ramkanth S, Uma Sankari K, Alagusundaram M, Gnanapraksah K, Devaki Devi P et al., Ethosomes - A noninvasive vesicular carrier for transdermal drug delivery . IntJRev Life Sci, 2011;1(1); 17-4.
13. Kumar R, Aslam MD,Tripathi A,Prasad D, Chaudhary V, Jain V et al., Ethosomes: Novel vesicular carriers in transdermal drug delivery. JGPT, 2009; 2(6): 1-7.
14.Vijayakumar MR, Abdul HS, Arun K. Formulation and evaluation of diclofanac potassium ethosomes.Int J PharmacyPharm Sci,2010; 2(4):82-6.
15.Vaibhav D, Dinesh M, Jain N. Melatonin loaded ethanolic liposomes: characterization and enhanced transdermal delivery. Eur J Pharm Biopharm 2007; 67(2):398-405.
9 / Signature of the candidate:
10 / Remarks of the Guide:
11 / Name and Designation of:

11.1 Institutional Guide: / Mrs.A Geetha lakshmi.
Asst.Professor
11.2 Signature:
11.3 Co-Guide:
11.4 Signature:
11.5 Head of the Department: /

Mr. Anup Kumar Roy

Asst. Professor & HOD
Dept. of Industrial Pharmacy
11.6 Signature
12 / 12.1 Remarks of the Principal
12.2 Signature /

Dr. Goli Divakar

Principal

ACHARYA & B.M.REDDY COLLEGE OF PHARMACY,
SOLDEVANAHALLI,
HESARAGHATTA MAIN ROAD,
BANGALORE-90.