Yoshiharu Deguchi

Objective The purpose of this study is to analyze the impact of the blood–brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) transport characteristics of drugs on their intracerebral distribution in male cynomolgus monkeys. Methods Steady-state concentrations of 15 drugs (13 drugs and 2 compounds) transported by passive diffusion or via solute carrier (SLC) and/or ATP-binding cassette (ABC) transporters were measured in the frontal cortex interstitial fluid (ISF), lateral ventricular cerebrospinal fluid (CSF) and lumbar CSF of monkey brain by means of microdialysis and lumbar puncture methods. The values of brain ISF (or CSF)/plasma unbound concentration ratio (K_p,uu) were calculated to quantify the intracerebral distribution characteristics. Results The K_{p,uu,ISF,cortex} values of substrates of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)were much lower than unity. The ISF concentration of these drugs were increased by the co-administration of elacridar, suggesting active efflux of these drugs at the BBB. Contrary to expectations, some of P-gp substrates were efficiently distributed into the brain with K_{p,uu,ISF,cortex} > 2. The lateral ventricular CSF concentrations of drugs tended to be higher than the ISF concentrations, while the lumbar CSF concentrations were comparable to the ISF concentrations. Conclusion The results of this study suggest that the intracerebral distribution of the test drugs from the blood in the monkey brain should consider the contribution of influx transporters as well as efflux transporters. In addition, the lumbar CSF concentrations of the test drugs appear to be a useful surrogate marker of the ISF concentrations.

Purpose Human choroid plexus papilloma (HIBCPP) cells derived from choroid plexus papilloma in the lateral ventricle are considered suitable model cells for the blood-cerebrospinal fluid barrier (BCSFB). Therefore, in this study, the transporters expressed in HIBCPP cells were identified, and the functions of representative transporters were evaluated. Methods Transporter protein and mRNA expression were analyzed in HIBCPP cells using quantitative proteomics and quantitative reverse transcription-PCR. Transporter functions were evaluated by cellular uptake and transcellular transport studies using typical substrates. Results The following solute carrier (SLC) and ATP-binding cassette (ABC) transporters involved in drug and nutrient transport were expressed in HIBCPP cells: glucose transporter 1 (GLUT1), monocarboxylate transporter 1 (MCT1), L-type amino acid transporter 1 (LAT1), cationic amino acid transporter 1 (CAT1), glutamate transporter (GLAST), SLC35F2, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP). Furthermore, the mRNA of organic anion/cation transporters, such as organic anion transporting polypeptide 1B3 (OATP1B3) and plasma membrane monoamine transporter (PMAT), were detected. Additionally, uptake of representative substrates of the SLC transporters LAT1, CAT1, GLAST, GLUT1, MCT1, and SLC35F2 in HIBCPP cells occurred in a time- and temperature-dependent manner and decreased in the presence of specific inhibitors. Furthermore, the representative substrates of these transporters were transported in a symmetric or asymmetric manner through HIBCPP cells between the apical (cerebrospinal fluid [CSF]) and basolateral (blood) sides. Conclusion Various nutrient and drug transporters are functionally expressed in HIBCPP cells. Therefore, HIBCPP cells could serve as a useful human BCSFB model to evaluate nutrient and drug transport between the CSF and blood.

Mesoridazine and metoclopramide are cationic drugs that are distributed in the human brain despite being substrates of multidrug resistance protein 1 (MDR1), an efflux transporter expressed at the blood–brain barrier (BBB). We investigated their transport mechanisms at the BBB using hCMEC/D3, a human cerebral microvascular endothelial cell line often used as an in vitro BBB model. The cells exhibited time- and concentration-dependent uptake of mesoridazine and metoclopramide, with Km values of 34 and 277 µM, respectively. The uptake of both drugs significantly decreased in the presence of typical inhibitors and/or substrates of the H+-coupled organic cation (H+/OC) antiporter but not in the presence of inhibitors or substrates of organic cation transporters (OCTs), OCTN2, OATPs, SLC35F2, or the plasma membrane monoamine transporter (PMAT). Furthermore, metoclopramide uptake by hCMEC/D3 cells was pH- and energy-dependent, whereas mesoridazine uptake was unaffected by intracellular acidification and treatment with metabolic inhibitors. These results suggest that the H+/OC antiporter is involved in the influx of mesoridazine and metoclopramide into the brain across the BBB.

Naltrexone is a mu-opioid receptor antagonist used in the treatment of opioid and alcohol dependence. The blood–brain barrier (BBB) transport characteristics of naltrexone was investigated by means of hCMEC/D3 cells, a human immortalized brain capillary endothelial cell line. In hCMEC/D3 cells, naltrexone is taken up in a concentration-dependent manner. Furthermore, naltrexone uptake significantly decreased in the presence of H⁺/organic cation (OC) antiporter substrates, during the little alteration exhibited by substrates of well-identified OC transporters classified into SLC22A family. Although naltrexone uptake by hCMEC/D3 cells was partially affected by changes of ionic conditions, it was markedly decreased in the presence of the metabolic inhibitor sodium azide. Furthermore, when treated by ammonium chloride, naltrexone uptake by hCMEC/D3 cells was altered by intracellular acidification and alkalization, suggesting the involvement of oppositely directed proton gradient in naltrexone transport across the BBB. The results obtained in the present in vitro study suggest the active transport of naltrexone from blood to the brain across the BBB by the H⁺/OC antiporter.

A proton-coupled organic cation (H⁺/OC) antiporter working at the blood–brain barrier (BBB) in humans and rodents is thought to be a promising candidate for the efficient delivery of cationic drugs to the brain. Therefore, it is important to identify the molecular entity that exhibits this activity. Here, for this purpose, we established the Proteomics-based Identification of transporter by Crosslinking substrate in Keyhole (PICK) method, which combines photo-affinity labeling with comprehensive proteomics analysis using SWATH-MS. Using preselected criteria, the PICK method generated sixteen candidate proteins. From these, knockdown screening in hCMEC/D3 cells, an in vitro BBB model, identified two proteins, TM7SF3 and LHFPL6, as candidates for the H⁺/OC antiporter. We synthesized a novel H⁺/OC antiporter substrate for functional analysis of TM7SF3 and LHFPL6 in hCMEC/D3 cells and HEK293 cells. The results suggested that both TM7SF3 and LHFPL6 are components of the H⁺/OC antiporter.

Purpose: The purpose of this study was to construct and validate an in vitro three-dimensional blood–brain barrier (3DBBB) model system equipped with brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs). Methods: The 3D-BBB system was constructed by seeding hiPS-BMECs onto the capillary lane of a MIMETAS OrganoPlate^® 3-lane coated with fibronectin/collagen IV. hiPS-BMECs were incubated under continuous switchback flow with an OrganoFlow^® for 2 days. The 3D capillary structure and expression of tight-junction proteins and transporters were confirmed by immunocytochemistry. The mRNA expression of transporters in the 3D environment was determined using qRT-PCR, and the permeability of endogenous substances and drugs was evaluated under various conditions. Results and Discussion: The expression of tight-junction proteins, including claudin-5 and ZO-1, was confirmed by immunohistochemistry. The permeability rate constant of lucifer yellow through hiPS-BMECs was undetectably low, indicating that paracellular transport is highly restricted by tight junctions in the 3D-BBB system. The mRNA expression levels of transporters and receptors in the 3D-BBB system differed from those in the 2D-culture system by 0.2- to 5.8-fold. The 3D-cultured hiPS-BMECs showed asymmetric transport of substrates of BCRP, CAT1 and LAT1 between the luminal (blood) and abluminal (brain) sides. Proton-coupled symport function of MCT1 was also confirmed. Conclusion: The 3D-BBB system constructed in this study mimics several important characteristics of the human BBB, and is expected to be a useful high-throughput evaluation tool in the development of CNS drugs.