Characterization of a novel brain barrier ex vivo insect-based P-glycoprotein screening model
Olga Andersson1, Liesbeth Badisco2, Ane Hakansson Hansen1, Steen Honore Hansen3, Karin Hellman1, Peter Aadal Nielsen1, Line Rørbæk Olsen3, Rik Verdonck2, N. Joan Abbott4, Jozef Vanden Broeck2 & Gunnar Andersson1.
In earlier studies insects were proposed as suitable models for vertebrate blood– brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efﬂux transporter in the brain barrier of the desert locust (Schistocerca gregaria). In an in vivo study on the locust, we found an increased uptake of the two well-known Pgp substrates, rhodamine 123 and loperamide after co-administration with the Pgp inhibitors cyclosporine A or verapamil. Furthermore, ex vivo studies on isolated locust brains demonstrated differences in permeation of high and low permeability compounds. The vertebrate Pgp inhibitor verapamil did not affect the uptake of passively diffusing compounds but signiﬁcantly increased the brain uptake of Pgp substrates in the ex vivo model. In addition, studies at 2°C and 30°C showed differences in brain uptake between Pgp-efﬂuxed and passively diffusing compounds. The transcriptome data show a high degree of sequence identity of the locust Pgp transporter protein sequences to the human Pgp sequence (37%), as well as the presence of conserved domains. As in vertebrates, the locust brain–barrier function is morphologically conﬁned to one speciﬁc cell layer and by using a whole-brain ex vivo drug exposure technique our locust model may retain the major cues that maintain and modulate the physiological function of the brain barrier. We show that the locust model has the potential to act as a robust and convenient model for assessing BBB permeability in early drug discovery.
In Vitro P-glycoprotein Assays to Predict the in Vivo Interactions of P-glycoprotein with Drugs in the Central Nervous System
Bo Feng, Jessica B. Mills, Ralph E. Davidson, Rouchelle J. Mireles, John S. Janiszewski, Matthew D. Troutman, and Sonia M. de Morais
Pharmacokinetics, Dynamics, and Metabolism Department, Pfizer Global Research and Development, Groton, Connecticut
Received June 26, 2007; accepted October 22, 2007
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 knock out (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 as
says. Data from transwell studies correlated well with the brain to-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 (R2 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.
An Insect-Based Ex Vivo Blood Brain Barrier Efflux Assay
By Sonia Al-Qadi * Morten Schiott† Steen Honore Hansen‡ Peter Aadal Nielsen + Lassina Badolo•
Drug efflux activity of ABC transporters, at the human blood brain barrier (BBB), constitutes a crucial challenge for central nervous system (CNS) drug development. Accordingly, early screening of CNS drug candidates is pivotal to sort out those whose brain uptake is substantially affected by efflux activity. In this context, affordable, simple, high-throughput and predictive screening models are required. It has recently been proposed that the grasshopper (locust) could be exploited as an ex-vivo model for drug BBB permeability assessment, as it has shown some similarities to vertebrate models. The p-glycoprotein (p-gp), encoded by the ABCB1 gene, is described as the most potent efflux pump that modulates drug brain disposition, so identification and characterization of such a transporter in the locust model is essential to demonstrate its utility and validity for drug development. The present work entails transcriptomic profiling followed by amino acid-based homology analysis of locust genes, in parallel to functional investigations using rhodamine 123 as a selective p-gp substrate. A protein with high sequence similarity to ABCB1 was found in the locust brain transcriptome, which indicates a conserved mechanism of brain efflux activity between insects and vertebrates. Functionally, the developed locust model showed a kinetic behavior comparable to those obtained from in vitro cell models such as the MDCKII cells expressing p-gp. Overall, the locust ex-vivo BBB model holds promise as a cheap model with a high-throughput screening potential in the early discovery phase of CNS drugs.