The Active Metabolite Hydroxyitraconazole Has Substantially Higher in Vivo Free Fraction and Free Concentrations Compared to Itraconazole

Itraconazole (ITCZ) is a triazole antifungal agent that is metabolized to many products. Hydroxyitraconazole (OH-ITCZ) is the major metabolite with antifungal activity comparable to that of ITCZ. Protein-free drug concentration has been reported to be a better biomarker for pharmacodynamics compared with total drug concentration. We developed an assay for quantification of free ITCZ and free OH-ITCZ concentrations using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) with equilibrium dialysis. The assay fulfilled the requirements of the US Food and Drug Administration guideline for assay validation, with lower limit of quantification of 0.025 and 0.25 ng/mL for free ITCZ and OH-ITCZ, respectively, than previous studies. Using this validated assay, we measured plasma free ITCZ and OH-ITCZ concentrations in 18 samples of 11 adult patients who received oral ITCZ between July 2016 and November 2022 at Oita University Hospital, and compared the in vivo percent free fraction and free concentration between the two compounds. Average plasma free concentrations and percent free fraction in 18 samples were, respectively, 0.188 ± 0.123 ng/mL and 0.024 ± 0.016 % for ITCZ, and 1.449 ± 1.017 ng/mL and 0.251 ± 0.109 % for OH-ITCZ, indicating that OH-ITCZ was 8.52-fold higher in percent free fraction and 10.42-fold higher in free concentration compared to ITCZ. Given that OH-ITCZ and ITCZ have similar in vitro antifungal activity, OH-ITCZ may contribute more to in vivo antifungal efficacy than ITCZ, suggesting that monitoring OH-ITCZ would be more beneficial.