Brain Delivery of a Potent Opioid Receptor Agonist, Biphalin during Ischemic Stroke: Role of Organic Anion Transporting Polypeptide (OATP)

Abstract

Transporters (expressed) at the blood-brain barrier (BBB) can play an essential role in the treatment of brain injury by transporting neuroprotective substance to the central nervous system. The goal of this study was to understand the role of organic anion transporting polypeptide (OATP1; OATP1A2 in humans and oatp1a4 in rodents) in the transport of a potent opioid receptor agonist, biphalin, across the BBB during ischemic stroke. Brain microvascular endothelial cells (BMECs) that were differentiated from human induced pluripotent stem cells (iPSCs) were used in the present study. The effect of oxygen-glucose deprivation (OGD) and reperfusion on the OATP1 expression, uptake, and transport of biphalin was measured in induced pluripotent stem cells differentiated brain microvascular endothelial cells (iPSC-BMECs) in the presence and absence of an OATP1 substrate, estrone-3-sulfate (E3S). Biphalin brain permeability was quantified while using a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. It was found that iPSC-BMECs expressed OATP1. In vitro studies showed that biphalin BBB uptake and transport decreased in the presence of an OATP1 specific substrate. It was also observed that OGD and reperfusion modulate the expression and function of OATP1 in BMECs. This study strongly demonstrates that OATP1 contributes to the transport of biphalin across the BBB and increased expression of OATP1 during OGD-reperfusion could provide a novel target for improving ischemic brain drug delivery of biphalin or other potential neurotherapeutics that have affinity to this BBB transporter.

Keywords: biphalin; blood-brain barrier; ischemic stroke; organic anion transporting polypeptide; transport mechanism.

Figure 1

(A) Immunocytochemistry indicates positive expression of organic anion transporting polypeptide (OATP)1 in three different brain endothelial cells; two human (induced pluripotent stem cells differentiated brain microvascular endothelial cells (iPSC-BMECs) and hCMEC/D3) and one mouse (bEnd.3). SHSY5Y cells were used as positive control. The image clearly shows that there are both perinuclear (predominant) and membranous expression of OATP1 in iPSC-MBECs. Beside human cell lines, bEnd.3 also expresses this transporter, however comparatively lower than iPSC-BMEC and hCMEC/D3 cells. (B) The expression of membrane OATP1 was confirmed while using flow cytometry. Mean fluorescence intensity (MFI) was measured using permeabilized and non-permeabilized cells. Flow cytometry analysis data confirm the expression of OATP1 in iPSC-BMECs on membrane as well as in perinuclear region (C) Barrier function of the cells was measured using transendothelial electrical resistance (TEER) (C-i) and [¹⁴C] sucrose permeability (C-ii) across cells monolayer. The iPSC-BMECs exhibit restrictive barrier properties as compared to hCMEC/D3 demonstrated by ten times higher TEER and ten times lower paracellular permeability. Data represented as Mean ± SD (n = 5). * p < 0.05, **** p < 0.0001.

Figure 2

Uptake (A) and transport (B) studies using iPSC-MBECs demonstrated that biphalin uptake in the endothelial cells (ECs) and transport across the blood-brain barrier (BBB) decreased in the presence of OATP1 inhibitors. (A) For uptake studies, the cells were pre-incubated with estrone-3-sulfate (E3S) for 10 min. before incubating with mixture of biphalin (10 µM) and inhibitor (E3S, 10 µM) for 20 min. (B) For transport studies, monolayer cells were pre-treated 30 min. with OATP1 inhibitor. A mixture of biphalin and inhibitor was added to the apical side and samples were collected from basolateral side (A to B transport). FITC-dextran 4kD was used to correct the paracellular permeability. Presented permeability coefficient (PC) is the net difference of PC of biphalin and FITC-dextran. Data represented as Mean ± SD (n = 4). * p < 0.05.

Figure 3

The effect of oxygen-glucose deprivation (OGD) exposure on biphalin uptake. (A) Biphalin uptake significantly increased after different times of OGD exposure; maximum after 2 h OGD (B) Biphalin uptake while reperfusion after 2 h OGD tend to increase time dependently, and it becomes significant at 2 h OGD and 6 h R. (C) Biphalin uptake increased after 30 min. reperfusion after 6 h OGD; however, it decreased during extended reperfusion times until reaching the basal normoxic level at 6 h reperfusion. Data represented as Mean ± SD (n = 4). * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 4

(A) Immunofluorescence images of iPSC-BMECs after staining with OATP1 antibody at different OGD)-reperfusion time points. (B) Quantitative measurement of the changes in OATP1 expression after normalizing mean fluorescence intensity of OATP1 with that of 4,6-diamidino-2-phenylindole (DAPI). The expression of OATP1 in iPSC-BMECs increased after 2 and 6 h OGD exposure which further increased during reperfusion. Data represented as Mean ± SD (n = 4). * p < 0.05, ** p < 0.01. a < 0.001, b < 0.0001 vs normoxic.

Figure 5

Biphalin uptake (A) into iPSC-BMECs and transport (B) across iPSC-BMECs monolayer decreased significantly when the cells were pre-incubated with competitive OATP1 substrate, E3S. Data represented as Mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.