In Vitro P-glycoprotein Assays to Predict the in Vivo Interactions of P-glycoprotein with Drugs in the Central Nervous System

Abstract

ABSTRACT:Thirty-one structurally diverse marketed central nervous system (CNS)-active drugs, one active metabolite, and seven non-CNSactive compounds were tested in three P-glycoprotein (P-gp) in vitro assays: transwell assays using MDCK, human MDR1-MDCK, and mouse Mdr1a-MDCK cells, ATPase, and calcein AM inhibition. Additionally, the permeability for these compounds was measured in two in vitro models: parallel artificial membrane permeation assay and apical-to-basolateral apparent permeability in MDCK. The exposure of the same set of compounds in brain and plasma was measured in P-gp knockout (KO) and wild-type (WT) mice after subcutaneous administration. One drug and its metabolite, risperidone and 9-hydroxyrisperidone, of the 32 CNS compounds, and 6 of the 7 non-CNS drugs were determined to have positive efflux using ratio of ratios in MDR1-MDCK versus MDCK transwell assays. Data from transwell studies correlated well with the brainto-plasma area under the curve ratios between P-gp KO and WT mice for the 32 CNS compounds. In addition, 3300 Pfizer compounds were tested in MDR1-MDCK and Mdr1a-MDCK transwell assays, with a good correlation (R 2 ‫؍‬ 0.92) between the efflux ratios in human MDR1-MDCK and mouse Mdr1a-MDCK cells. Permeability data showed that the majority of the 32 CNS compounds have moderate to high passive permeability. This work has demonstrated that in vitro transporter assays help in understanding the role of P-gp-mediated efflux activity in determining the disposition of CNS drugs in vivo, and the transwell assay is a valuable in vitro assay to evaluate human P-gp interaction with compounds for assessing brain penetration of new chemical entities to treat CNS disorders.Human P-glycoprotein (P-gp, MDR1) is known to be a determinant of drug absorption, distribution, and excretion of a number of clinically important drugs (Ambudkar et al., 1999;Fromm, 2000). P-gp is widely expressed in major organs, and, more specifically, P-gp is highly expressed in the capillaries of the blood brain barrier (BBB) and poses a barrier to brain penetration of its substrates (Schinkel, 1999). Given that P-gp efflux liability can be a major hurdle for CNS therapeutic drugs to cross the BBB and reach the target, the interactions of CNS compounds with P-gp may lead to the lack of CNS activity as a result of the decreased brain penetration. Thus, the prediction and understanding of the relevance of P-gp-mediated efflux transport have become important activities in the discovery and development of CNS drugs. In attempts to predict the effects of P-gp in vivo, a variety of in vitro P-gp assays have been developed to classify compounds as P-gp substrates. For instance, transwell-based assays using polarized cell lines such as the Madin-Darby canine kidney (MDCK) cell line. The MDCK cell line can be stably transfected with human MDR1 or mouse Mdr1a (MDR1-MDCK or Mdr1a-MDCK, respectively). Comparison of the efflux ratios between MDR1-MDCK and MDCK transwell assays can provide a measure of the specific human P-gp-mediated e...

Defining Desirable Central Nervous System Drug Space through the Alignment of Molecular Properties, in Vitro ADME, and Safety Attributes

“…In an effort to gain a better perspective on the ADME properties of drugs and candidates, we embarked on the in vitro profiling of these compounds in in-house HT assays to assess permeability, P-gp efflux liability, and metabolic stability. We have used the MDCK cell line to profile passive apparent permeability (P app ) (14). The MDCK cell line is an epithelial cell line that spontaneously forms a confluent polarized monolayer, well-suited for determination of compound transport across epithelial and barrier tissues.…”

“…In vitro ADME end points have become a main staple of information to guide medicinal chemists in drug design (7,14,15). In an effort to gain a better perspective on ADME space for the drugs and the candidates, these molecules were profiled utilizing high-throughput (HT) assays commonly employed by Pfizer and the industry.…”

Evaluation and Synthesis of Polar Aryl- and Heteroaryl Spiroazetidine-Piperidine Acetamides as Ghrelin Inverse Agonists

“…25 The latter two goals were set to minimize brain exposure. 26,27 With the discovery of 1, we had optimized the indane C(5) heteroaryl group as the 6-methyl-4-pyrimidine and chose to investigate polar replacements of the 4-methoxyphenyl group of 2.…”

“…g TPSA, topological polar surface area. h MDR efflux ratio (BA/AB) in MDCK/MDR1 cell line as described in ref 25. i Passive permeability measured using low efflux MDCKII cells as described in ref 34.…”

Predicting Efflux Ratios and Blood-Brain Barrier Penetration from Chemical Structure: Combining Passive Permeability with Active Efflux by P-Glycoprotein

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