Microsomal and Cytosolic Scaling Factors in Dog and Human Kidney Cortex and Application for In Vitro-In Vivo Extrapolation of Renal Metabolic Clearance

Abstract

In vitro-in vivo extrapolation of drug metabolism data obtained in enriched preparations of subcellular fractions rely on robust estimates of physiologically relevant scaling factors for the prediction of clearance in vivo. The purpose of the current study was to measure the microsomal and cytosolic protein per gram of kidney (MPPGK and CPPGK) in dog and human kidney cortex using appropriate protein recovery marker and evaluate functional activity of human cortex microsomes. Cytochrome P450 (CYP) content and glucose-6-phosphatase (G6Pase) activity were used as microsomal protein markers, whereas glutathione-S-transferase activity was a cytosolic marker. Functional activity of human microsomal samples was assessed by measuring mycophenolic acid glucuronidation. MPPGK was 33.9 and 44.0 mg/g in dog kidney cortex, and 41.1 and 63.6 mg/g in dog liver (n = 17), using P450 content and G6Pase activity, respectively. No trends were noted between kidney, liver, and intestinal scalars from the same animals. Species differences were evident, as human MPPGK and CPPGK were 26.2 and 53.3 mg/g in kidney cortex (n = 38), respectively. MPPGK was 2-fold greater than the commonly used in vitro-in vivo extrapolation scalar; this difference was attributed mainly to tissue source (mixed kidney regions versus cortex). Robust human MPPGK and CPPGK scalars were measured for the first time. The work emphasized the importance of regional differences (cortex versus whole kidney–specific MPPGK, tissue weight, and blood flow) and a need to account for these to improve assessment of renal metabolic clearance and its extrapolation to in vivo.

Targeted LC-MS/MS Proteomics-Based Strategy To Characterize in Vitro Models Used in Drug Metabolism and Transport Studies

“…The major advantage of this method is that it can simultaneously analyze multiple markers of subcellular organelle recovery, enrichment and purity with great sensitivity over other antibody- or activity-based assays. 2,13,36…”

“…Measurement of recovery and enrichment requires the use of markers of the corresponding subcellular fraction of interest, and estimation of purity needs the use of markers of other subcellular organelles other than subcellular fraction of interest. 12 CYP content, CYP reductase activity, glucose-6-phosphatese activity are commonly used as microsomal protein markers, and glutathione-S-transferase and ADH activity are used as cytosolic markers 13 . However, the results of these methods will be inaccurate if the subcellular fractions are not pure or the activities of the protein markers are weak 13 .…”

“…12 CYP content, CYP reductase activity, glucose-6-phosphatese activity are commonly used as microsomal protein markers, and glutathione-S-transferase and ADH activity are used as cytosolic markers 13 . However, the results of these methods will be inaccurate if the subcellular fractions are not pure or the activities of the protein markers are weak 13 . Though Western blot is often used to confirm the purity of the subcellular fractions, this method is not readily amenable to efficiently quantify multiple protein markers.…”

Advances in predictive in vitro models of drug-induced nephrotoxicity

“…Such analyses will require in vitro-to-in vivo extrapolations, involving mathematical models that can numerically simulate the behaviour of a drug in a complex system using results obtained experimentally in an in vitro system as the input parameter. The success of these mechanistic models is highly dependent on in-depth quantitative knowledge of renal physiology, such as relevant flow rates, the total number of nephrons and cells in a kidney, and absolute protein expression levels of transporters and enzymes 100,151 . In physiologically based pharmacokinetic modelling, drug pharmacokinetics or toxicokinetics can be predicted in silico by incorporating relevant drug disposition data such as renal drug metabolism and renal accumulation during excretion 152 .…”

Utility of Chimeric Mice with Humanized Liver for Predicting Human Pharmacokinetics in Drug Discovery: Comparison with <i>in Vitro</i>–<i>in Vivo</i> Extrapolation and Allometric Scaling

“…To predict renal clearance, IVIVE methods have been also evaluated. [52][53][54][55] Further, future studies will require human PK prediction methods which unify hepatic and extrahepatic clearances.…”