International Journal of Innovative Pharmaceutical Sciences and Research

Transcription

1 International Journal of Innovative Pharmaceutical Sciences and Research FORMULATION, DEVELOPMENT AND EVALUATION OF NON-IONIC SURFACTANT BASED ORGANOGEL FOR TRANSDERMAL DELIVERY OF ACYCLOVIR 1 Varsha Gupta*, 2 Meenu Nagpal, 3 Geeta Aggarwal, 4 Rupinder Kaur, 5 Sukhdev Singh, 6 Tapan Behl, 7 Upendra Kumar Jain Department of Pharmaceutics, Chandigarh College of Pharmacy, Landran, Mohali, Punjab, INDIA Abstract Transdermal drug delivery provides the promising delivery system for anti-viral drugs like Acyclovir. Acyclovir appears to be more active and effective as an antiviral agent than other molecules. Acyclovir has advantageous for topical treatment of Herpes Simplex Virus and Varicella-Zoster Virus infection that occurs at epidermis. NOs were evaluated for their physicochemical properties and in vitro release was carried out using modified Keshary-chien diffusion cell in saline phosphate buffer ph 7.4. Various materials such span 80, tween 80 and sunflower oil used to enhance the skin permeation of Acyclovir. The stability and the properties were found to be dependent on the proportion of the surfactant mixture and water. In general, when the ratio of SM: water was in the range of , the samples showed higher stability and improved properties.stability studies of NOs proved that there were no significant change in physical stability, drug content, ph and viscosity. Keywords: Non-ionic surfactant based organogels (NOs), transdermal drug delivery, Acyclovir, skin permeation study. Corresponding Author: Varsha Gupta Department of Pharmaceutics Chandigarh College of Pharmacy, Punjab, INDIA guptavarshaji@gmail.com Phone: Available online: July Issue 1297

2 INTRODUCTION: Transdermal drug delivery to the systemic circulation excels itself by avoidance of hepatic firstpass metabolism, potential of long-term controlled release with avoidance of the typical peak trough plasma-profiles associated with frequent dosage regiments, ease of administration and possibility of immediate withdrawal of the treatment. Transdermal drug delivery system (TDDS) provides a means to sustain drug release as well as reduce the intensity of action and thus reduce the side effects associated with its oral therapy. Transdermal drugs are self-contained, discrete dosage form. It delivers a drug through intact skin at a controlled rate into the systemic circulation. Delivery rate is controlled by the skin or membrane in the delivery system Drugs can be delivered across the skin to have an effect on the tissues adjacent to the site of application (topical delivery) or to have an effect after distribution through the circulatory system (systemic delivery). A gel may be defined as a semi-solid formulation having an external solvent phase, a polar (Organogels) or polar (hydrogel), immobilized within the spaces available of a three dimensional networked structure [1-7]. In the current review, attempts will be made to have an insight on the mechanism of formation and applications of the organogels as a delivery system. The organogels may be regarded as bi-continuous systems consisting of gelators and a polar solvent, which may or may not contain water-molecules entrapped within the self-assembled structures of the gelator. The gelators, when used in concentration < 15 % (approx.), may undergo physical or chemical interactions so as to form self-assembled fibrous structures which get entangled with each other resulting in the formation of a three-dimensional networked structure. The three-dimensional networked structure, hence formed, prevents the flow of external a polar phase [1, 8]. Some common examples of gelators include sterol, sorbitan monostearate, lecithin and cholesterol anthraquinone derivates. The thermo-reversible property of the organogels has generated much interest for the potential use of the organogels as drug delivery system. The thermodynamic stable nature of the organogels has been attributed to the spontaneous formation of fibrous structure by virtue of which the organogels reside in a low energy state. The occurrence of the gel-to-sol transition above room-temperature indicates that external energy has to be supplied to the organogels so as to disrupt the three-dimensional structure and subsequent transformation of the gelled state to the sol state. Apart from the temperature sensitivity, organogels are also sensitive to the presence of moisture which has also been explored to develop controlled delivery systems [9]. Acyclovir is a synthetic acyclic purine Available online: July Issue 1298

3 nucleoside analog that is currently used for the prevention and treatment of Herpes Simplex Virus (HSV) and Varicella-Zoster Virus (VZV) infection that occurs at epidermis [10]. The oral bioavailability of acyclovir is low, variable and species dependent. The pharmacokinetic properties of acyclovir are well established. The effects of dosage size on the extent of oral absorption are not well understood. Some reports suggest from the gastrointestinal tract may be a saturable, dose dependent process [11]. Acyclovir is also categorized class III drug according to the Biopharmaceutical Classification System (BCS) because of its high solubility and low permeability [12]. The US Food and Drug Administration (FDA) guidelines reported that a drug substance is considered highly soluble when the highest dose strength is soluble in 250 ml or less of the aqueous media over the ph range 1 to 7.5. The intrinsic solubility of acyclovir was 1.2mg/ml as measured by the acid base titration method. Acyclovir has dissociation constant (pka) of 2.34 and 9.23 and partition coefficient (P- Octanol) of This low partition coefficient may reflect the low membrane permeability of acyclovir. In humans, acyclovir showed poor and variable oral bioavailability (15%-30%), probably due to the relatively low lipophillicity of the drug. So the absorption rate limiting step is its poor permeability. Also systemic administration of acyclovir may cause mild to severe side effects for example nephrotoxicity. The availability of topical formulation is therefore desirable. The epidermal application is needed of acyclovir for the effective treatment of topical localized herpetic lesions. In the present study efforts were made to develop topical epidermal gel for treatment of HSV and VSV. MATERIALS AND METHODS Materials Acyclovir was a kind gift from Torrent Pharmaceuticals Pvt. Ltd., India. Span 80 (sorbitan monooleate) was procured from Loba chemie, Mumbai, India. Tween 80 (polyoxyethylene sorbitan monooleate), Edible sunflower oil was purchased from the local market. Equipments Melting point apparatus (PERFIT, India), Bath Sonicator (PCI analytical), Electronic weigh balance (Shimadzu, Japan), France diffusion assembly (Borosil), ph meter (Labindia Ltd., Mumbai,India) UV visible spectrosc (Shimadzu, Japan) vortex type mixer (PERFIT, India) Acyclovir characterization Available online: July Issue 1299

4 Characterization of acyclovir was done by development of calibration curve and also preformulation studies. This was mainly done to ensure that the acyclovir which is to be used is of the optimum quality and its properties could be judged. Calibration curve of Acyclovir Calibration curve of Acyclovir: Calibration curve was prepared for the acyclovir these were Methanol and Phosphate buffer (ph 7.4). Methanol 10mg was dissolved in 100ml methanol to prepare stock solution. From this stock solution were withdrawn samples for the range of 4ug/ml-40ug/ml. The calibration curve was formed using UV spectrophotometer. The base line was corrected and lambda max was determined on UV spectrophotometer (254nm). Absorbance of drug at different concentrations was calculated and graph was plotted. Phosphate buffer ph 7.4 For determining the absorbance maxima using phosphate buffer having ph 7.4, dissolve 10mg acyclovir in 100ml volumetric flask then take out 1ml volume up to 100ml with buffer now from this sub-stock prepared make dilutions in the range of 4ug/ml-40ug/ml. The calibration curve was formed using UV spectrophotometer. The base line was corrected and lambda max was determined on UV spectrophotometer (254nm). Absorbance of drug at different concentrations was calculated and graph was plotted. Pre-formulation Studies Before starting with the formulation development the pre-formulation studies were conducted to characterize the drug and the selection of excipients was also based on the pre-formulation studies. It mainly involved two parameters organoleptic and physicochemical properties of the API used. This was mainly done to check the purity of the drug and any deviation could also help to know if there is any detoriation involved. a) Organoleptic properties (Acyclovir) Appearance Colour Odour Available online: July Issue 1300

5 All the above studies were carried out by using no special equipment these were done by visual assessment. b) Physicochemical Properties (Acyclovir) Melting point: For determination of melting point of acyclovir a melting point apparatus of capillary type was used. The capillary filled with acyclovir was inserted and the temperature at which the acyclovir started to melt was noted down as melting point of the acyclovir. Partition coefficient: Shake flask method was used for determination of partition coefficient of acyclovir. This is a classical and most reliable method of log P determination. Partition coefficient is a measurement of drug s lipophilicity and its ability to cross cell membrane. Partition coefficient of acyclovir was determined at by taking 5ml of octanol and 5ml of methanol. About 10 mg of drug was added to this solution and was shaken.after shaking the system remained undisturbed for 24 hour. Two layers were separate through separating funnel and filterer through Whatman grade filter, and the amount of acyclovir solubilised, was determined by measuring the absorbance at 254nm against reagent blank through double beam UV/Vis spectrophotometer in both the solution. Partition coefficient was determined as ratio of concentration of drug in octanol to the concentration of drug in phosphate buffer (ph 7.4) and the value were reported as log P. Solubility Studies: K o/w = Concentration of drug in non aqueous phase Concentration of drug in aqueous phase These pre-formulation studies formed the most important part of the formulation development; as it formed the base for selection of excipients which had high solubility of the API. The procedure followed was the shake flask method. The solubility of acyclovir in various solvents was determined by dissolving excess amount of API in 2ml of each of the selected solvents in 5ml capacity stoppered vials separately. Each glass vial was then mixed for 10 min using a vortex mixer. The mixture vials were then kept at 37±1.0 C in a shaker bath for 72 h to get equilibrium. The equilibrated samples were removed from shaker and centrifuged at 3000rpm for Available online: July Issue 1301

6 15 min. The supernatant was taken and filtered. The concentration of acyclovir was determined in each solvent by UV spectrophotometer by with UV at nm (wavelength 254nm) [14]. Method of preparation Span 80, tween 80 and sunflower oil were mixed thoroughly in the proportion of 1:2 ratios (w/w) to obtain the surfactant mixture, which was used as gelator. Specified amount of the surfactant mixer was added to the sunflower oil, kept on stirring on a magnetic stirrer. The above mixture (gelator solution) was stirred for 20 min and add acyclovir drug. Subsequently, water was added drop-by-drop to the gelator solution using a burette until there was a formation of organogel or the total fraction of water has reached 80% of the volume of the gelator solution water mixture. Depending on the composition of the gelator solution-water mixture, the system either formed gelled structures or remained as liquid mixtures. A ternary plot depicting the proportions of surfactant mixer, water and sunflower oil was prepared to figure out the compositions, which formed organogels [15]. Evaluation of organogels was carried out by using following methods: Microscopic Study To understand the mechanism of organogels formation, water was slowly added to the until the formation of the organogel. The samples were observed under compound microscope as the proportion of water was varied. The microstructures were analyzed using Image. Gel-sol transition study Gel-sol transition temperature (Tgs) was found out by incubating the organogels in a water bath, whose temperature was varied from 30-70ºC. The temperature, at which the gels started to flow, when the glass vials were inverted, was noted as the gel-sol transition [16]. ph measurement: The ph of the organogels was measured using a digital ph meter. The ph was figure whether the ph of the organogels lies within the limits of skin ph [17]. FTIR Fourier Transform Infrared spectroscopy (FT-IR): measured to The compatibility between acyclovir and excipient was detected by IR spectra obtained on Perkin Elmer 1600 series, (USA). The spectra were recorded over the wave number range of 4000 to 500 cm-1. Drug content Drug content was the important criterion for optimization of formulation as it indicates the amount of drug entrapped of organogel. The solution was filtered through Whatman filter paper (No.1). The Available online: July Issue 1302

7 above filtrate (1 ml) was pipetted out and diluted to 10 ml with methanol. The sample was analysed with UV visible spectrophotometrer against blank at λmax254 nm. The drug was estimated using standard calibration curve constructed in the same solvent this study was carried out by diluting each ternary mixture with methanol and then centrifuged for 2 hrs at rpm which was then analysed using UV spectrophotometer [18]. RESULT AND DISCUSSION Calibration curve of Acyclovir Calibration curve was preparing Methanol and Phosphate buffer (ph7.4). Determination of absorbance maxima: Methanol- 10mg was dissolved in 100ml methanol to prepare stock solution. From this stock solution samples were withdrawn for the range of 4μg/ml-40μg/ml. The dilutions were prepared by using different concentrations as 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, and 40. The base line was corrected and it was scanned by using UV spectrophotometer by in the range nm. Absorbance of drug at different concentrations was calculated and graph was plotted. Fig 1: Calibration curve in methanol Phosphate buffer ph 7.4: For determining the determined in span 80, tween 80 and sunflower was found to be 18.72, and mg/ml respectively. Absorbance maxima using phosphate buffer having ph 7.4, 10mg API was dissolved in 100ml volumetric flask. From this stock solution samples were withdrawn for the range of 4ug/ml-40ug/ml. The dilutions were prepared by using different concentration as 0, 4, 8, 12, 16, 20, 24, 28, 32, 36 and 40. The base line was corrected and it was scanned by using UV spectrophotometer in the range of nm. Absorbance of drug at different concentrations was calculated and graph was plotted. Available online: July Issue 1303

8 Fig 2: Calibration curve in phosphate buffer ph 7.4 Pre-formulation studies The Preformulation studies were carried out for the acyclovir in use which involved the organoleptic and physicochemical properties of the drug. The results obtained were used in the formulation development process. a) Organoleptic properties: It was observed as a granular powder which was white in colour having no odour. b) Physico-chemical properties: The physicochemical properties were analysed to estimate its ability to be used in the formulation. The various properties studied were as follows Melting point: Melting point of Acyclovir was found to be C which is in accordance with the standard melting point of acyclovir. Drug Melting point observed Reported value Acyclovir C C Partition coefficient: Partition coefficient was determined as ratio of concentration of drug in octanol to the concentration of drug in water and the value were reported as log P. Drug Partition coefficient observed Reported value Acyclovir Available online: July Issue 1304

9 Solubility studies: Solubility of drug in various solvents, co-solvent and surfactants were carried out in order to screen for the components to be used for formulation development. Analysis of the drug was carried out on UV Spectrophotometer (Shimadzu UV-1601 UV/Vis) at 254 nm Phase studies While conducting the screening studies it was observed that 3 combinations involving three different co-surfactants which have same oil and surfactant combination have % Transmittance very close. Thus, it became necessary to finalize the final combination by construction of pseudo-ternary phase diagram which will indicate the highest region for formation of nanoemulsion will be the final combination selected having a fixed Smix ratio with fixed Smix ratio as 1:2 Ratio 1:1, 1:2, 2:1 Fig 3: Ternary diagrams for combination 1: span 80+ Tween 80+Sunflower oil From the Fig it can be concluded that the formation of organogel depends on the ratio s in which the oil, surfactant and co-surfactant are added. The pseudo-ternary diagram depicts the region (shaded as black) where there is the probability of formation of oraganogel ; from the fig. it s clear that in ratio 1:1,2:1 there is no formation of organogel and fig.depicts that in the ratio 1:2 the highest region of formation of organogel for this combination is formed. Hence, the ratio 1:2 was selected for this combination. Preparation of organogels The organogels were prepared by dissolving the surfactant mixer in sunflower oil followed by the addition of water. With the initial addition of water, the mixture turns into white turbid solution.as further amount of water was added, the samples either formed a gelled structure or remained as turbid solution. The samples were regarded as organogels, if the gelator solutionwater mixture did not flow when the culture bottles were inverted. The samples which formed Available online: July Issue 1305

10 gelled structures, released heat indicating an exothermic reaction as the gels were developed. This indicates that the samples attain a low energy state as they undergo transition into gelled structures and attain a thermodynamically stable state. Depending on the composition, colour of the gels varied from white to pale yellow. All the gels were opaque in nature. They were having slight odour and oily to touch. Fig 4: Organogel samples of different composition Evaluation of Organogel Microscopic study The microstructure of the gelator Solution was studied under microscope as different proportions of water were added to the gelator Solution. The micrographs suggested that gelator Solution showed some irregular structures. As water was added to the gelator Solution, there was formation of globular structures. There was an increase in the number of the globular structures as the proportion of water was increased in gelator Solution. The morphological analysis of the globular structure indicated that there was a decrease in the size of the globular as the proportion of water was increased in the gelator Solution. From the microscopic results it can be predicted that on addition of water in gelator Solution, there is a formation of globular reverse micellar structures having internal aqueous phase. Gel-sol transition analysis: The organogels were subjected to increasing temperature starting from 25ºC. An increment of 5ºC was made after 5 min of incubation at the previous temperature. The samples were considered to have undergone gel-sol transition, when they started to flow The gel-to-sol Available online: July Issue 1306

11 transition temperature of the organogels varied from 40ºC to 70ºC, depending on the composition of the organogels. as the temperature increased, there was a corresponding increase in the surface free energy with the subsequent increase in mobility of the self-assembled structures formed by the gelators. With further increase in temperature, the interactions amongst the self-assembled structures gets reduced which leads to the disruption of networked structure, thereby causing the gelled system to flow In general, the gel-sol transition was found to be >60ºC for the samples. S. No Sample Tgs (ºC) 1 A 50 2 B 60 3 C 65 ph measurement The ph of organogels was measured at 30ºC ± 2ºC by using electrode based digital ph meter. The ph values for all samples. The ph values of organogels varied in the range of 6.69 to The ph of the organogels were in accordance to the USP guidelines for topical and transdermal formulations. According to USP the ph of gels or ointments whichever to be used for topical or transdermal applications should lie within the limits of normal skin ph of FT-IR Fourier transform infrared spectroscopy indicates the presence of molecular interactions amongst the components present in a sample. Sample I was used as the representative blank organogel and sample ID was used as the drug loaded sample. The spectra of the samples I and ID organogels was found to be similar. A shallow broad peak was observed in the range of 3,700 cm-1 to 3,100 cm-1 wave numbers in both the cases suggesting the presence of stretched hydrogen bonded O-H groups in the samples. This indicates that presence of intermolecular hydrogen Available online: July Issue 1307

12 bonds amongst the water and the surfactants resulting in the formation of inverse tubular structures, which upon extension and physical crosslinking yields fluid matrix gel [19]. The spectra of sample ID indicated additional peak at 572 cm-1 in addition to the peaks at 468 cm-1 and 512 cm-1 which were also present in sample I organogel. CONCLUSION Preformulation studies on Acyclovir corroborate with the reported literature limits. The adopted method yielded translucent, smooth and viscous NOs with non-ionic surfactants. The viscosity, ph and drug content of NOs were uniform and reproducible. Decrease in viscosity was observed with subsequent increase in the concentration of penetration enhancers, irrespective of the sun flower oil used. Droplet size determination confirmed the formation of NOs and light microscopic studies revealed the existence of w/o type NOs necessary for better permeation of hydrophilic drug Acyclovir. FT-IR studies of NOs indicated no chemical interaction. The drug release through the human cadaver skin was directly proportional to the concentration of surfactant and inversely proportional to that of water. The absence of any allergic manifestations. NOs were found to be stable with respect to drug content, viscosity, and ph and release profile during the stability study period. Available online: July Issue 1308

13 REFERENCE 1. Vintiloiu, A. and J.-C. Leroux, Organogels and their use in drug delivery -- A review Journal of Controlled Release, (3): p Lim, P.F.C., et al., Physicochemical effects of terpenes on organogel for transdermal drug delivery. International Journal of Pharmaceutics, (1-2): p Pal, K., A. Banthia, and D. Majumdar, Biomedical evaluation of polyvinyl alcohol gelatin esterified hydrogel for wound dressing. Journal of Materials Science: Materials in Medicine, (9): p Pal, K., A.K. Banthia, and D.K. Majumdar, Polyvinyl Alcohol-Gelatin Patches of Salicylic Acid: Preparation, Characterization and Drug Release Studies. J Biomater Appl, 2006: p Pal, K., A.K. Banthia, and D.K. Majumdar, Preparation of Novel ph-sensitive Hydrogels of Carboxymethyl Cellulose Acrylates: A Comparative Study. Materials and Manufacturing Processes, (8): p Pal, K., A.K. Banthia, and D.K. Majumdar, Effect of heat treatment of starch on the properties of the starch hydrogels. Materials Letters, (2): p Pal, K., A.K. Banthia, and D.K. Majumdar, Polymeric Hydrogels: Characterization and Biomedical Applications. Designed Monomers & Polymers, : p Toro-Vazquez, J., et al., Thermal and Textural Properties of Organogels Developed by Candelilla Wax in Safflower Oil. Journal of the American Oil Chemists' Society, (11): p Wright, A. and A. Marangoni, Formation, structure, and rheological properties of ricinelaidic acid-vegetable oil organogels. Journal of the American Oil Chemists' Society, (6): p K.S. Erlich., West. J. Med., , S.A. Qureshi, M. Jiang, K.K. Midha & J.P. Skelly., J. Pharm. Pharm. Sci., , J. Luengo, T. Aranguiz & J. Sepulveda., J. Pharm. Sci., , Thomas NS and Panchagnula R. Transdermal delivery of Zidovudine: In vitro, exvivo and In vivo evaluation. Biopharm. Drug. Dispos : Jona JA, Dittert LW, Crooks PA and Hussain AA. Design of novel prodrugs for the enhancement of the transdermal penetration of Indomethacin. Int. J. Pharm : 127. Available online: July Issue 1309

14 15. Joshi H, Nand I and Bari M. Study of isopropyl myristate microemulsion systems containing cyclodextrins to improve the solubility of two model hydrophobic drugs. AAPS PharmaSci.Tech (1): Shaikh, I.M., et al., Aceclofenac Organogels: In Vitro and In Vivo Characterization. Current Drug Delivery, (1): p Bhatia, V.N. and R.H. Barber, The effect of ph variations of ointment bases on the local anesthetic activity of incorporated ethyl aminobenzoate. I. Hydrophilic ointment U. S. P. Journal of the American Pharmaceutical Association, (6): p Ahmed M, Manohara YN and Appala RS. Spectrophotometric determination of Aceclofenac in pharmaceutical dosage form. The Ind. Pharmacist : Available online: July Issue 1310