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Antifugal Action of Imidazole Derivatives fromNew Pharmaceutical Forms on Various Strains of Candida

MAGDALENA BIRSAN, ILEANA CORNELIA COJOCARU*, MONICA ILIUTA STAMATE, VLAD IOAN TEODOR, CRISTINA TUCHILUSFaculty of Pharmacy, Gr. T. Popa University of Medicine and Pharmacy, Iasi, 16, University Str., 700115, Iasi, Romania

The antifungal activity of imidazole derivatives was tested on three types of Candida, respectively C. albicans,C. sake, and C. glabrata. The antifungal activity was compared with the activity of miconazole nitrate in 16new formulations of oral biomucoadhesive tablets, with the purpose of being used in oral candidiasis. All the16 formulations of biomucoadhesive tablets which contain 25 mg miconazole have a good antifungalaction; the diameter of the inhibition area is over 20 mm in all the three strains of Candida. The second goalwas to compare the activity of miconazole nitrate with other antifungal substances: clotrimazole (benzylimidazole derivative), nistatin (polyenic macrolide), econazole (phenyl – ethyl – imidazole derivative), andfluconazole (triazole derivative). Good results, obtained by measuring the diameter of the inhibition area,were shown by econazole, with a diameter of over 22 mm, but this imidazole derivative does not penetratethe stratum corneum well enough, which implies a much longer treatment than miconazole. The third goalwas the assessment of the antifungal activity of the 16 formulations of biomucoadhesive tablets by meansof establishing the minimum inhibitory concentration (MIC) and of minimum fungicide concentration (MFC).

Keywords: first-generation azoles, biomucoadhesive tablets, miconazole nitrate, and Candida

Candidiasis is a frequent condition of mucosa andtegument, caused by various Candida species, which, inimmunodeficiency conditions, may become invasive.Usually, Candida species are pathogenic, isolatingthemselves from the microflora of mucosa in 10-15% ofnormal people. To become pathogenic, predisposingfactors are necessary, altering the immunity of the host:sugar diabetes, AIDS, people treated with cytostatic andcorticosteroids or antibiotics, and with mucosal ulcerations[1]. Candida species are part of man’s normal flora, beingthe main source of infection. The transmittance occurs viadirect contact with buccal, tegument, and vaginalsecretions that contain Candida [2].

Broad-spectrum antibiotic therapy significantlyincreased the life expectancy in patients with severeinfections with approximately 10 years, as some authorsstate. Unfortunately, these medical successes have broughtin the last 15 years the spread of fungal infections, followedby the increase of resistance to antifungal drugs, initially inpatients with severe immunodepression: after transplantor after chemotherapy in oncology [3]. Candidemia is moreand more signalled in long-term patients in Intensive Careunits, while the haematogenous fungal infections are thefourth cause of nosocomial infections in critical patients[4]. More and more patients with HIV/AIDS, neoplasic ortransplant are colonized with strains of fungi at thegateways, most often with Candida albicans, but also thestrains of Candida nonalbicans are significantly spread.

The most common substances that are used for theirantifungal actions are imidazole and triazole derivatives.Triazole derivatives are recommended by some studiesdue to their reduced toxicity in systemic administering anddue to their longer duration of action [5].

First-generation azoles are inhibitors of ergosterolbiosynthesis with a broad-spectrum activity, such asdermatophytes, unicellular, dimorphic and filamentousfungi. First-generation co-azoles have an exceptionaltolerance, with substantial reduction of duration of

* email: mail.icojocaru@yahoo.com

treatment and minimum side-effects, comparing withother antifungal drugs. They are lipophilic, little soluble inwater, reason why we chose the salt form of antifungaldrug, with a better solubility in water. Miconazole nitrate isan antimicotic drug, with a similar structure to econazole,differing only by the presence of 2 chlorine atoms (positions2 and 4) on benzyl radical.

Scheme 1. Miconazole Nitrate. 1-[2(2,4-dichlorophenil)-methoxy] -2-(2,4-dichlorophenyl)]-ethyl-1H-imidazole

Imidazole and triazole derivatives inhibit the synthesisand incorporation of ergosterol in the membrane of thefungal cell as a result of blocking sterol-14α-demethylase,a cytochrome P450 dependant enzyme, with a key role inbiosynthesis of ergosterol [6]. Accumulation of metyl-steroids affects the function of membrane phospholipidsand inhibits certain enzymatic systems membranedependant like ATPase and enzymatic transport systemswith inhibition of growth and development of fungi [7]. Itacts by affecting the permeability of the membrane of thefungi, selective inhibition of RNA and DNA precursors andits mucopolysaccharides.

The minimum fungicide concentration of miconazoletriggers an important growth of dispersion of theintracellular potassium, causing a cellular acidification thatwould favour the activity of autolytic enzymes.

Miconazole also acts by interfering with the fungalenzymes: cytochrome C oxidase, cytochrome Cperoxidase and catalase. It does not act directly on theenzymes, but it seems it interferes with the conversion of

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mitochondrial ATP and thus it helps synthetizing theseenzymes. NADH oxidase pathway is not affected, thereforeperoxides are produced in cytoplasm, at levels that areincompatible with the viability of the cells. The effect oncell morphology varies from the lysis of cell organelles,loss of plasmalemma and thickening of the cellular wall(in fungistatic doses) to progressive cytoplasmicdeterioration and loss of cellular functions with cellulardeath (in fungicide doses) [5].

Miconazole nitrate is incompletely absorbed from thegastrointestinal tract. When administered on skin andmucosa, it is absorbed in a limited proportion (~ 1%).Miconazole easily penetrates the stratum corneum, buildingeffective therapeutic concentrations, that last for more than4 days from its application on the skin. There is a 1.3%absorption on intravaginal application. It bonds in a 90%proportion to plasmatic proteins. It is metabolized in theliver in inactive compounds, which are eliminated throughfaeces (50% from the administered dose, as unmodifieddrug) and through kidneys, as inactive metabolites. Themedicine has a good penetrability also in inflamed joints,vitreous body, crystalline lens, and peritoneal cavity. Itreaches the saliva and sputum. It also crosses the blood-brain barrier. T½ is 20-30 min.

Miconazole nitrate is indicated for treatment of infectionsthat are provoked by Candida albicans, suprainfected ornot by a Gram positive bacterial flora, treatment ofdermatophytes, coccidiomicosis, paracoccidiomicosis,kryptoccocosis, systemic and mucocutaneous candidiasis(oropharyngeal candidiasis), and treatment of acne.

The main goal of this study was to test varioussubstances with antifungal action that are used in buccalcandidiasis, therefore we chose three different strains ofCandida. Recent studies have shown a very goodantimicotic activity by means of using polyenic macrolidesand semi synthesis antimicotic drugs, such as imidazoleand triazole derivatives [8-10].

We chose as testing samples 16 new formulations oforal biomucoadhesive tablets, conceived to guide therelease of the active substance in the buccal mucosa withthe goal to increase their effectiveness [11]. The use ofmucoadhesion concept with the purpose of controlledrelease of drug substance is based on the capacity of somenatural or synthetic polymers to interact with the layer ofmucus that covers the epithelial surface of the mucosa[12]. To improve the bioavailability of drugs that areadministered in oral cavity, oral biomucoadhesive tabletshave been developed in recent years, that may be appliedin various areas of the mouth: palate, mucosa of the cheek,between the gingiva and the upper lip [13]. The tabletadheres to the substrate through various mechanisms andit is kept in the same position until dissolution and/or thecompletion of release of the active substance. In recentyears, the market share of biomucoadhesive systems hassignificantly increased, according to a recent report thatwas published by Kalorama, with an estimated value of6.7 million dollars in 2006 [14] up to 7.9 million dollars in2010 [15]. This growth may be easily explained by thesuccess of these new pharmaceutical formulations due tothe advantages they have.

The second goal was to compare the activity ofmiconazole nitrate with clotrimazole (benzyl imidazolederivative), nistatin (polyenic macrolide), econazole(phenylethyl imidazole), and with fluconazole (triazolederivative).

The third goal was to assess the antifungal activity ofthe 16 biomucoadhesive formulations by establishing the

minimum inhibitory concentration (MIC) and minimumfungicidal concentration (MFC).

Experimental part16 new formulations of biomucoadhesive tablets with

miconazole nitrate were used as samples that were testedfor antifungal activity, in 25 mg concentration and variousexcipients [11].

The test microorganisms that we used were Candidaalbicans ATCC 10231, Candida sake, and Candida glabrataATCC MYA 2950. The strains we used in microbiologic testsare part of collection of microorganisms belonging todepartment of Microbiology, Faculty of Pharmacy, Gr. T. PopaUniversity of Pharmacy and Medicine, Iasi, and the culturemedium was Müller-Hinton Agar for fungi (HiMedia).

Testing the antimicrobial activityAntimicrobial activity of samples I-XVI was tested for

quality, by diffusimetric method [16, 17]. From the culturethat was incubated over night at the optimal temperature(300C), we prepared a suspension, with the density thatcorresponded the tube of 0.5 in Mc Farland turbidemetricscale.

The suspension was incorporated in 1/10 proportion inMuller-Hinton medium for fungi, melted and cooled downat 500C. After homogenization, the mixture was distributedin Petri dishes with 12 cm diameter, 25 mL each. Aftersolidification, stainless steel cylinders were disposed onthe surface of each Petri dish, in which 0.2 mL of the testingsurfaces were distributed. The results were read afterkeeping the dishes at thermostat, at 240C, for 48 h.

Each sample was simultaneously tested in three dishes.The diameter of the inhibition areas was measured andrecorded. The results are the average of three values thatwere obtained from measuring the diameter of theinhibition areas of the microbial growth.

We compared the antimicrobial activity of the sampleswith the activity of the martor microtablets: Econazole(50µg), Miconazole (50µg), Clotrimazole (50µg),Fluconazole (25µg), and Nistatin (100 mg/disc).

Quantitative antimicrobial activity was assessed byestablishing the minimum inhibitory concentration (MIC)and minimum fungicidal concentration (MFC). We usedmicrodilution in liquid Sabouraud medium method [17].We made double dilutions from samples in liquid Sabouraudmedium, obtaining thus concentrations between 0.06-12.5mg/ml. Each cup with double dilutions of each samplewas inoculated with 50 microliters from the solution ofeach test microorganism (with a density ofapproximatively 106 UFC/mL). The dishes were incubatedat 240C. After 24 h, MIC value was registered as the smallestsample concentration in which the visible growth ofmicroorganisms is absent.

Minimal fungicidal concentrations were established byharvesting micro cultures from double serial dilutions inwhich the visible microbial growth was absent. MFC wasgiven by the smallest product concentration that kills atleast 99.9% of inoculum.

Results and discussionsDiameters of the inhibition areas (in millimetres) that

correspond to the tested biomucoadhesive tablets areshown in table 1. Out of all the tested substances, theremarkable antifungal value belongs to miconazole nitrateout of the 16 new formulations, with miconazole as puresubstance and econazole as pure substance.

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Clotrimazole, a broad-spectrum antifungal agent,indicated in treatment of superficial mucocutaneousinfections, provoked by various species of pathogenicdermatophytes and yeast, builds a 21 mm diameter ofthe inhibition area for Candida albicans strain, 23 mm forCandida sake strain, and 18 mm for Candida glabratastrain. As antifungal activity, this azole has a good activityfor Candida albicans and Candida sake, and an averageaction for Candida glabrata. Unlike miconazole nitrate thatis stored in the stratum corneum up to 4 days afterapplication, clotrimazole is active at active therapeuticconcentrations up to 3 days. Topically administered,clotrimazole provokes itches, erythema, edema, rash ordesquamation, disadvantages that lead to the removal ofthe substance as a first choice in candidiasis.

Out of the 5 antifungal substances that we used,econazole has the best antimicrobial activity, by buildinga 24 mm diameter of the inhibition area on Candidaalbicans, 22 mm on Candida sake, and 27 mm on Candidaglabrata. Despite the fact that it makes a very gooddiameter of the inhibition area, this substance does notlast in the tissue as miconazole nitrate or clotrimazole,that is why the treatment with econazole needs a longtreatment, up to 2 weeks, to avoid relapse [17].

Fig. 1. Activity of the formulations tested on various strains of Candidaspp. (diffusimetric method) Table 1

ANTIFUNGAL ACTIVITY OF THE TESTED TABLETS.

Miconazole nitrate has a similar structureto econazole, differentiating itself from it bythe presence of 2 atoms of chlorine in position2 and 4 on benzyl radical. The presence ofchlorine atoms limits the activity of theantifungal with approximately 10%; inexchange, it has the advantage of easilypenetrating the stratum corneum, buildingconcentrations that last for more than fourdays from the topical application. Thediameter of the inhibition area that is largerthan 20 mm for all the three strains of Candida,together with the advantage of buildingconcentrations that last several days, avoidingthis way the risk for relapse, lead to a firstchoice of miconazole nitrate for the treatmentof buccal candidiasis.

Fluconazole, even if it has the advantageof a smaller toxicity in comparison withmiconazole nitrate or econazole, had a goodactivity on Candida albicans and Candida sakestrains, but a weak action for Candida glabratastrain (table 1).

Nistatin, polyenic macrolide, made a 19mm diameter of the inhibition area for the 3strains of Candida, a small diameter incomparison with miconazole or econazole,but if we consider the low toxicity, it has agood antifungal activity. This polyenicmacrolide offers an optimal diameter of theinhibition area for Candida glabrata ATCC MYA2950 strain.

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For Candida species that we used, the antifungal activityof the tested tablets is good, with large inhibition areas, ofover 20 mm.

Remarkable is the very good activity of formulations I-XI, with over 20 mm diameters of inhibition areas on testedCandida species.

The activity on Candida glabrata ATCC MYA 2950 waslower, proven by smaller values of the diameters of theinhibition areas (table 1). The formulated tablets provedhigher activity on the tested C. albicans, C. Glabrata, and C.sake.Conclusions

We tested the antifungal activity of the 16 newformulations of biomucoadhesive tablets with miconazolenitrate on various Candida strains, respectively Candidaalbicans ATCC 10231, Candida glabrata ATCC MYA 2950and Candida sake. One may notice a good activity of the16 formulations with miconazole nitrate on the strains ofCandida albicans ATCC 10231 and Candida glabrata ATCCMYA 2950, and a very good activity on Candida sake strain.

The best antifungal activity proven by the dimension ofthe diameter of the inhibition area of 23 mm is shown byformulations FV and FVIII. The antifungal action wascompared with the activity of the martor – miconazolenitrate pure substance and with the one of standardizedmicrotablets of econazole, miconazole, clotrimazole,fluconazole, and nistatin.

In our study, first-generation azoles presented a goodactivity, in comparison with the one of fluconazole, triazolederivative. Out of the results that were obtained on thethree strains of Candida spp., it is visible that the mostappropriate antifungal drug for buccal candidiasis is

miconazole nitrate, formulated in oral biomucoadhesivetablets.

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Manuscript received: 26.01.2016

Table 2MIC AND MFC FOR THE TESTED FORMULATIONS (mg/mL)