Actividad in vitro e in vivo de nuevos antifúngicos frente a hongos oportunistas

Abstract

Las terapias utilizadas actualmente para el tratamiento de infecciones fúngicas invasoras causadas por levaduras oportunistas están lejos de ser las óptimas. Estas infecciones afectan principalmente a pacientes inmunocomprometidos, dando lugar a tasas de mortalidad muy elevadas. Aunque varias especies de Candida son las responsables del mayor porcentaje de infecciones fúngicas invasoras causadas por levaduras oportunistas, otros géneros, como Cryptococcus, Trichosporon, Blastoschizomyces, Rhodotorula y Sporobolomyces ocasionan también infecciones de incidencia creciente y de difícil tratamiento, existiendo pocos estudios, tanto in vitro como in vivo, acerca de la eficacia de nuevos antifúngicos. El objetivo de la presente tesis es contribuir al desarrollo experimental de nuevos tratamientos o a la mejora de los tratamientos ya existentes. Para ello, se han realizado estudios in vitro para evaluar la eficacia in vitro de los antifúngicos, para posteriormente, comprobar la eficacia de los fármacos en modelos animales adecuados para cada estudio. <br/><br/> Las diferentes cepas de Trichosporon asahii estudiadas han mostrado ser resistentes in vitro a los antifúngicos usualmente utilizados en los tratamientos de las infecciones diseminadas por levaduras (la anfotericina B y el fluconazol), siendo por el contrario sensibles a los nuevos triazoles, voriconazol, ravuconazol y albaconazol.. Además el voriconazol ha resultado eficaz en el tratamiento de la tricosporonosis diseminada experimental en el cobaya inmunodeprimido, sugiriendo su potencial utilidad en la terapia de las infecciones humanas producidas por T. asahii. También se ha estudiado la actividad de varias combinaciones de antifúngicos, obteniéndose con la combinación de micafungina y anfotericina B un 100% de interacciones sinérgicas in vitro frente a T. asahii. Esta asociación de antifúngicos ha mostrado una excelente eficacia en el tratamiento experimental de la infección diseminada producida por T. asahii en el ratón.<br/><br/> Se ha desarrollado por primera vez un modelo animal de infección diseminada por Blastoschizomyces capitatus en el que se ha estudiado la eficacia del tratamiento con diferentes antifúngicos, clásicos y de reciente introducción. El fluconazol a dosis elevadas ha demostrado ser el tratamiento más efectivo de los ensayados frente a la infección experimental causada por B. capitatus. <br/> La combinación de la micafungina con la anfotericina B ha mostrado el mayor porcentaje de interacciones sinérgicas in vitro frente a 40 cepas estudiadas pertenecientes a las cuatro especies de Cryptococcus de mayor relevancia clínica. Se ha estudiado por primera vez el tratamiento con voriconazol en un modelo experimental de criptococosis cerebral en el ratón, demostrándose una eficacia excelente, lo que sugiere la potencial utilidad de este antifúngico en el tratamiento de la infección humana. <br/><br/> Se ha evaluado la actividad in vitro de la micafungina combinada con la anfotericina B frente a 115 cepas pertenecientes a las siete especies de Candida de mayor importancia clínica. La disparidad de resultados obtenida en función del criterio de lectura de la CMI, evidencia la necesidad de definir los puntos de corte adecuados para la lectura de las pruebas de sensibilidad in vitro frente a combinaciones de antifúngicos cuando se utiliza la técnica del damero.<br/><br/> Se ha estudiado la actividad in vitro de 8 antifúngicos y de las combinaciones de la micafungina con 5 de ellos frente a diferentes cepas de Rhodotorula glutinis y Sporobolomyces salmonicolor. Únicamente la anfotericina B, el ravuconazol y el albaconazol mostraron actividad in vitro frente a R. glutinis. Los triazoles itraconazol, voriconazol, ravuconazol y albaconazol y la terbinafina presentaron buena actividad in vitro frente a S. salmonicolor. Todas las combinaciones de antifúngicos estudiadas con excepción de la combinación de la micafungina con el fluconazol mostraron un alto porcentaje de interacciones sinérgicas frente a R. glutinis. La combinación de la micafungina con el itraconazol dio lugar al mayor porcentaje de interacciones sinérgicas frente a S. salmonicolor.

The conventional antifungal therapies commonly used for the treatment of systemic fungal infections due to opportunistic pathogenic yeasts are far to be the optimal. These infections affect mainly immunocompromised patients, causing high mortality rates. Although Candida spp. are the most common cause of fungal infections, other genera such as Cryptococcus, Trichosporon, Blastoschizomyces, Rhodotorula and Sporobolomyces, have also frequently reported. In general, these latter genera are less susceptible to conventional antifungal treatments and the number of in vitro and in vivo studies about the efficacy of new antifungal treatment against them is very scarce. The main objective of this thesis is to evaluate both in vitro and in animal models the efficacy of new therapeutical strategies. <br/> <br/>We have evaluated the in vitro activity of micafungin combined with amphotericin B against 115 strains belonging to the seven species of Candida most commonly found in clinical samples (20 of C. albicans, 20 of C. dubliniensis, 15 of C. glabrata, 20 of C. krusei, 10 of C. lusitaniae, 15 of C. parapsilosis and 15 of C. tropicalis).. When we used the MIC-2 endpoint criterion such combination showed synergism against all the species tested. <br/><br/>We have evaluated the in vitro activity of micafungin combined with each of the following drugs: amphotericin B, fluconazole, itraconazole, voriconazole and ravuconazole, against 37 strains belonging to the four most clinically relevant species of Cryptococcus i.e. C. neoformans, C. gattii, C. albidus and C. laurentii. The combination micafungin with amphotericin B showed the best synergism, with 70% of synergistic interactions. <br/><br/>Voriconazole showed an excellent activity in a murine model of central nervous system infection by Cryptococcus neoformans. We studied the efficacy of voriconazole administered at 10, 40 and 60 mg/kg/day, and amphotericin B administered at 1.5 mg/kg/day. All treatments significantly prolonged survival of mice infected with the three strains of C. neoformans tested with respect to the control group. In addition, all the treatments reduced significantly the fungal loads versus controls. However, the best results were obtained with voriconazole administered at 60 mg/kg/day. <br/><br/>Trichosporon asahii is an opportunistic fungus usually resistant in vitro to the conventional antifungals used in systemic infections caused by yeasts. On the other hand, the new triazoles, voriconazole, albaconazole and ravuconazole, have shown an excellent activity. We have evaluated the efficacy of voriconazole in a guinea pig model of trichosporonosis by T. asahii. Voriconazole was effective in resolving such infection. asahii and may represent an important advance in the therapy of this disease. We studied also the efficacy of combined therapy against T. asahii infections. In an in vitro study we tested micafungin combined with five antifungal drugs against ten strains of T. asahii. The combination micafungin with amphotericin B showed 100% of synergistic interactions. With the other combinations tested (micafungin combined with fluconazole, itraconazole, voriconazole or ravuconazole) we obtained between 40 and 20% of synergistic interactions. We selected the combinations that showed the best results i.e, micafungin plus amphotericin B or fluconazole, in the same murine model. Both combinations were effective in prolonging the mean survival time and also significantly reducing the fungal load respect the control group, but only the combination of micafungin with amphotericin B was able to improve the results obtained with monotherapies. <br/><br/>We established a reproducible murine model of disseminated blastoschizomycosis to evaluate the effectiveness of the most commonly used antifungal therapies against this infection. We evaluate the efficacy of amphotericin B at 1 and 3 mg/kg/day, fluconazole at 40 and 80 mg/kg/day, flucytosine at 60 mg/kg/day and voriconazole at 40 mg/kg/day, all them against two strains of B. capitatus. This study demonstrated the potential use of this murine model for evaluating therapies against systemic blastoschizomycosis and the highest efficacy of FLC administered at 80 mg/kg/day. Combined therapy has not yet been investigated in blastoschizomycosis and this could be a good alternative for those infections caused by azole-resistant isolates which are increasing among clinical yeasts in general. Since several cases of FLC failure in B, capitatus infections have been documented we decided to evaluate the efficacy of amphotericin B combined with voriconazole, flucytosine or micafungin, using the murine model developed in our previous study. In general, the combinations tested showed better results than the control group and their monotherapies, but they could not improve the efficacy of FLC at high doses. <br/><br/><br/><br/>We have also studied the in vitro activity of eight antifungals (amphotericin B, fluconazole, itraconazole, voriconazole, ravuconazole, albaconazole, micafungin and terbinafine) and the interactions of micafungin with five antifungals (amphotericin B, fluconazole, itraconazole, voriconazole or ravuconazole) against 10 strains of Rhodotorula glutinis and 10 of Sporobolomyces salmonicolor. Only amphotericin B, ravuconazole and albaconazole showed low MIC values against R. glutinis. For this reason, we consider interesting the finding that all the antifungal combinations showed a high percentage of synergic interactions (60-80%) with the exception of micafungin plus fluconazole that only showed 20%. Against S. salmonicolor, the amphotericin B MICs were very variable with a 6-fold difference between the lowest and the highest values (0.25-16 mg/L). The best combination was micafungin plus itraconazole with 60% synergic interactions. The other combinations only showed 20-40% synergic interactions. Although these percentages are not so high as for the other species tested, some of these combined therapies could be helpful when amphotericin B fails.