10.1 INTRODUCTION 10.2 EXPERIMENTAL WORK Preformulation Studies:

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1 10.1 INTRODUCTION Clotrimazole is an antifungal drug employed widely in the treatment of various fungal infections includes vaginal yeast infections, oral thrush, ringworm, athlete s foot, and jock itch, in case of both humans as well as other animals 1. It is also a promising agent for various diseases such as sickle cell anemia and cancer, anti-inflammatory effect in patients with rheumatoid arthritis and neuroprotectiveeffect. 2 It possesses broad antifungal activity against pathogenic yeasts as well as filamentous fungi. According to WHO, it is an effective antifungal agent in topical infections, safer than any other antifungal being listed in its essential drugs list currently and is generally recognized safe. 5 The commercially available cream of clotrimazole has the limitations of lower skin retention and deposition and poor residence ability at the targeted site. 2, 8 It has limitations such as poor water solubility, bioavailability, and the short half-life (2 h). Candida septicemia and urinary and pulmonary candidiasis have been cured with oral clotrimazole therapy. Results in other types of serious fungal infections, including pulmonary aspergillosis, have been disappointing. A limiting factor in oral clotrimazole therapy is the high incidence of gastro-intestinal disturbances and neurological reactions 9 Clotrimazole when administered orally exhibits poor bioavailability, due to low aqueous solubility and slow dissolution in water 3 Therefore to improve its bioavailability, an attempt was made to develop transdermal Clotrimazole. Clotrimazole topical preparations are generally well tolerated, but local irritation has necessitated withdrawal of therapy in a few cases. So, to overcome these limitations an attempt has been made to formulate transdermal gel and to optimize delivery through the skin upon the variation of different penetration enhancer EXPERIMENTAL WORK Preformulation Studies: Preformulation study is the preliminary step in the development of any drug delivery system. The objective of preformulation studies is to develop a portfolio of information about the drug substance to serve as a set of parameters against which detailed information design can be carried out. Melting point of the drug was analyzed Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 229

2 using digital melting point apparatus and observed value was compared with reported melting point. FT-IR spectral studies of (drug and excipients) were performed to analyze compatibility of drug and excipients. Drug-polymer purity and melting point were analyzed. A solution of CTZ was prepared in the phosphate buffer (ph7.4). λmax was determined by scanning the above solution between nm,using Shimadzu UV spectrophotometer. Standard plot of CTZ in phosphate buffer ph 7.4 Preparation of phosphate buffer solution (7.4): Dissolve 0.19gm of potassium dihydrogen phosphate, 2.38gm of disodium hydrogen phosphate and 8.0gm of sodium chloride in sufficient distilled water to produce 1000ml.Adjust the ph if necessary. Preparation of stock solution (100µg/ml): 10 mg of pure drug was taken in 10ml volumetric flask, volume was made up to marks with phosphate buffer (ph7.4)(stock solution A).5ml of the stock solution A was taken in another volumetric flask and volume was made up to 50ml with phosphate buffer 7.4(stock solution B i.e. 100µg/ml solution was obtained).from this solution,aliquots of 1.0ml, 2.0ml, 3.0ml, 4.0ml and 5.0ml were taken and diluted up to 10ml in order to get the concentration ranging from 10-50µg/ml. These concentrations were used to determine absorbance at ʎmax 262nm against blank using UV-VIS spectrophotometer Formulation of carbopol 940 gel containing 1% CTZ Procedure: About 0.1gm of CTZ was weighed and dissolved in 10gm of ethanol, to this solution, specified quantity of glycerin and propylene glycol was added and dissolved (solution A). Weighed quantity of carbopol 940 was added to distilled water, added permeation enhancer and stirred to dissolve the same. The solution was then neutralized and made viscous by addition of triethanolamine (solution B).Solution was then added drop wise in solution A with constant stirring and weight was made up to 100gm to get the final gel preparation.11 formulations were prepared without and with % of tulsi oil and oleic acid as permeation enhancers. Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 230

3 Table 10.1: Composition formula of carbopol 940 formulations by using different concentrations of different enhancers S. no. Composition CTZ Carbopol Propylene glycol 4 Polyethylene glycol Glycerin Peppermint oil Ethanol Triethanolami ne Distil. Water Tulsi oil Oleic acid Characterization method for Clotrimazole gel: The prepare carbopol gel, having different permeation enhancer were characterized by in-vitro dissolution studies (release rate). Physical appearance and homogeneity: Gels were tested for their appearance and presence of any aggregates. 14,15,16 Clarity: The clarity of various formulation was determined by visual inspection under black and white background and it was graded as follows; turbid:+,clear:++,very clear(glassy): Measurement of ph: The ph of various gel formulations were determined by using digital ph meter. 1gm of gel was dissolved in 100ml distilled water and store for two hours. The measurement of ph of each formulation as done in triplicate and average values are calculated. 13,14,15,16 Spreadability: 0.5gm gel was placed within a circle of 1cm diameter premarked on a glass plate over which a second glass plate was placed. A weight of 500gm Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 231

4 was allowed to rest on the upper glass plate for 5 min. The increase in the diameter due to spreading of the gel was noted. 17 Extrudability: The formulations were filled in to collapsible aluminium tubes. The tubes were pressed to extrude the 0.5 cm ribbon of the gel in 10 second and the extrudability of formulations was checked. 13 Drug content: A specific quantity(100mg) of gel was taken and dissolved in 100ml of phosphate buffer of ph 7.4.the volumetric flask containing gel solution was shaken for 2hr on mechanical shaker in order to get complete solubility of drug. This solution was filtered and estimated. 14,15,16 In-vitro dissolution studies Dissolution study was carried out across egg membranes by using USP apparatus- I, basket type for 8 hr and the rotating rate was 50 rpm. Phosphate buffer ph 7.4 and ethanol in the ratio (6:4), was used as medium (900 ml) and was maintained at 37 ± 0.50 C. Samples (5ml) were collected periodically at 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 hr and assayed for dissolution spectroscopically at 252 nm. Each sample was replaced with equal volume of fresh dissolution medium, and dissolution rate test was repeated thrice and average values were reported. In-vitro antifungal activity 11,12 The antifungal efficacy study against Candida albicans was determined by agar diffusion method employing cup plate technique. Sterile solutions of Clotrimazole in DMSO (standard solution) and the developed gel having the ph adjusted to 7.0, were poured into cups (0.1 ml of 0.1% w/v) bored into sterile malt yeast agar previously seeded with test organism. After allowing diffusion of the solutions for 2 h, the agar plates were incubated at 37 for 48 h. The zone of inhibition (ZOI) was measured and compared with that of pure drug. The entire operation was carried out in aseptic condition throughout the study. Each solution was tested in triplicate. Both positive and negative controls were maintained throughout the study. Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 232

5 10.3: RESULTS AND DISCUSSION: Preformulation Studies Description of drug: Sample of CTZ was found white and crystalline powder Melting point determination: Melting point was analyzed using digital melting point apparatus and found to be C λ max scanning: λ max scanning helps in identifying the drug s purity. The scanned graph was according to reported literature and hence confirmed that the obtained drug sample was clotrimazole. λ max =262nm FTIR Spectral Studies. Figure 10.1: λ max Scan of CTZ Figure 10.2: FTIR of pure drug Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 233

6 Calibration curve of clotrimazole in phosphate buffer (7.4) and ethanol Table 10.2: Calibration data of clotrimazole in phosphate buffer (7.4) S. No. Concentration (µg/ml) Absorbance Figure 10.3: Calibration curve of CTZ in phosphate buffer (ph 7.4) Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 234

7 The obtained R 2 value is high (0.998), close to 1. From this study, it was concluded that there was a good correlation between the experimental and theoretical values : Characterization of GEL: In vitro dissolution study was carried out for carbopol gel, having different permeation enhancers in phosphate buffer ph 7.4. All studies were carried out in triplicate and average values were recorded (table 10.3). Physical appearance and homogeneity All the gel formulations showed good homogeneity with absence of lumps.all gels were found to be transparent and were free from presence of particles. ph The ph of the gel formulations was in the range of 6.2 to 6.8, which lies in the normal ph range of the skin and would not produce any skin irritation. Spreadability Spreadability diameter for different formulations showed good spreadability i.e. gel is easily spreadable. Extrudability The extrudability of formulations was found to be good. Drug Content analysis The drug content of the gel formulations was in the range of to , showing content uniformity. In-vitro dissolution studies In vitro dissolution studies of 11formulations were performed using ph 7.4 phosphate buffer as medium and measuring drug concentration spectrophotometrically at 262 nm. The cumulative percent of drug release (% CDR) at different time intervals are shown in table Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 235

8 Table 10.3: Evaluation of formulated batches of carbopol gel Formula- Clarity Homogeneity ph Spreadi- Drug Extrudability tion Code bility Content (cm) analysis 1 ++ Homogenous Homogenous Homogenous Homogenous Homogenous Homogenous Homogenous Homogenous Homogenous Homogenous Homogenous NOTE: + Satisfactory, ++ Good, +++ Excellent Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 236

9 Table 10.4: % Cumulative Drug Release from formulation batches 1 6 Time (hr) % Cumulative drug release (% CDR)*±S.D ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.76 *Average of three readings Figure 10.4: Graphical representation of % CDR for batches 1 6 Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 237

10 Table 10.5: % Cumulative Drug Release from formulation batches 7 11 Time (hr) % Cumulative drug release (% CDR)*±S.D ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.00 *Average of three determinations Figure 10.5: Graphical representation of % CDR for Batches 7 11 Table 10.6: % Cumulative Drug Release of formulations batches Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 238

11 1-11 (In-vitro study) S. No. Formulation Code % CDR (in 8hr) Figure10. 6: Graphical representation of % CDR for formulation1 11 In-vitro antifungal activity Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 239

12 In the present study, optimized batches CG1 (Plain gel), CG5, CG10 and pure clotrimazole drug were screened for their antifungal activity. All the tested optimized formulations possessed variable antifungal activity against (Candida albicans). Positive controls produced significantly sized inhibition zones against the tested yeast. However, negative control produced no observable inhibitory effect against any of the test organism as shown in Table Table 10.7: In-vitro antifungal activity against Candida albicans of compounds transdermal gel and drug through agar with diffusion method Formulation Code Zone of inhibition %minimum inhibitory concentration Clotrimazole pure drug DMSO(Negative control) - - Figure 10.7: Comparison of % minimum inhibitory concentration On the basis of minimum inhibitory activity shown against the yeast, one formulation is having tulsi oil 5 and other formulation is consisting of 10, was found to be Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 240

13 most effective with zone of inhibition of 24.6 mm. However in case of test control (pure Clotrimazole) zone of inhibition was found 27.6 mm (Table-10.7 and Fig.17). MIC of 5 showed good result than CD1 plain gel formulations. 5 showed good activity against the tested fungal pathogen, thus this formulation can be further used as an antimicrobial agent in pharmaceutical industry, after testing its toxicity. Figure 10.8: Photographs showing zone of inhibition on agar plate CONCLUSION: Clotrimazole is a broad spectrum imidazole antifungal drug. It is lipophilic in nature. It crosses the SC but for permeation through viable epidermis which is hydrophilic in nature it needs permeation enhancers. From above study it was concluded that tulsi oil (10.5%) gave best result. Tulsi oil is natural antiseptic and anti-inflammatory agent which agonistically enhanced the result. In-vitro antifungal activity as compared to pure drug, gel without permeation enhancer and DMSO as negative control revealed further the same findings. Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 241

14 REFRENCES: 1. Mallu UR, Reddy KH, Bobbarala V, Penumajji S. Determination of beclomethasonedipropionate, clotrimazole, chloramphenicol and lidocaine in pharmaceutical formulations using a novel RP-HPLC method. Int J Pharmacol Biol Sci. 2011; 2(3): Ning M, Gu Z, Pan H, Yu H, Xiao K. Preparation and In vitro evaluation of liposomal/niosomal delivery systems for antifungal drug clotrimazole. Indian J Exp Biol. 2005;43(2): Pedersen M. Effect of hydrotropic substances on the complexation of clotrimazole with b-cyclodextrin. Drug Development and Industrial Pharmacy. 1993; 19: Burgess MA, Bodey GP. Clotrimazole (Bay b 5097): In vitro and clinical pharmacological studies. Antimicrob Agents Chemother. 1972;2(6): Bero LA. Review of application of clotrimazole for topical or intravaginal use in vulvovaginal candidiasis. WHO Reproductive Health Guidelines. 2005: Spiekermann PH, Young MD. Clinical evaluation of clotrimazole. A broadspectrum antifungal agent. Arch Dermatol. 1976; 112(3): Mahgoub ES. Clinical trial with clotrimazole cream in dermatophytosis and onychomycosis.mycopathologia ; 56(3): Hashem FM., Shaker DS., Ghorab MK., Nasr M., Ismail A. Formulation, characterization and clinical evaluation of microemulsion containing clotrimazole for topical delivery.pharm Sciences Tech. 2011; 12(3): Phyllis R., Sawyer R.K., Brogden M., Pinder K.M,. Speight T.M, Avery G.S. Clotrimazole: A Review of its Antifungal Activity and Therapeutic Efficacy. Drugs.1975; 9(6): BachhavYG,Patravale VB. Microemulsion-based vaginal gel of Mclotrimazole: formulation, In vitro evaluation, and stability studies. AAPS Pharm Sci Tech. 2009; 10(2): HarishNM, PrabhuP,Chargulu RM, Gulzar M.A and Subrahmanyam EVS. Formulation and Evaluation of in situ Gels Containing Clotrimazole for Oral Candidiasis.Indian Journal Pharma Sciences.2009;7(14): Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 242

15 12. Murleedhara KK, Kumar SK, ParthibhanS.Formulation and evaluation of mucoadhesivethermosensitivepuronic lecithin organogel of clotrimazole for vaginal candidiasis.international journal of current pharmaceutical and clinical research.2013;3(2): Rashmi. Topical Gel: A Review. Pharmainfo.net Khandre P. Formulation and evaluation studies on nimesulide topical gel.rajiv Gandhi University of Health Sciences. Karnataka, Bangalore. 2010: Gupta A., Mishra AK., Singh AK., Gupta V and Bansal P. Formulation and Evaluation of Topical Gel of Diclofenac sodium using different polymers. Drug Invention Today. 2010; 2 (5): Shivhare UD, Jain KB,Mathur VB, Bhusari KP, Roy AA. Formulation Development and Evaluation of diclofenac Sodium gel using water soluble polyacrylamide polymer. Journal of Nanomaterials and Biostructures.2009; 4 (2): Nair R, Sevukarajan M, Mohammed B, Kumar J. Formulation of microemulsion based vaginal gel-in vitro and In vivo evaluation. Der Pharmacia Lettre. 2010; 2 suppl 6: Shakeel F, Baboota S, Ahuja A, Aqil M, Shafiq S. Nanoemulsion as vehicle for transdermal delivery of aceclofenac. American Journal of Phamaceutical. Sciences. 2007; 8(4): 104. Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 243