Our aim was to detect the presence of an alternative oxidase (AOX) in Candida krusei clinical strains and its influence on fluconazole susceptibility and in reactive oxygen species (ROS) production. Candida krusei clinical isolates were tested to evaluate the presence of AOX. Debaromyces hansenii 2968 (AOX positive) and Saccharomyces cerevisiae BY4742 (AOX negative) were used as control strains. Measurements of oxygen consumption were performed in the presence of 1 mM KCN, an inhibitor of the classical respiratory chain, and 5 mM salicylhydroxamic acid (SHAM). AOX expression was monitored by Western blotting using an AOX monoclonal antibody. Interactions between fluconazole and SHAM were performed using checkerboard assay. ROS production was evaluated in the presence of SHAM plus fluconazole, H(2) O(2) , menadione, or plumbagin. AOX was present in all C. krusei tested. The combination of fluconazole with SHAM resulted in an indifferent effect. In the presence of SHAM, the treatment with ROS inductors or fluconazole increased ROS production, except in the AOX-negative strain. An alternative respiratory pathway resistant to cyanide is described for the first time as a characteristic of C. krusei species. This AOX is unrelated to fluconazole resistance; however, it protects C. krusei from oxidative stress.

Mechanoactivation has attracted considerable attention in the pharmaceutical sciences due to its ability to generate amorphous materials and solid-state synthetic products without the use of solvent. Although some studies have reported drug degradation during milling, no studies have systematically investigated the use of mechanoactivation in predicting drug degradation in the solid state. Thus, this work explores the autoxidation of drugs in the solid state by comilling amorphous mifepristone (MFP):polyvinylpyrrolidone vinyl acetate (PVPVA) and amorphous olanzapine (OLA):PVPVA. MFP was amorphized by ball milling and OLA by quench cooling techniques. Subsequently, comilling the amorphous drugs in the presence of a 10-fold weight ratio of PVPVA (the excipient containing reactive free radicals) was performed at several milling frequencies to identify the kinetics of mechano-autoxidation over milling durations. Overall, milling led to the degradation of up to 5% drug in the solid state. The autoxidation mechanism was confirmed by performing a stress study in the solution at 50 °C for 5 h, by using a 10 mM azo-bis(isobutyronitrile) (AIBN) as a stressing agent. By deconvoluting the effect of milling frequency and the energy on the extent and kinetics of milling-induced autoxidation of amorphous drugs, it was possible to fit an extended Arrhenius model that allowed extrapolation of mechanoactivated degradation rates (K m) to zero milling frequencies. Further, the autoxidation rates of drugs stored at high temperatures were observed to follow an Arrhenius behavior. A good degree of agreement was observed between the model predictions obtained by mechanoactivation (K m) to the reaction rates observed under accelerated temperatures. Additionally, the impact of adding an antioxidant (e.g., butylated hydroxytoluene) to the mixture during comilling was also examined. This study can be helpful in evaluating the stability of amorphous solids stored in accelerated (non-hermetic) conditions, in screening solid-state autoxidation propensity of drugs, and for the rational selection of antioxidants.