RESUMEN
In vitro susceptibility assays of antifungal activity do not always accurately predict in vivo efficacy. As well as having a clear clinical importance, the ability to predict efficacy is also essential for effective screening of novel drug compounds. Initial screening of novel compounds must often be based on in vitro data. The present report describes the use of serum-MIC, an in vitro test of antifungal susceptibility, to accurately predict in vivo efficacy of echinocandin drugs in a mouse model of disseminated candidiasis. The basis of the serum-MIC method was to measure the inhibitory activity of a test compound against Candida albicans hyphal growth in the presence of pooled mouse serum. For 13 previously uncharacterized echinocandin compounds, as well as for the known echinocandin drugs, micafungin and caspofungin, serum-MIC determinations were shown to give better correlation to efficacy in the animal model than conventional, CLSI standard, in vitro antifungal susceptibility tests. The most accurate prediction of efficacy was obtained when the serum-MIC was adjusted in relation to the serum concentration at 30 min post-treatment. Furthermore, when the efficacy of micafungin was determined by measuring C. albicans kidney burden in the mouse model of infection, the adjusted serum-MIC consistently reflected the effective serum concentrations. Our data indicate that determination of serum-MIC values will facilitate prediction of the in vivo potency of new antifungal compounds such as novel echinocandins.
Asunto(s)
Antifúngicos , Candida albicans , Candidiasis/tratamiento farmacológico , Equinocandinas , Pruebas de Sensibilidad Microbiana/métodos , Suero/microbiología , Animales , Antifúngicos/farmacocinética , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Candidiasis/microbiología , Candidiasis/mortalidad , Equinocandinas/farmacocinética , Equinocandinas/farmacología , Equinocandinas/uso terapéutico , Hifa/crecimiento & desarrollo , Lipopéptidos , Lipoproteínas/farmacocinética , Lipoproteínas/farmacología , Lipoproteínas/uso terapéutico , Masculino , Micafungina , Ratones , Ratones Endogámicos ICR , Valor Predictivo de las Pruebas , Resultado del TratamientoRESUMEN
The aim of this study was to compare the pharmacodynamics of the azole antifungal drugs fluconazole, itraconazole and ketoconazole, and the polyene antifungal amphotericin B, in a mouse model of disseminated Candida albicans infection. In order to directly compare effective serum concentrations of these antifungals, drug concentrations were assayed microbiologically by measuring inhibition of C. albicans mycelial growth (mMIC) in a mouse serum-based assay (serum antifungal titer). Efficacy in the mouse infection model was determined using an organ-based (kidney burden) endpoint. For all four drugs, the serum antifungal titers, 8 hr after administration of single doses of drugs at a range of drug concentrations, correlated closely with C. albicans kidney fungal burden in the mouse model. The results showed that determining serum antifungal titer may be used to accurately represent kidney fungal burden in a mouse model of disseminated candidiasis and allowed direct comparison of the pharmacodynamics of differing classes of antifungal drugs.
Asunto(s)
Antifúngicos/farmacocinética , Candidiasis/metabolismo , Anfotericina B/farmacocinética , Animales , Antifúngicos/sangre , Candida albicans/efectos de los fármacos , Candidiasis/sangre , Modelos Animales de Enfermedad , Fluconazol/farmacocinética , Itraconazol/farmacocinética , Riñón/metabolismo , Ratones , Pruebas de Sensibilidad MicrobianaRESUMEN
To establish an in vitro method of predicting in vivo efficacy of antifungal drugs against Candida albicans and Aspergillus fumigatus, the antifungal activities of fluconazole, itraconazole, and amphotericin B were determined in mouse serum. The minimum inhibitory concentration (MIC) of each drug was measured using mouse serum as a diluent. For C. albicans, the assay endpoint of azoles was defined as inhibition of mycelial extension (mMIC) and for A. fumigatus, as no growth (MIC). The MICs of amphotericin B for both pathogens were defined as the MIC at which no mycelial growth occurred. Serum MIC or mMIC determinations were then used to estimate the concentration of the drugs in serum of mice treated with antifungal drugs by multiplying the antifungal titer of the serum samples by the serum (m)MIC. The serum drug concentrations were also determined by HPLC. The serum concentrations estimated microbiologically showed good agreement with those determined by HPLC, except for itraconazole. Analysis of the serum samples from itraconazole-treated mice by a sensitive bioautography revealed the presence of additional spots, not seen in control samples of itraconazole. The bioautography assay demonstrated that the additional material detected in serum from mice treated with itraconazole was an active metabolite of itraconazole. The data showed that the apparent reduction in the itraconazole serum concentration as determined by HPLC was the result of the formation of an active metabolite, and that the use of a microbiological method to measure serum concentrations of drugs can provide a method for prediction of in vivo efficacy of antifungal drugs.
Asunto(s)
Antifúngicos/sangre , Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Candida albicans/efectos de los fármacos , Itraconazol/sangre , Itraconazol/farmacología , Animales , Antifúngicos/farmacocinética , Biotransformación , Itraconazol/farmacocinética , Masculino , Ratones , Ratones Endogámicos ICR , Pruebas de Sensibilidad MicrobianaRESUMEN
We studied the anti-Aspergillus activity of micafungin by using two fluorescent dyes to detect cell viability. Micafungin induced flattened hyphae, caused by the bursting of cells, which had lost their viability. Micafungin has killing activity against actively growing hyphae, even though it is not fungicidal against the whole burden of Aspergillus fumigatus.
Asunto(s)
Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Lipoproteínas/farmacología , Péptidos Cíclicos/farmacología , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/metabolismo , Barbitúricos/metabolismo , Equinocandinas , Fluoresceínas/metabolismo , Colorantes Fluorescentes , Hifa/efectos de los fármacos , Hifa/crecimiento & desarrollo , Hifa/metabolismo , Isoxazoles/metabolismo , Lipopéptidos , Micafungina , Microscopía Fluorescente , Microscopía de InterferenciaRESUMEN
The in vivo micronucleus test using mouse colonic epithelial cells was evaluated as the 11th collaborative study organized by the Collaborative Study Group on the micronucleus test (CSGMT) with three model chemicals that were known to induce chromosome damage in mouse colonic cells. Five laboratories participated in this validation study. All three model chemicals, i.e. 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), N-methyl-N-nitrosourea (MNU), and mitomycin C (MMC), induced micronucleated colonic epithelial cells in a 4-day exposure protocol in all participating laboratories. We confirmed that the present single cell suspension method could be used to detect the model chemicals as micronucleus inducers in mouse colonic epithelial cells. Advantages of this method are that experiments are easy to perform and that intact cells can be analyzed. The present study suggested that the colon micronucleus assay proposed here is useful for mechanistic studies of colon carcinogenesis.