SUMMARY Breastfeeding has multiple beneficial effects on maternal and neonatal health; however, the statistics indicate that up to 96% of lactating women in the US take one or more medications while breastfeeding. Medications consumed by lactating women may be transferred into breast milk to a significant extent, resulting in unintentional infant exposure of medications and in some cases adverse health outcomes for the infants. Quantifying drug transfer into human breast milk is important for rational risk assessment balancing the toxicity risk of drug exposure to infants and the benefits of breastfeeding. However, clinical pharmacokinetic (PK) studies in the population of lactating women are challenging and logistically not possible for every drug taken by lactating women, necessitating the use of prediction methods to address this issue. One historical approach is the prediction of drug concentrations (or drug AUC) in breast milk based on maternal plasma concentration (or AUC) and the milk-to-plasma (M/P) concentration or AUC ratio. The M/P ratio itself can be predicted using both physicochemical characteristics of drugs and physiological parameters of breast milk. While this approach may predict the M/P ratios of drugs that enter the milk predominantly by passive diffusion, no methods are currently available to accurately predict milk secretion of drugs via transport mechanisms. Nonetheless, milk secretions of many drugs, xenobiotics and endogenous substances are known to be mediated by transporters expressed in mammary epithelial cells (MECs). In this application, we propose a systems pharmacology approach to predict transporter-mediated milk secretion of drugs. Our hypothesis is that the transporter-mediated drug PK in human breast milk can be predicted using in vitro experimental data combined with Physiologically Based Pharmacokinetic (PBPK) modeling and simulation (M&S). Specifically, we propose an innovative approach which utilizes human MECs and transporter-transfected cells or plasma membrane vesicles expressing individual transporters of interest (i.e. OCT1, BCRP). Using quantitative targeted proteomics, the human MECs will allow us to determine the protein abundance of these transporters in the mammary gland. The transporter-transfected cell or plasma membrane vesicle studies will allow us to determine the in vitro intrinsic transport clearance of a drug by a single transporter. Then, the in vitro intrinsic transporter-mediated clearances will be extrapolated to in vivo in the mammary gland for PBPK M&S. PBPK model predictions will be verified using the drug PK data in human breast milk obtained from a clinical study conducted with a transporter substrate. Combined, these data will allow us to predict transporter-mediated drug PK in the milk of lactating women. These studies will address a critical gap in our understanding of drug PK in human breast milk during lactation. Since our approach can be applied to other drugs that are subs...