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.

Qian Liu,Dan Zhang,Jinfeng Hou,Jinlan Zhang,Hua Sun,Xiaoguang Chen,Gengtao Liu,Muzamil Ahmad

doi:10.1371/journal.pone.0102442

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.

Highlights

As the main functional interface between the circulatory system and brain, the blood-brain barrier (BBB) is a major challenge for effective delivery of therapeutics to the brain [1,2]
Samples were initially seeded at approximate cell density to both normal and Parkinson’s disease (PD) rat cerebral microvessel endothelial cells (rCMECs) obtained by the puromycin purification method [18], in primary culture 3 days after treated with 4 mg/ml puromycin, the initial outgrowth morphology of normal (Figure 3Ab) and PD (Figure 3Bb) rCMECs colonies were similar to an elongated swirling pattern described before [19]
Our study represents the first to compare normal rCMECs with PD rCMECs isolated from Parkinson animals following 6-OHDA substantia nigra (SN) lesion which has never been formally isolated before

Summary

Introduction

As the main functional interface between the circulatory system and brain, the blood-brain barrier (BBB) is a major challenge for effective delivery of therapeutics to the brain [1,2]. 98% of small molecule drugs and all large molecule neurotherapeutics are barely able to cross BBB [3], unless they are actively taken up into the brain. For this reason, most drugs presently in clinical use for CNS therapy are lipophilic compounds with molecular weight less than 500 Da. a variety of small lipophilic therapeutics which were predicted to permeate the brain were obstructed by the BBB due to the presence of drug efflux transporters localized on surface of the cerebral microvessel endothelial cells [4]. The interactions of FLZ with both P-gp and BCRP were examined through in vitro BBB models

Methods

Results

Conclusion