{"title":"The active metabolite hydroxyitraconazole has substantially higher in vivo free fraction and free concentrations compared to itraconazole","authors":"Motoshi Iwao , Ryota Tanaka , Yosuke Suzuki , Ryosuke Tatsuta , Takehiro Hashimoto , Kazufumi Hiramatsu , Hiroki Itoh","doi":"10.1016/j.jpba.2025.116775","DOIUrl":null,"url":null,"abstract":"<div><div>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 <em>in vivo</em> 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 <em>in vitro</em> antifungal activity, OH-ITCZ may contribute more to <em>in vivo</em> antifungal efficacy than ITCZ, suggesting that monitoring OH-ITCZ would be more beneficial.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"259 ","pages":"Article 116775"},"PeriodicalIF":3.1000,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of pharmaceutical and biomedical analysis","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0731708525001165","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/2/25 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
引用次数: 0
Abstract
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.
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This journal is an international medium directed towards the needs of academic, clinical, government and industrial analysis by publishing original research reports and critical reviews on pharmaceutical and biomedical analysis. It covers the interdisciplinary aspects of analysis in the pharmaceutical, biomedical and clinical sciences, including developments in analytical methodology, instrumentation, computation and interpretation. Submissions on novel applications focusing on drug purity and stability studies, pharmacokinetics, therapeutic monitoring, metabolic profiling; drug-related aspects of analytical biochemistry and forensic toxicology; quality assurance in the pharmaceutical industry are also welcome.
Studies from areas of well established and poorly selective methods, such as UV-VIS spectrophotometry (including derivative and multi-wavelength measurements), basic electroanalytical (potentiometric, polarographic and voltammetric) methods, fluorimetry, flow-injection analysis, etc. are accepted for publication in exceptional cases only, if a unique and substantial advantage over presently known systems is demonstrated. The same applies to the assay of simple drug formulations by any kind of methods and the determination of drugs in biological samples based merely on spiked samples. Drug purity/stability studies should contain information on the structure elucidation of the impurities/degradants.
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