Subash Sandhya, Singh Dilip K, Ahire Deepak S, Khojasteh S Cyrus, Murray Bernard P, Zientek Michael A, Jones Robert S, Kulkarni Priyanka, Smith Bill J, Heyward Scott, Cronin Ciarán N, Prasad Bhagwat
Department of Pharmaceutical Sciences, Washington State University (WSU), Spokane, Washington (S.S., D.K.S., D.S.A., B.P.); Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (S.C.K., R.S.J.); Drug Metabolism, Gilead Sciences, Foster City, California (B.P.M., B.J.S.); Drug Metabolism and Pharmacokinetics, Takeda Development Center Americas, San Diego, California (M.A.Z.); Drug Metabolism and Pharmacokinetics, Takeda Development Center Americas, Cambridge, Massachusetts (P.K.); BioIVT Inc., Baltimore, Maryland (S.H.); and Structural Biology and Protein Sciences, Pfizer Global Research & Development and Medical, La Jolla, California (C.N.C.).
Department of Pharmaceutical Sciences, Washington State University (WSU), Spokane, Washington (S.S., D.K.S., D.S.A., B.P.); Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California (S.C.K., R.S.J.); Drug Metabolism, Gilead Sciences, Foster City, California (B.P.M., B.J.S.); Drug Metabolism and Pharmacokinetics, Takeda Development Center Americas, San Diego, California (M.A.Z.); Drug Metabolism and Pharmacokinetics, Takeda Development Center Americas, Cambridge, Massachusetts (P.K.); BioIVT Inc., Baltimore, Maryland (S.H.); and Structural Biology and Protein Sciences, Pfizer Global Research & Development and Medical, La Jolla, California (C.N.C.)
Drug Metab Dispos. 2023 Oct;51(10):1362-1371. doi: 10.1124/dmd.123.001379. Epub 2023 Jul 10.
We investigated the effect of variability and instability in aldehyde oxidase (AO) content and activity on the scaling of in vitro metabolism data. AO content and activity in human liver cytosol (HLC) and five recombinant human AO preparations (rAO) were determined using targeted proteomics and carbazeran oxidation assay, respectively. AO content was highly variable as indicated by the relative expression factor (REF; i.e., HLC to rAO content) ranging from 0.001 to 1.7 across different in vitro systems. The activity of AO in HLC degrades at a 10-fold higher rate in the presence of the substrate as compared with the activity performed after preincubation without substrate. To scale the metabolic activity from rAO to HLC, a protein-normalized activity factor (pnAF) was proposed wherein the activity was corrected by AO content, which revealed up to sixfold higher AO activity in HLC versus rAO systems. A similar value of pnAF was observed for another substrate, ripasudil. Physiologically based pharmacokinetic (PBPK) modeling revealed a significant additional clearance (CL; 66%), which allowed for the successful prediction of in vivo CL of four other substrates, i.e., O-benzyl guanine, BIBX1382, zaleplon, and zoniporide. For carbazeran, the metabolite identification study showed that the direct glucuronidation may be contributing to around 12% elimination. Taken together, this study identified differential protein content, instability of in vitro activity, role of additional AO clearance, and unaccounted metabolic pathways as plausible reasons for the underprediction of AO-mediated drug metabolism. Consideration of these factors and integration of REF and pnAF in PBPK models will allow better prediction of AO metabolism. SIGNIFICANCE STATEMENT: This study elucidated the plausible reasons for the underprediction of aldehyde oxidase (AO)-mediated drug metabolism and provided recommendations to address them. It demonstrated that integrating protein content and activity differences and accounting for the loss of AO activity, as well as consideration of extrahepatic clearance and additional pathways, would improve the in vitro to in vivo extrapolation of AO-mediated drug metabolism using physiologically based pharmacokinetic modeling.
我们研究了醛氧化酶（AO）含量和活性的变异性及不稳定性对体外代谢数据缩放的影响。分别使用靶向蛋白质组学和卡巴泽兰氧化测定法测定了人肝细胞溶质（HLC）和五种重组人AO制剂（rAO）中的AO含量和活性。AO含量高度可变，不同体外系统中的相对表达因子（REF，即HLC与rAO含量之比）范围为0.001至1.7。与无底物预孵育后进行的活性相比，在有底物存在的情况下，HLC中AO的活性降解速率高10倍。为了将rAO的代谢活性缩放至HLC，提出了一种蛋白质标准化活性因子（pnAF），其中活性通过AO含量进行校正，结果显示HLC中的AO活性比rAO系统高多达六倍。对于另一种底物瑞巴派特，观察到了相似的pnAF值。基于生理的药代动力学（PBPK）模型显示出显著的额外清除率（CL；66%），这使得能够成功预测其他四种底物（即O-苄基鸟嘌呤、BIBX1382、扎来普隆和佐尼普明）的体内CL。对于卡巴泽兰，代谢物鉴定研究表明直接葡萄糖醛酸化可能导致约12%的消除。综上所述，本研究确定了蛋白质含量差异、体外活性的不稳定性、额外AO清除的作用以及未考虑的代谢途径是AO介导的药物代谢预测不足的合理原因。在PBPK模型中考虑这些因素并整合REF和pnAF将有助于更好地预测AO代谢。意义声明：本研究阐明了醛氧化酶（AO）介导的药物代谢预测不足的合理原因，并提供了应对这些原因的建议。研究表明，整合蛋白质含量和活性差异、考虑AO活性的损失以及考虑肝外清除和额外途径，将使用基于生理的药代动力学模型改善AO介导的药物代谢从体外到体内的外推。
