Daan W. Van Valkengoed1, Vivi Rottschäfer2,3, Elizabeth C.M. de Lange1
1Division of Systems Pharmacology and Pharmacy, Leiden Academic Center for Drug Research, Leiden University, 2Mathematical Institute, Leiden University, 3Korteweg-de Vries Institute for Mathematics, University of Amsterdam
Objectives Active transporters like P-glycoprotein (P-gp) expressed at the Blood-Brain-Border (BBB) limit the distribution of drugs to the central nervous system (CNS) [1]. Measuring pharmacokinetics (PK) in the human CNS is ethically restricted, hindering development and optimization of CNS-based drugs. In vitro based methods for the prediction of drug distribution to the CNS are therefore sought after. In vitro derived measures of transport, like drug efflux ratios or Michaelis-Menten parameters, are commonly used in physiologically-based pharmacokinetic (PBPK) models for prediction of PK profiles of transporter substrates [2]. Such models have shown promise for the mechanistic prediction of P-gp substrate PK in the CNS [3-7]. PBPK models extrapolate transporter activity from in vitro to in vivo (IVIVE) based on differences in protein expression, assuming a directly proportional and drug-independent relationship [5-9]. This does not always work, sometimes requiring empirical scaling factors to match observed data [6, 7, 10, 11]. Some studies have shown the relationship between P-gp expression and activity to be linear [8, 11-13], while others were unable to quantify this relationship [14-16]. As such, uncertainty exists regarding this crucial assumption for mechanistic PK modelling of P-gp activity. Our objective was to investigate the P-gp concentration-activity relationship (CAR) through a kinetic model of P-gp binding and efflux. We aimed to see whether, and under which conditions, P-gp activity scales linearly with its concentration. Methods For this simulation study, a previously published kinetic binding model that mechanistically describes drug interaction with P-gp during transwell permeability assays was used [17]. The kinetic rate constants describing the interaction with P-gp (kon [association], koff [dissociation] and ke [efflux]) were available for seven P-gp substrates. Simulations were performed for a 1 µM administration of these drugs, first with a P-gp concentration of 1000 µM (reference P-gp concentration). The cumulative amount of drug effluxed by P-gp was determined for this reference condition (Aeff,cum,100%). Subsequently, the P-gp concentration was varied from 2% to 300% of the reference condition, and the Aeff,cum,x% was determined at every P-gp concentration of x%. The relative CAR was then calculated as rCARx% = (Aeff,cum,x% / Aeff,cum,100%) * 100 for every P-gp concentration. To better understand the P-gp CAR, virtual drugs with a wide range of koff and ke were simulated. The rCAR at 50% P-gp concentration (rCAR50%) was determined for each condition. The impact of the reference P-gp concentration and drug concentration on the CAR was also included. Results Simulations of the P-gp substrates show that the P-gp CAR is drug-dependent. Ketoconazole and verapamil showed a linear CAR, with ~50% of the reference P-gp activity present at 50% P-gp concentration (rCAR50% = 53% and 54%, respectively). Amprenavir, digoxin, quinidine and vinblastine followed a non-linear CAR, as >90% of the reference P-gp activity remained at 50% P-gp concentration (rCAR50% ranged from 91% to 96%). Loperamide showed an intermediate, but still non-linear, CAR (rCAR50%= 79%). We explored whether passive diffusion (Papp) and the membrane partitioning coefficient (KCP) correlated to the distinct CAR between the drugs. Lower Papp and higher KCP shifted the CAR towards non-linearity. However, the differences in these parameters between the seven drugs did not explain the corresponding CARs. Instead, this was explained by the ratio koff/ke. Expanding the simulations to drugs with a range in koff (10³-107 /s) and ke (0.1 – 30 /s), as well as different reference P-gp concentrations (10¹-105 µM) and drug exposures (0.1-100 µM), highlighted two main trends. The P-gp CAR moves away from linearity whenever the ratio koff/ke becomes smaller, or when the ratio between reference P-gp and drug concentration increases. Conclusions This study shows that the P-gp CAR depends on the drug and system. A non-linear CAR is expected when a drug is efficiently effluxed (low ratio koff/ke) and/or when P-gp is in excess compared to the drug. The a priori assumption that P-gp activity scales linearly with P-gp concentration might therefore work for some but not all drugs and scenarios. This is crucial information not only for in vitro informed PBPK predictions of CNS PK, but for predictions of transporter-mediated PK in general.
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Reference: PAGE 33 (2025) Abstr 11727 [www.page-meeting.org/?abstract=11727]
Poster: Drug/Disease Modelling - Absorption & PBPK