Shivhare UD and Wasnik SV./ International Journal of Biopharmaceutics. 2013; 4(3): International Journal of Biopharmaceutics
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1 231 e- ISSN Print ISSN International Journal of Biopharmaceutics Journal homepage: IJB FORMULATION DEVELOPMENT AND EVALUATION OF NIOSOMAL GEL FOR TRANSDERMAL DELIVERY OF AN ANTIHYPERTENSIVE DRUG Shivhare UD* and Wasnik SV Sharad Pawar College of Pharmacy, Wanadongri, Hingna road, Nagpur (M.S.), India. ABSTRACT The present study aimed at the formulation development and evaluation of niosomal gel as a vesicular drug carrier system. Carvedilol has an oral bioavailability 25% due to high first pass metabolism (80%) and presence of food decreases its absorption rate in gastrointestinal tract. Short half life of the Carvedilol (4 to 6 h) indicates need of controlled release delivery. To overcome these limitations Carvedilol was incorporated in niosomes for transdermal delivery. Niosomal drug delivery system was preferred due to improved stability and it carries significant quantity of drug across the skin thus enhancing the systemic absorption of drug. Niosomal gels were prepared by thin film hydration method using different ratios of span60 and cholesterol. Optimization of these formulations was done by 3 2 full factorial designs. The niosomal gel formulations of Carvedilol were characterized for vesicles size, entrapment efficiency, in vitro release study, stability study, ph, viscosity and drug content uniformity of the gel. The effects of span60 and cholesterol on entrapment and release profile of Carvedilol were also assessed. Transdermal patch of the above formulation was prepared using different polymers such as Eudragit RS 100 and PVP which is used as device to hold the gel formulation. Optimized formulation gave entrapment upto 65.42% and release 94.38% upto 12 h. Thus niosomes was found to be a promising carrier system for Carvedilol because of ease in preparation and stability for prolonged period. Key words: Carvedilol, Transdermal, Niosome, Optimization. INTRODUCTION Transdermal delivery of drug through skin to the systemic circulation provides convenient root of administration for variety of clinical indications. For transdermal delivery of drug, stratum corneum is main barrier layer for permeation of drug. Hence to increase the flux through the skin membrane, different approaches of penetration enhancement are used. Drug vehicle based enhancement methods such as liposomes, prodrugs and ion-pair are used in transdermal research as better Corresponding Author U. D. Shivhare udshivhare@gmail.com alternative methods to enhance the permeation of drug through skin. Many approaches involving chemical penetration enhancement are extensively studied but vesicle based enhancement approach is not exploited too much (Flynn GL et al., 1985). Niosomes are a novel drug delivery system, in which vesicles are microscopic lamellar structures formed on admixture of non ionic surfactant of the alkyl or dialkyl polyglycerol ether class and cholesterol with subsequent hydration in aqueous media. The vesicles is composed of bilayers of nonionic surface active agents and hence the name niosomes. Niosomes can entrap both hydrophilic and lipophilic drugs, either in aqueous layer or in vesicular membrane made of lipid material. It is reported to attain better stability than liposomes. It can prolong the circulation of entrapped drugs. Because of
2 232 the presence of non ionic surfactants with the lipid, there is better targeting of drugs to the tumor, liver and brain. It may prove very useful for targeting the drug for treating cancer, parasitic, viral and other microbial disease more effectively (Alok N et al., 1996). Hypertension is a very common disorder, particularly past middle age. It is not a disease in itself, but is an important risk factor for cardiovascular mortality and morbidity. The cutoff manometric reading between normotensive and hypertensives is arbitrary. For practical purposes hypertension could be that level of BP at or above which long-term antihypertensive treatment will reduce cardiovascular mortality. The JNC7* (2003) and guidelines (2003) have defined it to be 140 mmhg systolic and 90 mmhg diastolic, though risk appears to increase even above 120/80 mmhg. Epidemiological studies have confirmed that higher the pressure (systolic or diastolic or both) greater is the risk of cardiovascular disease (Tripathi KD, 2008). MATERIALS AND METHODS Materials Carvedilol was Gift sample from Zim Laboratories, Kalmeshwar, Nagpur. Eudragit RS-100 gift sample from Evonik Degussa Pvt., Ltd., Mumbai. All other ingredients used throughout the study were of analytical grade and were used as received. METHOD OF PREPARATION OF NIOSOMES Thin film hydration method The accurately weighed quantities of surfactant (span-60) and cholesterol in different ratios were dissolved in 10 ml of the chloroform: methanol mixture (2:1v/v) along with Carvedilol in a round-bottom flask. The organic solvents were removed under vacuum in a rotary evaporator at 50 0 C for 1 hour to form a thin film on the wall of the flask and kept under vaccum for 2 hours to ensure total removal of trace solvents. The hydration of the film was carried out using 10 ml ph 7.4 phosphate buffer at 60 0 C for 1 hour which is above the gel liquid transition temperature (Tc) of surfactant. The resulting suspension was mechanically shaken for 1 hour using the horizontal mechanical shaking water bath. The dispersion was left for 4 hours at room temperature for complete hydration and stored at 4 0 C overnight before use, and then it was sonicated in 3 cycles of 1min on and 1min off leading to the formation of multilamellar niosomes(azeem A et al., 2008). EVALUATION OF PREPARED NIOSOMES Formulation of preliminary trial batches and their optimization by entrapment efficiency Primary trial batches of niosomal formulation were prepared for optimizing the formulation components like span 60, cholesterol and drug concentration (Table 1). Microscopic study The microscopic study of niosomes was performed by using motic digital microscope model no. (DMW 201). The niosomal suspension was placed on a glass slide covered with the cover slip and examined for the structure under microscope at magnification power of (10X and 45X) and photomicrographs were recorded (Mokhtar M et al., 2008). Vesicle size analysis The particle size of niosomes was determined by using motic digital microscope. All the prepared batches of niosomes were viewed under microscope to study their size. Size of niosomal vesicles from each batch was measured at different location on slide by taking a small drop of niosomal dispersion on it and average size of niosomal vesicles were determined (Mokhtar M et al., 2008). Determination of entrapment efficiency (%) Entrapment Efficiency of niosomal formulation was determined by centrifugation method. Aliquots of niosomal dispersion subjected to centrifugation on a laboratory centrifuge (Remi R4C) at 3500 for a period of 2 hr. The clear supernatant was seperated, filtered and sufficiently diluted with ethanol and the absorbance recorded at 241nm with UV-Visible spectrophotometer. The percentage of drug entrapment was calculated(moazeni E et al., 2010). Percentage entrapment of the drug was calculated by the by the following formula: % Drug Entrapped (PDE) = Amt. Of drug in sediment 100 Total amount of drug In-vitro release study The In-vitro diffusion study was performed in a modified Franz diffusion cell of capacity 15 ml using cellophane membrane. The cellophane membrane was activated by keeping it in the mixture of phosphate buffer 7.4 and ethanol for overnight. A section of membrane was cut, measured, and placed on the receiver compartment. The donor compartment was filled with niosomal formulation. A 15 ml of aliquot of 4:6 (v/v) Ethanol: ph7.4 phosphate buffer was used as receptor medium. The receptor medium was maintained at 37 0 C and stirred by a magnetic bar at 300 rpm. Aliquots of 0.5 ml of the receptor medium were withdrawn and immediately replaced by equal volume of fresh receptor solution at appropriate interval of an hour up to 12 h. The 0.5 ml sample is diluted to 10 ml receptor medium and observed spectrophotometrically at λ max 241 nm (Ning M, 2005). Optimization The runs or formulations, which are designed based on 3 2 full factorial designs, are evaluated for the
3 233 response variables. The response values are subjected to multiple regression analysis to find out the relationship between the factors used and the response values obtained. The response values subjected for this analysis are; 1. Entrapment efficiency (%) 2. In-vitro cumulative drug release (%) Solution obtained from the design expert software was used for optimization of % cumulative drug release and % entrapment efficiency. Above solution was used for validation of optimized formulation on basis of desirability. Five different batches of formulation were prepared and studied for evaluation % entrapment efficiency and % cumulative drug release. Stability study The optimized niosomal formulation OF22 were sealed in 30 ml clear glass vials and kept for stability study at refrigeration temperature (4 ± 2 0 C), room temperature (25 ± 2 0 C), and hot condition (45 ± 2 0 C). After 7, 15, 30, 45 days, the entrapment efficiency of each sample was determined and compared to the freshly prepared niosomes (Hao Y et al., 2002). PREPARATION OF GEL The 0.6 g of carbopol 934 was dispersed in the small quantity of distilled water to prepare an aqueous dispersion. The dispersion was allowed to hydrate overnight and subjected to stirring. The required quantity of niosomal suspension was incorporated under gentle stirring. The quantity of methyl paraben and propyl paraben equal to 0.2 and 0.02 g respectively were solubilized in small amount of water and incorporated in dispersion. The final weight of gel was optimized by distilled water. The ph of the gel was adjusted to by addition of triethanolamine. The gel was allowed to stand overnight to remove entrapped air. PREPARATION OF COMPLETE DEVICE (PATCH) Transdermal patch was prepared by mercury substrate method using different ratios of polymer and plasticizer. Polymeric solution (10 % w/v) was prepared dissolving Eudragit RS 100 and PVP K 30 in different ratios into isopropyl alcohol: dichloromethane (40:60) ratio. 130 mg of PEG 400 (13 % w/v) was added to this solution as a plasticizer and finally poured on mercury substrate glass petridish. The rate of evaporation of solvent was controlled by placing the inverted funnel over the petridish. The solvent was allowed to evaporate at 50 0 C for 6 h. The O ring shaped spacer was used to hold the gel in the device. The spacer was adhere to the protective impermeable backing membrane using an adhesive to obtain an empty device. The spacer was filled with appropriate quantities of niosomal gel containing carvedilol (as prepared above). Aluminium foil was used as a backing membrane and wax paper as a release liner. EVALUATION OF GEL FORMULATION IN PATCH Consistency and clarity After preparation, the gel formulations were visually inspected by naked eyes. The consistency and clarity of the gel formulation were observed. Viscosity The viscosity of the gel formulations were determined by Brookfield viscometer using spindle no. 7. The gel sample was taken in a beaker and the dial reading was noted at 100 rpm for 60 sec and at temperature of 30 0 C. ph determination The ph of the gel formulation containing Carvedilol was measured using digital ph meter. The gel formulations were diluted in ratio 1:25 using distilled water. Standard buffer solution of ph 4, 7 and 10 were used for calibration of ph meter. The gel formulation was tested in triplicate to obtain mean ph value. The diluted gel was in contact with ph electrode for 10 minutes to allow the ph values to stabilize. The electrode was thoroughly washed between each sample (Head AA et al., 2008). Drug content uniformity Fixed quantity (50 mg) of the gel samples were collected from different sites of transdermal patch and accurately weighed into a 10 ml volumetric flask each. Around 8ml receptor medium was added and flask was shaken vigorously to disperse the gel. The sample was then subjected to sonication for about 10 minutes to effect complete extraction of the drug. Volumes were made then to 10 ml and then these stock solutions were filtered and further diluted. These samples were analyzed by UV spectroscopy (Panigrahi L et al., 1997). Skin irritation test Skin irritation is defined as a nonimmunological local inflammatory reaction which is usually reversible and characterized by erythema and oedema, following single or repeated application of a chemical to the same cutaneous site. Skin irritation test was performed to evaluate the deleterious effect of the transdermal patch that may have on the skin when applied on the skin. Areas of the back of the rats were clean shaved 24 h prior to testing. Transdermal patch was applied to the cleaned areas and held in its place by glue tapes. After every h, up to 8 consecutive h, the transdermal patch was removed and observed visually for any signs of oedema or erythema and scored according to Draize s scoring index. Transdermal patch without drug was used as control patch (CDER, 1999).
4 234 Table 1. Effecet Of Variables At Different Concentration On Entrapment Eficiency Formulation code Drug (mg) Span 60 (mg) Cholesterol (mg) Entrapment (%) F ±0.06 F ±0.21 F ±0.34 F ±0.51 F ±0.25 F ±0.42 F ±0.15 F ±0.38 F ±0.10 F ±0.07 F ±0.20 F ±0.19 Table 2. Factors And Levels Of Variables Factors Low level (-1) Medium level (0) High level (1) Span Cholesterol Table 3. Composition Of Niosomal Formulation Using 3 2 Factorial Designs Formulation code Carvedilol Span 60 Cholesterol F F F F F F F F F All quantities in mg Table 4. Mean Particle Size Sr. No Formulation Mean particle size (µm)±s.d. 1 F ± F ± F ± F ± F ± F ± F ± F ± F ±0.07 * All values are expressed as mean ± SD (n=3) Table 5. % Entrapment Efficiency And % Cumulative Drug Release Formulation % Entrapment Efficiency % cumulative drug release F ± ±0.27 F ± ±0.45 F ± ±0.25 F ± ±0.34 F ± ±0.52
5 235 F ± ±0.16 F ± ±0.27 F ± ±0.45 F ± ±0.62 * All values are expressed as mean ± SD (n=3). Table 6. Annova for the selected factorial model (% entrapment) Source Sum of Square Df Mean square F Value p-value Prob>F Model Significant A-Span B-Cholesterol AB A B Residual Cor total Table 7. Annova For The Selected Factorial Model (% Cumulative Release) Source Sum of Square Df Mean square F Value p-value Prob>F Model Significant A-Span B-Cholesterol AB A B Residual Cor total Table 8. Solution Of % Entrapment Efficiency And % Cumulative Release For Optimization Number Span 60 (mg) Cholesterol (mg) %Entrapment Efficiency %Cumulative Release Desirability Table 9. % Entrapment Efficiency and % Cumulative Drug Release For Optimized Formulation (Of22) Formulation code Entrapment Efficiency (%) Cumulative drug release (%) OF22-A 63.96± ±0.24 OF22-B 64.71± ±0.44 OF22-C 64.67± ±0.26 OF22-D 63.88± ±0.31 OF22-E 65.42± ±0.60 * All values are expressed as mean ± SD (n=3) Table 10. Viscosity and ph determination of gel formulation Sr. No Evaluation parameter Mean 1 Viscosity 6300± ph 6.70±0.020
6 236 GRAPHICAL ANALYSIS Graphical analysis of % Entrapment efficiency Fig 1. Effect of factors on % entrapment efficiency Fig 2. Surface response plot of % entrapment efficiency Graphical analysis of % cumulative drug release Fig 3. Effect of factors on cumulative drug release (%) Fig 4. Surface response plot of cumulative drug release (%) Fig 5. Stability study of formulation OF22
7 237 RESULT AND DISCUSSION Microscopic study of the niosomal formulation was performed by using motic digital microscope which revealed the formation of vesicular structure with entrapped Carvedilol. Particle size analysis by microscopy showed the particle size 4.18 µm of niosomal formulation F17 i.e. it forms multilamellar vesicles. As the concentration of cholesterol increases there is increase in the particle size due to formation of rigid bilayers structure but upto a specific concentration. The entrapment efficiency was found to be maximum with formulation containing span 60 and cholesterol in the ratio 80:60 (F17). Formulation F17 containing 80 mg of span 60 and 60mg cholesterol showed drug entrapment of 66.23% with satisfactory stability and rigidity. However, when span 60 was further increased to 90 mg keeping cholesterol in low concentration there was decrease in % drug entrapment with slight decrease in stability and rigidity of niosomal structure. From the results of In-vitro release study it was observed that, the order of release was found to be: F13<F15<F19<F14<F21<F20<F16<F18<F17. The drug release kinetics showed majority of the batches governed by peppas model. All the batches showed release up to 12 h and above 79% of drug released with each formulation. Formulation F17 (96.24%) showed maximum release while other formulation showed less amount of drug release in 12 h. Formulation F17 has highest correlation coefficient ( R 2 =0.9697) value and follows drug release by peppas model. The runs or formulations, which are designed based on 3 2 full factorial designs, are evaluated for the response variables. The response values are subjected to multiple regression analysis to find out the relationship between the factors used and the response values obtained. The response values subjected for this analysis are; 1. Entrapment efficiency (%) 2. In-vitro cumulative drug release (%) In case of Entrapment efficiency response, the Model F-value of implies the model is significant. Values of "Prob > F" was which is less than indicate model terms are significant. In this case A, B, AB are significant model terms. In case of % cumulative response, The Model F- value of implies the model is significant. Value of "Prob > F" was which is less than indicate model terms are significant. In this case A, B, AB are significant model terms. Final Equation in Terms of Coded Factors R1- % Entrapment Efficiency = *A *B *A*B *A *B 2 Final Equation in Terms of Actual Factors R1-%Entrapment Efficiency = * span * cholesterol * span60* cholesterol * span * cholesterol 2 Final Equation in Terms of Coded Factors R2- % Cumulative Release = *A *B 0.040*A*B 9.55*A *B 2 Final Equation in Terms of Actual Factors R2-% Cumulative Release = * span * cholesterol * span60* cholesterol * span * cholesterol In the present study, the optimized formulation OF22 was selected for stability studies. Stability studies of drug formulation performed to ascertain whether the drug undergoes degradation during its shelf life. From the stability data it can be concluded that the drug is stable in the optimized formulation for the study period. Validation was performed for OF22 formulation. The niosomal formulation was incorporated in carbopol gel and this gel was placed on transdermal patch which was used as device to hold the gel formulation. This was then evaluated for ph, consistency and clarity, viscosity, drug content and skin irritation test. The entire evaluated test shows good results. CONCLUSION From all experiments we can be concluded that, Entrapment increases with increase in concentration of span 60 upto 80 mg. Entrapment increases with increase in concentration of cholesterol upto 60 mg. Formulation have maximum stability at 4 0 C while better stability at 25 0 C. Transdermal patch of formulated niosomal gel with faster onset of action can be fabricated. Thus the drug in novel delivery system form i.e. niosomal form deliver drug at faster rate and for prolonged period of time and also may be of commercial value if progressed accordingly. REFERENCES Alok N, Jain NK. Niosomes as drug carriers. Ind J Pharm Sci. 1996; 58(2): Azeem A, Ahmad FJ, Khan ZI, Talegaonkar S. Non-ionic surfactant vesicles as a carrier for transdermal delivery of frusemide. J Dispers Sci Tech. 2008; 29: Flynn GL, Maibach HI, Bronargh RL. In percutaneous absorption. Eds. New York, Marcel Dekkar, Inc. 1985; Hao Y, Zhao F, Li K, Yang Y. Studies on high encapsulation of colchicine by a niosome system. Int J Pharm, 2002; 244:
8 238 Head AA, Puranik PK. Formulation development and evaluation of transdermal patches of antiemetic drug. Government college of pharmacy, Aurangabad, 2008; Moazeni E, Gilani K, Sotoudegan F, Pardakhty A, Galendari R. Formulation and evaluation of ciprofloxacin containing niosomes for pulmonary delivery. J Microencap. 2010; Mokhtar M, Sammour OA, Hammad MA, Megrab NA. Effect of some formulation parameters on flubiprofen encapsulation and release rates of niosomes prepared from proniosomes. Int J Pharm. 2008; 361: Ning M. Preparation and in-vitro and in-vivo evalution of liposomal/niosomal gel delivery system for clotrimazole. Drug Dev and Ind Pharm. 2005; 31: Panigrahi L, John T, Shariff A, Shobanirani RS. Formulation and evalution of lincomycin hydrochloride gels. Ind J Pharm Sci. 1997; 59(6): Skin irritation and Sensitization Testing of Generic Transdermal Drug Products. U. S. Department of Health and Human and Services. Food and Drug Administration Centre for Drug Evalution and Research (CDER), 1999, 1-8. Tripathi KD. Essentials of medical pharmacology, 2nd ed., Jaypee Brothers Publishers Ltd., Delhi, 2008, 53.
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