Project Summary The overarching goal of the proposed research is to predict the intracellular and extracellular concentration- time profiles using models that include membrane partitioning, membrane permeability, organ blood flow, active transport, and metabolism. In the funding period from 2018-2022, we have made significant progress in developing models to predict drug volume of distribution, and models to predict drug absorption. We have used the basic principles underlying permeability and partitioning to build a new framework for PBPK models (termed PermQ). This framework now allows us to incorporate permeability-limited distribution, partitioning, organ blood flow, and active transport into PBPK models with explicit membrane kinetics. We have started to build upon our current work to develop novel frameworks to predict drug clearance, a new focus of this renewal. These new modeling paradigms, together with our time- and distance- dependent continuous absorption models, will provide markedly better predictions of intracellular concentrations in the presence of drug metabolizing enzymes and transporters, and will address an unmet critical need for cost effective drug development by providing novel predictive tools for drug pharmacokinetics in humans. Three specific aims are proposed. 1) New in vitro and mathematical methods will characterize the time-course of cellular permeability and partitioning, to inform mechanisms underlying drug distribution, absorption, and intracellular concentrations that drive drug clearance. Experiments in artificial membrane environments at various pH values will define the pH partitioning – membrane permeability relationship. In vitro microdialysis and transwell techniques will capture the time-course of drug partitioning into cells including MDCK, Caco-2, adipocytes, and hepatocytes. Partitioning into erythrocyte glycocalyx will be measured. Resulting data will be used as inputs to develop mathematical models to predict drug permeability across single vs. multiple membranes across a cell, and drug partitioning into membranes. 2) Excipient effects on oral absorption will be predicted in humans and rodents. Effect of excipient dose-dependent (Polysorbate 80 and PEG400) inhibition of intestinal drug metabolizing enzymes and transporters (DMETs) will be evaluated with a refined rat intestinal model. A continuous intestinal mouse absorption model will be developed and refined. The human and rat intestinal models will be interfaced with species-specific PermQ models. 3) New in vitro and mathematical methods will improve predictions of drug clearance. Rat data will be collected with microfluidics in hepatocytes and in vivo, with regional drug quantification with MALDI imaging. Three standard liver models – well-stirred (WSM), parallel-tube (PTM), and dispersion (DM) – will be evaluated within human and rat PermQ. Enzyme zonation within the liver sinusoid will be modeled with both literature (discretized) and new parti...