Diphenyl Diselenide and Clotrimazole Co-loaded into Eudragit® RS 100 Nanocapsules Formulation Has Superior Antioxidant Potential and Promising Anti-candida Activity

Abstract In the current study, nanocapsules (NC) formulations containing a co-load of clotrimazole (C), a highly prescribed antifungal drug, and diphenyl diselenide [(PhSe)2], an organoselenium compound with a promising scope of pharmacological actions, were prepared. Formulations were characterized as well as the potential toxicity, antioxidant action, and antifungal effect were assessed using in vitro techniques. The NCs were prepared employing Eudragit® RS 100 as polymeric wall and medium chain triglycerides or virgin coconut oil (CO) as core. All NC suspensions had pH around acid range, compound content close to theoretical value (1 mg/mL/drug), average diameter in nanometric range, positive values of zeta potential as well as high encapsulation efficacy and mucoadhesive property. Physicochemical stability was performed over a 30-day period and showed no modification in the aforementioned parameters to all samples. Preliminary screening of toxicological potential performed by the hen's egg test chorioallantoic membrane technique classified the formulations as non-irritant. The DPPH radical assay revealed that nanoencapsulated compounds had superior antioxidant action in comparison to their free forms (concentration range tested 1.0-25.0 µg/mL). Importantly, the formulation composed of CO and containing C and (PhSe)2 showed the highest antioxidant potential and was selected for further investigation regarding antifungal effect against some Candida spp strains. Results of in vitro antifungal assay demonstrated that the C and (PhSe)2 co-encapsulation had a minimum inhibitory concentration (MIC) values around 60. Thus, our study supplies additional data about advantages achieved by encapsulating active compounds.

Comparison of the Minimum Fungicidal Concentration of Clotrimazole, Ketoconazole, Miconazole and Terbinafine Against Clinical Isolates of Dermatophytes.

Superficial fungal infections affect millions of people worldwide. Earlier most dermatophyte strains had relatively restricted geographical distribution. But currently, dermatophytosis has become one of the most common human infectious diseases worldwide. Fungal infections are common in hot and humid climate of tropical countries like India. Topical and systemic therapies are commonly used to treat dermatophyte infections.Clotrimazole is one of the most commonly used topical antifungal drugs. This study compared the minimum fungicidal concentration (MFC) of Clotrimazole with Miconazole, Ketoconazole and Terbinafine in skin dermatophytes. The study demonstrated that Clotrimazole had lower MFCs as compared to Ketoconazole and Miconazole against Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. Clotrimazole had comparable MFCs versus Terbinafine against Trichophyton rubrum but it had lower MFCs against Trichophyton mentagrophytes and Microsporum canis. Thus, Clotrimazole is an effective antifungal agent for dermatophytosis even today.The efficacy of Clotrimazole even against strains with intermediate resistance or resistance to the older azole anti fungal drugs reiterate the current decisions of empirical treatment with topical Clotrimazole for the management of superficial dermatophyte infections.

Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.

Disturbance in hemoglobin metabolism and in vivo antimalarial activity of azole antimycotics / Alteración en el metabolismo de la hemoglobina y actividad antimalárica in vivo de azoles antimicóticos

Plasmodium parasites degrade host hemoglobin to obtain free amino acids, essential for protein synthesis. During this event, free toxic heme moieties crystallize spontaneously to produce a non-toxic pigment called hemozoin or ß-hematin. In this context, a group of azole antimycotics, clotrimazole (CTZ), ketoconazole (KTZ) and fluconazole (FCZ), were investigated for their abilities to inhibit ß-hematin synthesis (IßHS) and hemoglobin proteolysis (IHbP) in vitro. The ß-hematin synthesis was recorded by spectrophotometry at 405 nm and the hemoglobin proteolysis was determined by SDS-PAGE 12.5 percent, followed by densitometric analysis. Compounds were also assayed in vivo in a malaria murine model. CTZ and KTZ exhibited the maximal effects inhibiting both biochemical events, showing inhibition of β-hematin synthesis (IC50 values of 12.4 ± 0.9 µM and 14.4 ± 1.4 µM respectively) and inhibition of hemoglobin proteolysis (80.1 ± 2.0 percent and 55.3 ± 3.6 percent, respectively). There is a broad correlation to the in vivo results, especially CTZ, which reduced the parasitemia ( percentP) of infected-mice at 4th day post-infection significantly compared to non-treated controls (12.4 ± 3.0 percent compared to 26.6 ± 3.7 percent, p = 0.014) and prolonged the survival days post-infection. The results indicated that the inhibition of the hemoglobin metabolism by the azole antimycotics could be responsible for their antimalarial effect.

Los parásitos del género Plasmodium degradan la hemoglobina hospedera obteniendo aminoácidos libres para su síntesis proteica. Durante este evento, unidades de hemo libre tóxicas cristalizan espontáneamente formando un pigmento no tóxico denominado ß-hematina. En este trabajo, se investigó la capacidad de un grupo de azoles antimicóticos: clotrimazol (CTZ), ketoconazol (KTZ) y fluconazol (FCZ), en inhibir la síntesis de ß-hematina y la proteólisis de la globina. La síntesis de ß-hematina se registro por espectrofotometría a 405 nm y la proteólisis de la hemoglobina se determino por SDS-PAGE 15 por ciento seguido por análisis densitométrico de las bandas de hemoglobina intactas. Los compuestos fueron también ensayados in vivo en un modelo de malaria murina. CTZ y KTZ inhibieron la síntesis de ß-hematina con CI50 entre 10 y 15 µM y bloquearon la proteólisis de la hemoglobina (80.01 ± 2.04 por ciento y 55.33 ± 3.57 por ciento, respectivamente). En relación directa con los resultados encontrados in vitro, el CTZ redujo la parasitemia de ratones infectados en forma significativa, así como prolongó lo días de sobrevivencia post-infección en comparación con animales controles no tratados. Se sugiere así que la inhibición del metabolismo de la hemoglobina por los antimicóticos azólicos pudiera ser el mecanismo responsable de su actividad antimalárica.

[In vitro susceptibility of Fluconazole, Clotrimazole and Toucrium Polium smoke product on Candida isolates of vaginal candidiasis]

It has been estimated that up to 75% of women in their child-bearing age have been affected by vulvovaginal candidiasis at least once in their life time. Almost 45% of women experience this infection two or more times. The antifungal azole group, in topical and oral forms, is the common way of therapy. Herbal products are often used for vulvovaginal therapy. Nowadays, Toucrium polium [TP] products are being used as traditional medicine to reduce signs of Candida vaginitis. There is no study regarding to antifungal activity of TP smoke product in Iran. The aim of this study was to evaluate the In vitro activity of TP smoke product against Candida, isolated from women with Candida vaginitis, compared with antifungal drugs which are ordinary used to cure Candida vaginitis. The present study was conducted at the University of Medical Sciences of Shiraz in 1387 [2008]. During seven months, samples were taken from 450 patients suffering from urogenital infections and 105 Candida vaginitis were detected. Germ tube test was used for identification of fungal species. TP smoke product was prepared in suitable potency. Antifungal activity of fluconazole, clotrimazole and TP product were evaluated by disk diffusion method. Sterile blank disks were loaded by TP smoke product in potency of 10-240 microliter/disk. Inhibition zone around the disks were measured and compared with each other. 105 Candida species were isolated from the patients. Candida species were identified by germ tube test as Candida albicans 74 [70.5%] and Candida non-albicans 31[29.5%].The mean of inhibition zone around the clotrimazole disks was 22 +/- 5.39 along with one case of resistance. Forty seven species had resistance to fluconazole while 94% and 55.2% of all samples were sensitive to clotrimazole and fluconazole respectively. All of the clinical isolates and standard Candida species were sensitive to TP smoke product. Considering the resistance of Candida species to antifungal drugs and good antifungal activity of TP smoke product, it is necessary to analyze the main compounds and chemical contents of TP extract for better evaluation of its antifungal activity

Susceptibilidad in vitro de aislamientos vaginales de Candida frente a clotrimazol y nistatina

Se determinó la susceptibilidad in vitro frente a clotrimazol y nistatina de 123 aislamientos de Candida, obtenidos mediante exudados vaginales de 404 mujeres que asistieron al Hospital Ginecoobstétrico "Ramón González Coro" de Ciudad de La Habana. De acuerdo con el número de colonias obtenidas en el aislamiento primario, las cepas fueron separadas en 2 categorías: colonización e infección. Para cada cepa se determinó la concentración mínima inhibitoria (CMI) de cada antifúngico, mediante un método de microdilución en caldo casitona. Las medias geométricas de los valores de CMI fueron mayores frente a nistatina (1,08 mg/mL) que frente a clotrimazol (0,22 mg/mL), aunque los rangos fueron similares (£ 0,125-16 mg/mL). Para Candida albicans, que fue la especie aislada con mayor frecuencia (54,5 %); las medias geométricas de los valores de la CMI fueron de 0,17 y 0,16 mg/mL para clotrimazol y de 0,71 y 0,92 mg/mL para nistatina, en ambas categorías. C. glabrata mostró el valor de CMI más elevado (16 mg/mL) frente a ambos antifúngicos. Solo los aislamientos de C. lusitaniae mostraron diferencia significativa (p < 0,01) entre los valores de CMI de nistatina de acuerdo con el número de colonias en el aislamiento primario. En aislamientos procedentes de mujeres tratadas con clotrimazol y/o nistatina en episodios anteriores de candidiasis, se observaron valores más elevados de las medias de CMI frente a nistatina que frente a clotrimazol; aunque esta diferencia no fue estadísticamente significativa.

The susceptibility in vitro of 123 isolates of Candida against clotrimazole and nystatin was determined. The isolates were obtained by vaginal smears from 404 women that attended "Ramón Gonzalez Coro" Gynecoobstetric Hospital, in Havana City According to the number of colonies obtained in the primary isolation, the strains were separated into 2 categories: colonization and infection. The inhibitory minimum concentration was determined for each antigunfal by a method of microdilution in casitone broth. The geometrical means of the IMC values were higher against nystatin (1.08 mg/mL) than against clotrimazole (0.22 mg/mL), eventhough the features were similar ( £ 0.125 -16 mg/mL) For Candida albicans, that was the most frequently isolated species (54.5 %), the geometrical means of the IMC values were 0.17 and 0.16 mg/mL for clotrimazole and 0.71 and 0.92 for nystatin, in both categories. C. glabrata showed the highest IMC values (16 mg/mL) against antifungals. Only the isolates of C. lusitaniae showed a significant difference (p < 0.001) between the IMC values of nystatin according to the number of colonies in the primary isolation. In isolates from women treated with clotrimazole and/or nystatin in previous episodes of candidiasis, there were observed more elevated values of the means of IMC against nystatin than against clotrimazole, although this difference was not statistically remarkable.

Efectos de ketoconazol, clotrimazol, nistatina y cicloheximida sobre el crecimiento de phaffia rhodozyma / Effects of ketoconazole, clotrymazole, nystatin and cycloheximide on the growth of phaffia rhodozyma

Debido a la importancia biotecnológica en la producción de pigmentos, se determinaron los niveles de sensibilidad a antifúngicos de 3 cepas de Phaffia rhodozyma, con la finalidad de obtener mutantes resistentes, característica usada como marcador en el análisis genético de diferentes microorganismos. El desarrollo de las tres cepas, fue inhibido por ketoconazol, clotrimazol, nistatina y cicloheximida respectivamente. La cepa UCD 67-210 resultó ser la más sensible al ketoconazol, siendo inhibida por concentraciones de 5 ug/ml de dicho compuesto. Para las cepas UCD 67-383 y UCD 67-385 se requieren concentraciones de 20 ug/ml. El cotrimazol, inhibió el crecimiento de las cepas UCD 67-210 y UCD 76-385, a una concentración de 10 ug/ml y a la cepa UCD 67-383 a 20 ug/ml. La nistatina inhibe el crecimiento de las cepas UCD 67-383 y UCD 67-385 a una concentración de 3 ug/ml y a la cepa UCD 67-210 a 5 ug/ml. La cicloheximida impide el desarrollo de las cepas UCD 67-210 y UCD 67-385 a una concentración de 2 ug/ml. La cepa UCD 67-383 resultó ser la más resistente, requiriéndose concentraciones sobre 6 ug/ml para inhibir su crecimiento

Study of combination effects of nystatin and clotrimazole with some antibiotics on candida albicans

Binary combinations between 2 antifungal agents nystatin and clotrimazole and 4 antibiotics tetracyclines, chloramphenicol, rifampicin and gentamicin were tried using 7 Candida albicans strains isolated from clinical specimen using the checker board method. The best combinations were obtained with nystatin-tetracyclines and clotrimazole- gentamicin. However, synergistic activity between the 2 antifungal agents and these antibiotics was found only at high concentrations of the latter 500 ug - 125 ug/ml, thus limiting its application in vivo

Un estudio comparativo de isoconazol vs clotrimazol en micosis vaginal / A comparative study of isoconazole vs clotrimazole in vaginal mycosis

Se estudiaron 60 pacientes en la Unidad Sanitaria y Clínica GUERRA MAS de Puerto Cabello, en pacientes sintomáticas y con verificación mediante cultivos de micosis vaginal a Cándida Albicans. Mediante procedimiento randomizado se constituyeron dos grupos a ser tratados con Isoconazol un óvulo vaginal-dosis única en 30 pacientes y un segundo grupo de 30 pacientes tratados con Clotrimazol una tableta vaginal, dosis única. Se incluyeron pacientes portadores de factores predisponentes con anticonceptivos y embarazos. Los resultados obtenidos revelaron desaparición de síntomas en ambos grupos y los cultivos micológicos de control a las 3 ó 4 semanas fueron negativos en el 90% como promedio. Resalta la mayor aceptabilidad de la paciente al óvulo que a la tableta vaginal. Se concluye que ambos medicamentos cumplen un nivel excelente de efectividad en el tratamiento de la micosis vaginal

Susceptibilidad "in vitro" de cepas de dermatófitos frente a drogas utilizadas en micoticoterapia: método cuantitativo para hongos filamentosos / In vitro susceptibility of dermatophyte strains to drugs used in mycosis therapy: quantitative method for filamentos fungi

Teniendo en cuenta las diversas técnicas que se emplean para determinar el poder antimicótico de algunas drogas sobre hongos filamentosos, se realizó un modelo experimental que permitió evaluar cuantitativamente la acción fungicida de distintos quimioterápicos en diferentes cepas de dermatofitos (aislados de dermatomicosis). Para determinar cuantitativamente el desarrollo de un hongo filamentoso se lo disgregó en solución fisiológica estéril con cidrio molido o utilizando un homogeneizador de tejidos. En el caso de dermatofitos los inóculos fueron llevados a concentración final de 400 macroconidias/ml para las distintas cepas de Microsporum, 44 macroconidias/ml en las cepas de Epidermophyton y 125 microconidias/ml en las cepas de Trichophyton, utilizando para tal fin una cámara de Neubauer. Con los inóculos obtenidos de esta manera se realizaron las diluciones apropiadas para contar en placas con Sabourad-dextrosa 160-180 unidades formadoras de colonias (UFC) para las cepas pertenecientes al género Microsporum, 100-150 UFC para las pertenecientes al género Epidermophyton y 160 UFC en las cepas de Trichophyton, al cabo de 7 días de incubación a 28-C. Al utilizar este modelo experimental con concentraciones crecientes de diferentes antifúngicos se puede determinar cuantitativamente la acción de éstos "in vitro", de acuerdo a la disminución del número de UFC de las cepas de dermatofitos estudiadas. Para evaluar esta metodología de cuantificación se utilizaron los dermatofitos. Puede ser aplicable a otros hongos filamentosos inclusive a los productores de micoses sistémicas