BY: PATEL MINAL NARAN U29/35243/2010 SUPERVISOR: DR. P. M. NJOGU MICROBIOLOGICAL ASSAY OF ANTIFUNGAL CLOTRIMAZOLE AND MICONAZOLE CREAMS IN THE KENYAN MARKET BY: PATEL MINAL NARAN U29/35243/2010 SUPERVISOR: DR. P. M. NJOGU

INTRODUCTION Drug therapy is by far the commonly used form of treatment. Unfortunately it cannot be supported only by the innovator drugs especially in the developing countries. Hence the need for generic drugs due to their low costs. Topical antifungal azoles such as clotrimazole and miconazole are widely used for the treatment of tinea pedis, tinea cruris and candidiasis. Various generics are available in the Kenyan market hence the need to carry out tests and determine whether the generic drugs are pharmaceutically equivalent to the innovator drug.

OBJECTIVES To carry out identification tests of the innovator and generic brands of clotrimazole and miconazole creams according to the British Pharmacopeia 2008. To compare the potency of innovator and generic brands of clotrimazole and miconazole creams using microbiological assay method.

EXPERIMENTAL Three samples of clotrimazole and miconazole creams each were obtained from the pharmacies within Nairobi City. They comprised of the innovator drugs Canestan® (clotrimazole) and Daktarin® (miconazole) and their respective generics that were either locally manufactured or imported. Ultra-violet (UV) spectroscopy was used for identity tests. All samples complied with the British Pharmacopoeia (2008) specifications for identity of clotrimazole and miconazole.

MICROBIOLOGICAL ASSAY Agar diffusion method was employed. Sabouraud dextrose agar was used as growth media and Candida albicans was used as test micro-organism. The assay comprised of two dose levels of each standard and sample. Means of responses to standard test solutions were designated S(0.25mg/ml) and S1(0.5mg/ml) for low and high dose, respectively. Similarly, mean responses to the sample of unknown potency were designated T(0.25mg/ml) and T1(0.5mg/ml).

Standard and Sample preparation Approximately 5g of clotrimazole cream (1% w/w) samples and 2.5g of the miconazole nitrate cream (2% w/w) samples were dissolved in 100ml of dimethylformamide (DMF) to obtain a concentration of 0.5mg/ml and filtered. The resulting solutions were diluted with DMF to obtain a second concentration of 0.25mg/ml. Standards were prepared by dissolving approximately 0.05g of clotrimazole and miconazole in 100ml to obtain a concentration of 0.5mg/ml. Concentration levels of standard solutions were prepared by diluting with DMF to obtain another concentration of 0.25mg/ml.

Sample application Figure 1. S (0.25mg/ml) and S1 (0.50mg/ml) represent the standard solutions; T (0.25mg/ml) and T1 (0.50mg/ml) represent the test solutions; C is the negative control (DMF). S T S1 C T1

Figure 2. Zones of inhibition by Daktarin®

POTENCY DETERMINATION Potency of each sample was calculated according to the formula: E= ½ [(T1 – T) + (S1 – S)] F= ½ [(T1 + T) – (S1 + S)] b = E/I antilog M = F/b Where: T- Diameter of zones of inhibition for test samples. S- Diameter of zones of inhibition for standards. E- Mean difference in response between adjacent dose levels. F- Difference in response between two preparations. I- Logarithm of the ratio of adjacent dose levels (0.3) b- Theoretical mean difference in response arising from a 10-fold increase in dose. M- Logarithm of the ratio of potencies of test solutions, sample:standard.

RESULTS Drug Sample Potency Label claim (%) Clotrimazole Canestan® 0.712 0.72   Sample A 0.698 0.70 Sample B 0.657 0.66 Miconazole Daktarin® 0.806 1.61 Sample C 0.754 1.49 Sample D 0.649 1.29

DISCUSSION The innovator drugs and generic samples of clotrimazole had similar potency of approximately 0.7. this implies the generic drugs are pharmaceutically equivalent to the innovator drug and hence can be substituted for the innovator drug. The innovator drug Daktarin® and one of the generic drugs (sample C) had potencies that were similar of approximately 0.8. This shows that sample C is bioequivalent to the innovator drug. The second generic of miconazole (sample D) had a potency lower than the innovator drug. This may imply that Sample D is not bioequivalent to Daktarin®.

CONCLUSION The generic samples of clotrimazole had potencies equivalent to that of the innovator drug implying that they are equally potent and hence serve as cheaper substitutes for treatment of fungal infections. One of the miconazole generic drug had a lower potency compared to the brand drug suggesting it might not be bioequivalent to the innovator drug.

RECOMMENDATIONS Since microbiological assay for antifungal azole creams is not an official method, it could be one of the methods that could be validated for use in determining the potencies and hence used as a method to study the bioequivalence of various generics that are available in the market. Various other fungi could be used to assess the potency of the creams. Example Saccharomyces cerevisiae, Aspergillus niger, Trichophyton rubrum, Trichophyton mentagrophytes and Epidermophyton floccosum.