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Antifungal activity of topical microemulsion containing a thiophene derivative

Abstract

Fungal infections have become a major problem of worldwide concern. Yeasts belonging to the Candida genus and the pathogenic fungus Cryptococcus neoformans are responsible for different clinical manifestations, especially in immunocompromised patients. Antifungal therapies are currently based on a few chemotherapeutic agents that have problems related to effectiveness and resistance profiles. Microemulsions are isotropic, thermodynamically stable transparent systems of oil, water and surfactant that can improve the solubilization of lipophilic drugs. Taking into account the need for more effective and less toxic drugs along with the potential of thiophene derivatives as inhibitors of pathogenic fungi growth, this study aimed to evaluate the antifungal activity of a thiophene derivative (5CN05) embedded in a microemulsion (ME). The minimum inhibitory concentration (MIC) was determined using the microdilution method using amphotericin B as a control. The formulations tested (ME- blank and ME-5CN05) showed physico-chemical properties that would allow their use by the topical route. 5CN05 as such exhibited moderate or weak antifungal activity against Candida species (MIC = 270-540 µg.mL-1) and good activity against C. neoformans (MIC = 17 µg.mL-1). Candida species were susceptible to ME-5CN05 (70-140 µg.mL-1), but C. neoformans was much more, presenting a MIC value of 2.2 µg.mL-1. The results of this work proved promising for the pharmaceutical industry, because they suggest an alternative therapy against C. neoformans.

antifungal susceptibility; Cryptococcus neoformans; microemulsion; thiophene derivatives


RESEARCH PAPER

Antifungal activity of topical microemulsion containing a thiophene derivative

Geovani Pereira GuimarãesI; Mysrayn Yargo de Freitas Araújo ReisII; Dayanne Tomaz Casimiro da SilvaII; Francisco Jaime Bezerra Mendonça JuniorI, III; Attílio ConvertiIV; Adalberto Pessoa JrV; Bolívar Ponciano Goulart de Lima DamascenoI, II; José Alexsandro da SilvaI, II

IPrograma de Pós-Graduação em Ciências Farmacêuticas, Universidade Estadual da Paraíba, Campina Grande, PB, Brazil

IIDepartmento de Farmácia, Universidade Estadual da Paraíba, Campina Grande, PB, Brazil

IIILaboratório de Síntese e Vetorização de Moléculas, Departamento de Ciências Biológicas, Universidade Estadual da Paraíba, João Pessoa, PB, Brazil

IVDepartment of Civil, Chemical and Environmental Engineering, Chemical Engineering Pole, Genoa University, Genova, Italy

VDepartmento de Tecnologia Bioquímico-Farmacêutica, Universidade de São Paulo, São Paulo, SP, Brazil

Correspondence Correspondence: J.A. Silva Graduation Program in Pharmaceutical Sciences Universidade Estadual da Paraíba Rua Juvêncio Arruda S/N, Bairro Universitário 58429-600 Campina Grande, PB, Brazil E-mail: alexuepb@pq.cnpq.br

ABSTRACT

Fungal infections have become a major problem of worldwide concern. Yeasts belonging to the Candida genus and the pathogenic fungus Cryptococcus neoformans are responsible for different clinical manifestations, especially in immunocompromised patients. Antifungal therapies are currently based on a few chemotherapeutic agents that have problems related to effectiveness and resistance profiles. Microemulsions are isotropic, thermodynamically stable transparent systems of oil, water and surfactant that can improve the solubilization of lipophilic drugs. Taking into account the need for more effective and less toxic drugs along with the potential of thiophene derivatives as inhibitors of pathogenic fungi growth, this study aimed to evaluate the antifungal activity of a thiophene derivative (5CN05) embedded in a microemulsion (ME). The minimum inhibitory concentration (MIC) was determined using the microdilution method using amphotericin B as a control. The formulations tested (ME- blank and ME-5CN05) showed physico-chemical properties that would allow their use by the topical route. 5CN05 as such exhibited moderate or weak antifungal activity against Candida species (MIC = 270-540 µg.mL-1) and good activity against C. neoformans (MIC = 17 µg.mL-1). Candida species were susceptible to ME-5CN05 (70-140 µg.mL-1), but C. neoformans was much more, presenting a MIC value of 2.2 µg.mL-1. The results of this work proved promising for the pharmaceutical industry, because they suggest an alternative therapy against C. neoformans.

Key words: antifungal susceptibility, Cryptococcus neoformans, microemulsion, thiophene derivatives.

Introduction

Fungal infections have become an issue of great concern around the world; it was estimated that over 40 million people do in fact suffer fungal infections both in developed and in developing countries (Güngõr, Erdal and Aksu, 2013). The incidence and prevalence ofinvasive fungal infections have increased since the 1980s, especially in immunocompromised patients and those hospitalized with serious underlying diseases (Espinel-Ingroff, 2009).

Candida yeasts are microorganisms belonging to the normal microbiota of healthy individuals, mainly in the oral mucosa, the gastrointestinal tract and the female genitourinary tract (Shao, Sheng and Zhang, 2007). However, these fungi are responsible for different clinical manifestations, especially in immunocompromised patients, ranging from skin and mucous infections to systemic infections (Sardi et al., 2013). Their importance comes from the high frequency with which they colonize and infect the human host (De Bernardis et al., 2004), being the fourth most common pathogens associated with cases of nosocomial infection (Wisplinghoff et al., 2004).

Cryptococcus neoformans is an encapsulated fungus capable of causing infections in immunocompromised patients (Carroll et al., 2012; Guerra et al., 2012). Inhalation of its basidiospores or cells present in the environment can result in lung infection and its subsequent spread to the central nervous system, causing meningoencephalitis, recognized as one of the most important opportunistic infections in patients with HIV with a worldwide incidence of approximately 957,000 cases per year (Leongson et al., 2013).

Conventional treatments of fungal diseases are currently based on a few chemotherapeutic agents such as az-oles and polyenes, which have serious problems related to effectiveness, activity spectrum, toxicity, low power and inadequate pharmacokinetics (Scotti et al., 2012). Moreover, the occurrence of microbial resistance to antimicrobial agents is shown to be a growing public health problem worldwide and the biggest obstacle to the success of a treatment, as it continues to reduce the number of drugs available in the market (Oliveira and Silva, 2008; Ben-ghezal et al., 2007). This scenario demonstrates the need for the development of new antifungal agents as therapeutic alternatives to control fungal infections.

Organic compounds containing aromatic hetero-cyclic rings such as thiophene are widely distributed in nature and are generally of great importance in many biochemical processes, having a broad spectrum of pharmacological properties (Mohammad et al., 2012). The versatile applicability in syntheses and the biological activity of thes e heterocycles make them important structural fragments in synthetic medicinal chemistry, serving as a basis for planning and implementation of new therapeutic agents (Meotti et al., 2003). For the present work, it was synthesized a derivative of 2-amino thiophene, namely the 2-[(3,4-dichloro-benzylidene)-amino]-5,6-dihydro-4H-cy-clopenta[è]thiophene-3-carbonitrile, referred to as 5CN05 (Figure 1). This compound belongs to a series of synthetic tiophene derivatives with well-proven antifungal activity in vitro (Mendonça Junior et al., 2011). Its biopharmaceutical characteristics are not yet well understood, especially with respect to permeability. However, the high value of its partition coefficient (logP = 5.98) suggests that it has poor water solubility and permeability.


A microemulsion (ME) is a thermodynamically stable and optically transparent system made up of a mixture of two immiscible liquids stabilized by an interfacial surfactant film. The specific properties of microemulsioned systems are primarily related to their low interfacial tension, large interfacial area, small size of droplets, low viscosity and high solubilization power for lipophilic, hydro-philic and amphiphilic drugs (Damasceno et al., 2011; Fanun, 2012; Silva etal., 2009b). MEs usually contain high concentrations of surfactants, an essential factor for successful incorporation of poorly water soluble drugs (Da-masceno et al., 2012; Djekic et al., 2012). These systems have the ability to deliver water insoluble compounds by carrying the drugs in their oil core or on the water-oil interface. This biological property allows the use of MEs as bio-technological carriers for large water insoluble molecules such as those of the tiophene derivatives.

The treatment of fungal infections by topical dosage forms shows some advantageous features such as possibility of action at the application site and reduction of the risk of systemic side effects, increased efficacy of the therapy and its acceptance by the patients (Güngõr, Erdal and Aksu, 2013). In topical formulations, MEs have proven effective in increasing the absorption of both hydrophilic and lipo-philic drugs by the skin when compared to conventional systems such as emulsions and aqueous solutions (Gram-purohit, Ravikumar and Mallya, 2011). The use of MEs may also increase the local or systemic action of drugs by different mechanisms, thereby representing an interesting alternative to antifungal drugs (Jadhav, Shetye and Kadam, 2010).

In this context, the present study aims to evaluate the antifungal activity of 5CN05 embedded in a ME for topical use, by determination of its minimum inhibitory concentration against pathogenic yeast species.

Materials and Methods

Preparation of microemulsion formulation and incorporation of 5CN05

To obtain microemulsions (MEs), pseudoternary phase diagrams (PTPD) were constructed using a surfactant blend of LAS® (Brasquim, São Paulo - SP, Brazil) and Plurol Oleique® (Gattefossé, Saint-Priest, France), and iso-propyl myristate as oil phase (Via Farma, São Paulo - SP, Brazil). Purified water was produced by a reverse osmosis purification system, model OS10LX (Gehaka, São Paulo - SP, Brazil).

For 5CN05 incorporation in ME, such a drug was initially dispersed in the oil phase, and the remaining components were added, including water. This multiphase mixture was then subjected to three 1 min-alternate homogeni-zation cycles by an ultrasound probe, model DES500 (Unique, Indaiutaba - SP, Brazil), and the excess air bubbles were removed by 1 min-treatment in an ultrasound bath, model USC-3300 (Unique). The formulation obtained, called ME-5CN05, was characterized for its macroscopic appearance, pH, electrical conductivity and refractive index, and compared to that without drug (ME-blank).

Target microorganisms

The antifungal activity of ME-5CN05 and ME-blank systems was assessed using standard strains from the American Type Culture Collection (ATCC), namely Candida albicans ATCC 18804, Candidaparapsilosis ATCC 22019 and Candida tropicalis ATCC 13803, provided by the Laboratory of Drug Development and Testing (LABDEM) belonging to the State University of Paraíba. Their activity was also assessed against Cryptococcus neoformans (LM10), provided by the Mycology Laboratory of the Federal University of Paraíba.

Determination of Minimum Inhibitory Concentration

The Minimum Inhibitory Concentration (MIC) of MEs was determined by the microdilution method using 96-well microplates, consisting of serial dilution either of drug and or the ME-5CN05 and ME-blank formulations, prepared following the guidelines of the Clinical and Laboratory Standards Institute, rule CLSI M27-A3 for yeasts. 5CN05 was dissolved in LAS® at a concentration of 2175 µg.mL-1 and then tested at concentrations ranging from 8 to 1088 µg.mL-1. Amphotericin B (AmB) was used as reference at concentrations from 2 to 0.015 µg.mL-1. The possible activity of LAS® was evaluated as well, using the broth without inoculum as a negative control.

To prepare inocula, microbial suspensions were standardized in tubes containing 5 mL of 0.9% sterile saline solution and adjusted spectrophotometrically to 90% trans-mittance at 530 nm, corresponding to a concentration of 106 cfu.mL-1. Samples were serially diluted in the micro-dilution wells containing 100 µL of Sabouraud-Dextrose broth (Hi Media, Sasti - Maharashtra, India). After addition of inoculum (100 µL) in separate plates for each yeast, the plates were incubated at 35 ± 1 °C for 24 h and visually evaluated. The MIC corresponded to the lowest sample dilution capable of providing growth inhibition of yeasts. All tests were performed in duplicate.

Results

Figure 2 shows the phase diagram characterizing the phase behavior of surfactants blends, namely the oil and water phases. It is noteworthy the existence of a variety of states, highlighting the presence of microemulsion formation areas, which are regions where the energy used and the components proportions were suitable to reduce the interfa-cial tension until the formation of a homogeneous, clear and translucent system (Silva et al., 2009a).


The composition of MEs was 35.3% w/w LAS®, 17.5% w/w Plurol Oleique®, 5.9% w/w isopropyl myristate and 41.2% w/w water. The ME-blank formulation was shown to be a translucent, slightly yellowish solution with slightly viscous appearance, while the addition of the drug at a concentration of 560 µg.mL-1 to prepare the ME-5CN05 formulation did not modify its translucent appearance and consistency (as confirmed by rheological characterization studies - data not shown), differing mainly in the color. Table 1 summarizes the main results of physico-chemical characterization tests performed on both formulations.

As expected, the tests of Minimum Inhibitory Concentration (MIC) either for the drug or MEs, whose results are listed in Table 2, showed a clear growth of all the yeasts only in the controls containing the inocula in addition to the culture medium, hence confirming the suitability of the selected conditions.

All the microorganisms studied proved sensitive to 5CN05, which showed moderate activity against Candida species (MIC = 270-540 µg.mL-1) and an activity more than one order of magnitude greater against Cryptococcus neoformans (MIC = 17 µg.mL-1), while the MIC value for the positive control (AmB) was only 0.5 µg.mL-1.

As far as the MEs are concerned, microorganisms did not show any sensitivity to ME-blank, while ME-5CN05 proved excellent activity against Candida species, mainly C. albicans and C. parapsilosis (MIC = 70 µg.mL-1), and excellent activity against C. neoformans (MIC = 2.2 µg.mL-1).

Discussion

All MEs were handled and maintained at room temperature for 48 h before their characterization. Under these conditions, all formulations exhibited a homogeneous appearance, without any evidence of phase separation or precipitate formation. As is well known, the optimum pH of a topical formulation must be selected so as to maximize the stability of active components and the tolerance of the skin, being acceptable values between 5.5 and 8.0 (Silva et al., 2009a). The pH values shown for ME-blank and ME-5CN05 were within this range, which would allowthe topical applicationofthe ME-5CN05 sys- tem. Moreover, this parameter was not influenced by the addition of 5CN05.

The use of topical MEs to improve the antifungal activity of drugs is a strategy adopted by some researchers. For instance, Jadhav, Shetye and Kadam (2010), developing MEs containing topical fluconazole, observed greater activity of their formulations than conventional gels available in the market, a fact that was attributed to the better diffusion of the drug provided by the presence of surfactants and oil phase. El-Hadidy et al. (2012), investigating the application of MEs as topical carriers for voriconazole, observed that their antifungal activity against C. albicans was considerably higher than that of a supersaturated solution of the same drug.

5CN05, either as such or in ME, showed greater activity against C. neoformans than against Candida spp. This activity profile is in agreement with the results of Mendonça Junior etal. (2011), who found that tiophene derivatives have moderate or weak antifungal activity against Candida strains and high activity against C. neoformans.

Nowadays, fungal infections are treated using a limited number of therapeutic agents, among which the main are echinocandins, polyenes (AmB), antimetabolites (flucytosine) and azoles (fluconazole), but only the last three have activity against C. neoformans (Hast et al., 2011). Despite its wide use, AmB has high nephrotoxicity and hepatotoxicity and may cause severe anemia (Tonomura et al., 2009). Azoles are the drugs of choice in the therapy of infections caused by Cryptococcus spp., but, although there is no report, as far as we are aware, on C. neoformans resistance to usual antifungals yet, it is certain that less susceptible strains will arise as a result of the extensive use of fluconazole in long-term or maintenance therapies (Guerra et al., 2012).

One of the most important factors of C. neoformans virulence is its polysaccharide capsule that has in its composition two different polysaccharides (90-95% of glucu-ronoxylomannan and 5-8% of galactoxylomannan) and mannoproteins (Vecchiarelli and Monari, 2012). This fungus has the ability to modulate the structure and size of its capsule during the infection so as to allow its adaptation to different environments and to make its dissemination in different tissues easier (Charlier et al., 2005). Such structural changes in the capsule have been reported as an important factor for the microorganism crossing of the blood-brain barrier (Guerrero et al., 2006; Jain, Guerrero and Fries, 2006). Therefore, one can assume that the activity of 5CN05, after its incorporation into a ME, is probably related to the interaction between lipid components of the system and fungal capsule, thereby increasing the contact area and making the drug access to the microorganism easier. Finally, we believe that the ME-5CN05 formulation developed in this work may act as a facilitator of drug permeation through the capsular material of C. neoformans.

The results of this work could be of some concern for the pharmaceutical industry, because they suggest an alternative therapy against C. neoformans.

Acknowledgments

This work was supported by PROCAD, Project N°: 552652/2011-3, Development of Processes and Pharmaceuticals and Biotechnological Processes Using the Sus-tainability Potential of Semiarid. The authors also acknowledge the Coordination of High-Level Personnel Training (CAPES), linked to the Brazilian Ministry of Education, for financial support of Prof. A. Converti as Special Visiting Researcher (Science without Borders Program, project n. 2609/2013), the State of São Paulo Research Foundation (FAPESP- Process n°: 2013/22212-0), the National Council for Scientific and Technological Development (CNPq) and the Center for Strategic Technology in the Northeast (CETENE) and Edital PROPESQ-UEPB n° 02/2010.

Submitted: April 11, 2013

Approved: September 9, 2013

All the content of the journal, except where otherwise noted, is licensed under a Creative Commons License CC BY-NC.

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  • Correspondence:
    J.A. Silva
    Graduation Program in Pharmaceutical Sciences
    Universidade Estadual da Paraíba
    Rua Juvêncio Arruda S/N, Bairro Universitário
    58429-600 Campina Grande, PB, Brazil
    E-mail:
  • Publication Dates

    • Publication in this collection
      30 Sept 2014
    • Date of issue
      June 2014

    History

    • Accepted
      09 Sept 2013
    • Received
      11 Apr 2013
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