P-Glycoprotein Mediated Efflux Limits the Transport of the Novel Anti-Parkinson's Disease Candidate Drug FLZ across the Physiological and PD Pathological In Vitro BBB Models

Abstract

FLZ, a novel anti-Parkinson's disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson's conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport.

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“…Transport studies were also performed, where only P-gp blocker effectively inhibited the efflux of FLZ. Thus, from this study it is possible to conclude that P-gp is the main responsible for poor brain penetration of FLZ and low BBB permeability (Liu et al, 2014).…”

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“…In addition, it was observed that in vitro BBB model facilitates the manipulation of some parameters that affects the barrier such as aglycemia, hypoxia, among others [32]. For decades, two-dimensional or three-dimensional in vitro models of BBB have been developed by cultivating either as a monolayer or in cocultivation with mouse brain microvascular endothelial cells and murine or human endothelial cells with pericytes or astrocytes or glial tissue among others in a way that mimics the barrier under physiological or pathological conditions such as Alzheimer's and Parkinson's diseases or stroke [33][34][35][36][37]. Models of BBB based on stem cells are also reported in the literature [38,39].…”

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“…[25][26][27] Our previous study investigated the cellular transport of nimesulide and the para-methoxy substituted analog 1d across Caco-2 cells, which provide a valid surrogate model for the BBB efflux behavior of pharmaceutical molecules. 28 This study indicated that neither nimesulide nor 1d were P-gp substrates in vitro.…”