Elevation of antidermatophytic action of mefenamic acid by cobalt ions.

OBJECTIVES: To evaluate the antifungal property of mefenamic acid, which is a member of non-steroidal anti-inflammatory drugs (NSAIDs) group.
MATERIALS AND METHODS: In order to evaluate the antifungal property of mefenamic acid on dermatophytes, it was mixed with cobalt (Co) in culture media. Two species related to two genera of dermatophytes were tested for their susceptibility to mefenamic acid and its complex with Co by using colony diameter measurement method.
RESULTS: The inhibitory action of mefenamic acid on fungal strains was increased in the presence of Co. Epidermophyton floccosum showed more susceptibly to either mefenamic acid or its complex with Co than Trichophyton mentagrophytes variant mentagrophytes.
CONCLUSIONS: Mefenamic acid showed potential ability to prevent growth of dermatophytes. This ability increased due to the presence of Co.

Introduction

Mefenamic acid has pharmacologic actions similar to those of other non-steroidal anti-inflammatory drugs (NSAIDs). The drug exhibits anti-inflammatory, analgesic, and antipyretic activity.[1] Large amounts of mefenamic acid are consumed every year due to these therapeutic properties, especially during summer.[2]

Mefenamic acid is demonstrated to have antibacterial effects based on the susceptibility of seven pathogenic bacteria to this agent.[3] However, many fungal species showed resistance to or no affect of mefenamic acid. Candida albicans is affected by this drug. It is considered to be an important opportunistic pathogenic fungus.[4] Cobalt (Co) is considered as one of important metals for fungal development through its association with Ni transporter system into the cells.[5] On the other hand, Co may have antimicrobial activities, especially when it complexes with many synthetic chemical structures. These complexes could exhibit a moderate inhibitory effect on many microbial proliferations.[6] Dermatophytes are special group of pathogenic fungi that can infect human skin and cause lesions in cutaneous layer.[7] They are not influenced by the presence of mefenamic acid in their media (data not shown), while its complexes may have such influences. Epidermophyton floccosum, one species of dermatophytes, is found to be inhibited at concentrations ranging from 6 to 50 μg/mL of Co complex (thiophene).[8] However, Edrissi et al.[9] illustrated that mefenamic acid can react with Co(II) ions to form highly stable complex at ambient temperature. Thus, mixing of mefenamic acid with Co in culture media was studied to detect any possible effects of such mixture on dermatophytes.

Materials and Methods

Organisms

Dermatophytes were clinically isolated from a old infected with dermatophytoses (Tinea corporis) at AL-Hussein General Hospital of Karbala province in February 2009. Skin scales of fungal lesion were cultured on Sabouraud’s glucose agar comprising of the following components: glucose 20 g, peptone 10 g, agar 15 g, chloramphenicol 0.05 g, cycloheximide 0.5 g, and 1000 mL of distilled water. Cultures were incubated at 28°C for 2 weeks. Grown fungi isolated from the same lesion were diagnosed according to criteria reported by Rippon[10] and Emmons et al.[11] which also include biochemical tests.

The isolated strains were related to two different genera (Trichophyton and Epidermophyton): Trichophyton mentagrophytes variant mentagrophytes and E. floccosum.

Chemical agents

Mefenamic acid was supplied from Brown and Burk Pharmaceutical Limited, Sipcot, Hosur India. Cobalt nitrate (Co(II)) was purchased from Hopkin and Williams Ltd., Chadwell Heath, ESSEX, England. Griseofulvin was supplied by Arabic Drug Industry (Baghdad, Iraq).

Antidermatophytic assay

Colony diameter method recorded by Kücüc and Kivan[12] was used. Various concentrations of mefenamic acid (0.156, 0.3125, 0.625, 1.25, and 2.5 mg/mL) were mixed with melted Sabouraud’s glucose agar containing 0.5% dimethyl sulfoxide (DMSO). Co of 0.5 mg/mL was mixed with each previous concentration of mefenamic acid. Then, prepared media were poured in Sterile Petri dishes. In addition to free media as control, either Co, mefenamic acid, or griseofulvin (10 μg/mL) was used as control after mixed with prepared media. A disk (9 mm) of old grown fungi (at 28°C for 1 week) was inoculated on the center of culture media. Plates were incubated at 28°C for 1 week. Perpendicular colony diameters (mm) of grown strains were measured and percentage inhibition calculated according to the formula:

where C is colony diameter (mm) of the control and T is colony diameter (mm) of the test plate.

Results

Activity of mefenamic acid complex with Co was investigated against dermatophytes. Compared to control (free media) and media containing Co only, incorporation of various concentrations of mefenamic acid in culture media showed decrease in fungal colony diameter of isolated strains. These results are indicated by high percentage inhibition of grown strains [Figures 1 and 2], which showed an increase in the antidermatophytic activity of mefenamic acid due to the presence of Co ions.

Figure 1

Effect of mefenamic acid, its complex with Co, and griseofulvin (10 μg/mL) on T. mentagrophytes variant mentagrophytes.

Figure 2

Effect of mefenamic acid, its complex with Co, and griseofulvin (10 μg/mL) on E. floccosum.

E. floccosum exhibited more susceptibility to mefenamic acid and its complex with Co (high ratio of inhibition) than T. mentagrophytes variant mentagrophytes [Figure 2]. However, neither mefenamic acid alone or with Co exceeded the effect of standard antidermatophytic agent (griseofulvin) [Figures 1 and 2].

Discussion

Wide spread dermatophyte infections among people over the world and failure of most drugs to cure such infections are a stimulus for continuous search for new drugs effective against dermatophytes. Thus, many chemical compounds are being tested for determining their activity on dermatophytes.

Cobalt has the ability to enhance the action of some antimicrobial drugs. Antibacterial action of ampicillin and chloramphenicol was increased when mixed with Co ions.[13] This encourages us to test the interaction of Co and mefenamic acid against dermatophytes. Mefenamic acid was chosen from NSAID group n the basis of prior reported interaction of this agent with Co ions.[9] Thus, any weakness in antifungal action of mefenamic acid could be diminished through mixing with such type of metal. Furthermore, low inhibitory action of Co had been increased due to mix with mefenamic acid. Therefore, enhancement the action of mefenamic acid and Co on dermatophytes could be proposed.

Like other NSAIDs, mefenamic acid shows its therapeutic activity through inhibition of prostaglandin synthesis in body tissues. This action is performed by inhibition of cyclooxygenase (COX) enzyme which is important for prostaglandin synthesis.[14] Dermatophytes are proven to have the ability to produce prostaglandin which is responsible for chronic fungal colonization.[15] Furthermore, COX enzyme was also demonstrated to be present in fungal cells.[16] Thus, mefenamic acid may act against dermatophytes through inhibition of COX enzyme. Co may play a role in entry of mefenamic acid into cell through cell membrane of dermatophytes in mechanism similar to that with Ni transport.[5] Therefore, mefenamic acid can easily access its site of action in fungal cells. Moreover, an important property of mefenamic acid is its inability to dissolve in water.[17] Thus, interaction of mefenamic acid with Co may increase solubility rate of this member of NSAIDs.

E. floccosum exhibited more susceptibility to mefenamic acid and its complex. This may be related to the absence of microconidia in this species compared with other species of dermatophytes.[1011] Thus, E. floccosum has less efficiency in reduction of the toxic action of mefenamic acid due to lower fungal mass. T. mentagrophytes has a heavy density of fungal elements that is composed of macroconidia, microconidia, and fungal hyphae.

Increasing the concentrations of mefenamic acid led to enhanced inhibitory action of this agent on two isolated dermatophytes either added alone or with Co ions in culture media. However, low concentrations of mefenamic acid complex continued to show high degree of inhibition than Co.

In conclusion, mefenamic acid showed ability to prevent dermatophytes. This ability was enhanced by the presence of Co. High percentage inhibition was noted distinctly after mixing of mefenamic acid with Co. Furthermore, E. floccosum exhibited more susceptibility to mefenamic acid than T. mentagrophytes.