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Abstract The purpose of this study was to clarify the mechanism of the blood–brain barrier (BBB) transport of H-Tyr- d-Arg-Phe-β-Ala-OH (TAPA), which is a novel dermorphin analog with high affinity for the µ[sub 1]-opioid receptor. The in vivo BBB permeation influx rate of [[sup 125]I]TAPA after an i.v. bolus injection (7.3 pmol/g body weight) into mice was estimated to be 0.265 ± 0.025µL/(min · g of brain). The influx rate of [[sup 125]I]TAPA was reduced 70% by the coadministration of unlabeled TAPA (33 nmol/g of brain), suggesting the existence of a specific transport system for TAPA at the BBB. In order to elucidate the BBB transport mechanism of TAPA, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The acid-resistant binding of [[sup 125]I]TAPA, which represents the internalization of the peptide into cells, was temperature- and concentration-dependent with a half-saturation constant of 10.0 ± 1.7 µ m. The acid-resistant binding of TAPA was significantly inhibited by 2,4-dinitrophenol, dansylcadaverine (an endocytosis inhibitor) and poly- l-lysine and protamine (polycations). These results suggest that TAPA is transported through the BBB by adsorptive-mediated endocytosis, which is triggered by binding of the peptide to negatively charged sites on the surface of brain capillary endothelial cells. Blood–brain barrier transport via adsorptive-mediated endocytosis plays a key role in the expression of the potent opioid activity of TAPA in the CNS. [ABSTRACT FROM AUTHOR]

Gastric mucoadhesive drug delivery systems are very promising for eradication of Helicobacter pylori (H. pylori), a spiral bacterium that resides in the gastric mucus layer and at the mucus- epithelial cell interface. New positively charged biodegradable microspheres were prepared using aminated gelatin by surfactantfree emulsification in olive oil, followed by a cross-linking reaction with glutaraldehyde. The amino group contents of the modified gelatin and the microspheres were determined using a 2,4,6-trinitrobenzenesulfonic acid method. With the increase of glutaraldehyde concentration, the amino group content of the microspheres decreased accordingly. The influence of glutaraldehyde concentration, cross-linking reaction time, drug-loading patterns, and type of release media on the in vitro release characteristics of amoxicillin from the microspheres was investigated. Amoxicillin release rate from the modified gelatin microspheres was significantly reduced compared with that from gelatin microspheres. Furthermore, the release was decreased with the increase of glutaraldehyde concentration and/or cross-linking time. On the other hand, a faster release was observed in a lower pH release medium and/or using a lower pH solution for amoxicillin loading. The gastric mucoadhesive properties of the microspheres were evaluated using RITC-labeled microspheres in an isolated rat stomach. The gastric mucoadhesion of the modified gelatin microspheres was markedly improved compared with that of gelatin microspheres. The modified gelatin microsphere proves to be a possible candidate delivery system for the effective eradication of H. pylori. [ABSTRACT FROM AUTHOR]

Endothelial fenestrae are transcellular pores that pierce the capillary walls in endocrine glands such as the pituitary. The fenestrae are covered with a thin fibrous diaphragm consisting of the plasmalemma vesicle–associated protein (PLVAP) that clusters to form sieve plates. The basal surface of the vascular wall is lined by basement membrane (BM) composed of various extracellular matrices (ECMs). However, the relationship between the ECMs and the endothelial fenestrae is still unknown. In this study, we isolated fenestrated endothelial cells from the anterior lobe of the rat pituitary, using a dynabeads-labeled antibody against platelet endothelial cell adhesion molecule 1 (PECAM1). We then analyzed the gene expression levels of several endothelial marker genes and genes for integrin α subunits, which function as the receptors for ECMs, by real-time polymerase chain reaction (PCR). The results showed that the genes for the integrin α subunit, which binds to collagen IV, fibronectin, laminin-411, or laminin-511, were highly expressed. When the PECAM1-positive cells were cultured for 7 days on collagen IV-, fibronectin-, laminins-411-, or laminins-511-coated coverslips, the sieve plate structures equipped with probably functional fenestrae were maintained only when the cells were cultured on fibronectin. Additionally, real-time PCR analysis showed that the fibronectin coating was effective in maintaining the expression pattern of several endothelial marker genes that were preferentially expressed in the endothelial cells of the fenestrated capillaries. These results indicate that fibronectin functions as the principal factor in the maintenance of the sieve plate structures in the endothelial cells of the fenestrated capillary. [ABSTRACT FROM AUTHOR]

There is increasing evidence that a proton-coupled organic cation (H+/OC) antiporter facilitates uptake of various central nervous system-active drugs, such as the histamine H1 receptor antagonist diphenhydramine, into the brain. The purpose of this study was to clarify the structural requirements for H+/OC antiporter-mediated uptake into hCMEC/D3 cells, an established in vitro model of the human blood-brain barrier, by using a series of diphenhydramine analogs. For this purpose, we synthesized seven tertiary amine analogs and three amide analogs. Uptake of all the amines was facilitated by an outwardly directed H+ gradient and inhibited by pyrilamine, a typical substrate and a strong inhibitor of the H+/OC antiporter. Further, uptake of most of the amines was trans -stimulated by pyrilamine. Uptake of the amines was 21 times faster than that of the amides on average, even though the lipophilicity (log D 7.4) of the amines is lower than that of the amides. Amines containing a pyrrolidine or piperidine ring showed the highest uptake rates. Our results suggest that an amine moiety, especially a heterocyclic amine moiety, is important for recognition and transport by the H+/OC antiporter. [ABSTRACT FROM AUTHOR]

Varenicline is a selective partial α 4 β 2 nicotinic acetylcholine receptor agonist, which is used to help achieve smoking cessation. Here, we investigated varenicline transport at the blood-brain barrier by means of in vivo microdialysis, in situ brain perfusion, and brain efflux index measurements in rats, and in vitro uptake studies in human brain capillary endothelial cells. Microdialysis demonstrated that varenicline is actively transported from blood to brain in rats. Blood-to-brain uptake transport of varenicline, as measured by the in situ brain perfusion technique, was strongly inhibited by diphenhydramine, a potent inhibitor of proton-coupled organic cation (H + /OC) antiporter. However, brain efflux index study showed that brain-to-blood efflux transport of varenicline was not inhibited by diphenhydramine. In human brain capillary endothelial cells, varenicline was taken up time- and concentration-dependently. The uptake was dependent on an oppositely directed proton gradient, but was independent of extracellular sodium and membrane potential. The uptake was inhibited by a metabolic inhibitor, and by substrates of H + /OC antiporter, but not by substrates or inhibitors of OCTs, OCTNs, PMAT, and MATE1, which are known organic cation transporters. The present results suggest that the H + /OC antiporter contributes predominantly to varenicline uptake at the blood-brain barrier. [ABSTRACT FROM AUTHOR]

Tramadol is a centrally acting analgesic whose action is mediated by both agonistic activity at opioid receptors and inhibitory activity on neuronal reuptake of monoamines. The purpose of this study was to characterize the blood-brain barrier ( BBB) transport of tramadol by means of microdialysis studies in rat brain and in vitro studies with human immortalized brain capillary endothelial cells (h CMEC/ D3). The Kp,uu,brain value of tramadol determined by rat brain microdialysis was greater than unity, indicating that tramadol is actively taken up into the brain across the BBB. Tramadol was transported into h CMEC/ D3 cells in a concentration-dependent manner. The uptake was inhibited by type II cations (pyrilamine, verapamil, etc.), but not by substrates of organic cation transporter OCTs or OCTN2. It was also inhibited by a metabolic inhibitor but was independent of extracellular sodium or membrane potential. The uptake was altered by changes of extracellular pH, and by ammonium chloride-induced intracellular acidification, suggesting that transport of tramadol is driven by an oppositely directed proton gradient. Thus, our in vitro and in vivo results suggest that tramadol is actively transported, at least in part, from blood to the brain across the BBB by proton-coupled organic cation antiporter. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3335-3341, 2014 [ABSTRACT FROM AUTHOR]

The online version of the original article can be found at https://doi.org/10.1007/s11095-022-03249-3 B Correction to: Pharmaceutical Research b https://doi.org/10.1007/s11095-022-03249-3 This article was updated to correct the phrase "in vitro" in the Abstract and in three instances in the text. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Correction to: Construction and Functional Evaluation of a Three-Dimensional Blood-Brain Barrier Model Equipped With Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells. [Extracted from the article]

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 K m 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.

In order to establish an in vitro model of the human blood-brain barrier (BBB), MDR1-overexpressing human induced pluripotent stem cells (hiPSCs) were generated, and they were differentiated to MDR1-expressing brain microvascular endothelial-like cells (MDR1-expressing hiPS-BMECs). MDR1-expressing hiPS-BMECs monolayers showed good barrier function in terms of tight junction protein expression and trans-epithelial electrical resistance (TEER). In sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS), MDR1 protein expression was markedly increased in MDR1-expressing hiPS-BMECs, whereas other ABC and SLC transporters showed almost identical expression between MDR1-expressing hiPS-BMECs and mock hiPS-BMECs, suggesting that MDR1 overexpression had little or no knock-on effect on other proteins. The basolateral-to-apical transport of MDR1 substrates, such as quinidine, [ 3 H]digoxin and [ 3 H]vinblastine, was higher than the apical-to-basolateral transport, and the efflux-dominant transport was attenuated by PSC833, an MDR1-specific inhibitor, indicating that MDR1-mediated efflux transport is functional. The robust MDR1 function was also supported by the efflux-dominant transports of [ 3 H]cyclosporin A, loperamide, cetirizine, and verapamil by MDR1-expressing hiPS-BMECs. These results suggest that MDR1-expressing hiPS-BMECs can be used as an in vitro model of the human BBB., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

Aim: Identification of blood-brain barrier (BBB) uptake transporters is a major challenge in the research and development of central nervous system (CNS) drugs. However, conventional methods that consider known drug uptake characteristics have failed at identifying the responsible transporter molecule. The present study aimed at identifying aripiprazole uptake transporters in BBB model hCMEC/D3 cells using a knockdown screening study targeting various transporters, including uncharacterized ones., Methods: We evaluated the effect of 214 types of siRNA targeting transporters on the uptake of aripiprazole, an atypical antipsychotic drug, in hCMEC/D3 cells. Aripiprazole uptake was determined using Xenopus oocytes expressing the candidate genes extracted from the siRNA screening assay., Results: The estimated unbound brain to plasma concentration ratio (K p,uu,brain ) of aripiprazole was estimated as 0.67 in wild-type mice and 1.94 in abcb1a/1b/abcg2 knockout mice, suggesting the involvement of both uptake and efflux transporters in BBB permeation. According to siRNA knockdown screening studies, organic cation/carnitine transporter 2 (OCTN2) and long-chain fatty acid transporter 1 (FATP1) were identified as candidate genes. The uptake of aripiprazole by hCMEC/D3 cells was decreased by OCTN2 inhibitors, but not by FATP1 inhibitors. A partially increased uptake of aripiprazole was observed in OCTN2-expressing Xenopus oocytes. Finally, to evaluate transporter-mediated BBB permeation of drugs, the reported and estimated K p,uu,brain values were summarized., Conclusions: A knockdown screening study in combination with K p,uu,brain values showed that aripiprazole was a potential substrate of OCTN2. The technique described in this study can be applied to identifying novel BBB transporters for CNS drugs., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The likelihood of reoccurrence of acute lymphoblastic leukemia is influenced by the cerebral concentration of the therapeutic agent 6-mercaptopurine (6-MP) during treatment. Therefore, it is important to understand the blood-brain barrier (BBB) transport mechanism of 6-MP. The purpose of this study was to characterize this mechanism using human induced pluripotent stem cell-derived microvascular endothelial cells (hiPS-BMECs). The permeability coefficient of 6-MP across hiPS-BMECs monolayer in the basal-to-apical direction (B-to-A) was significantly greater than that in the opposite direction (A-to-B). The inhibition profiles of 6-MP transport in the A-to-B direction were different from those in the B-to-A direction. Transport in the A-to-B direction was mainly inhibited by adenine (an inhibitor of equilibrative nucleobase transporter 1; ENBT1), while transport in the B-to-A direction was significantly reduced by inhibitors of multidrug resistance-associated proteins (MRPs), especially zaprinast (an MRP5 inhibitor). Immunocytochemical analyses demonstrated the expression of ENBT1 and MRP5 proteins in hiPS-BMECs. We confirmed that the cellular uptake of 6-MP is decreased by ENBT1 inhibitors in hiPS-BMECs and by knockdown of ENBT1 in hCMEC/D3 cells. These results suggest that ENBT1 and MRP5 make substantial contributions to the transport of 6-MP in hiPS-BMECs and hCMEC/D3 cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Prostaglandin E 2 (PGE 2 ) in the hypothalamus is a principal mediator of the febrile response. However, the role of organic anion transporting polypeptide 2A1 (OATP2A1/ SLCO2A1 ), a prostaglandin transporter, in facilitating this response is unknown. Here, we investigated the effect of Slco2a1 deficiency on the body core temperature (Tc) and on the PGE 2 concentration in hypothalamus interstitial fluid ( C isf ) and CSF ( C csf ) of lipopolysaccharide (LPS; 100 μg/kg, i.p.)-treated mice of both sexes. Slco2a1 -/- mice did not develop a febrile response. C csf was increased in Slco2a1 +/+ and Slco2a1 -/- mice, and C csf of Slco2a1 -/- mice was well maintained at 5 h after LPS injection (1160 pg/ml) compared with Slco2a1 +/+ mice (316 pg/ml). A microdialysis study revealed that C isf peaked at 2 h after LPS injection in Slco2a1 +/+ mice (841 pg/ml), whereas the increase in C isf was negligible in Slco2a1 -/- mice. The PGE 2 plasma concentration in Slco2a1 -/- mice (201 pg/ml) was significantly higher than that in Slco2a1 +/+ mice (54 pg/ml) at 1 h after LPS injection, whereas the two groups showed similar PGE 2 concentrations in the hypothalamus. Strong Oatp2a1 immunoreactivity was observed in F4/80-positive microglia and perivascular cells and in brain capillary endothelial cells. The changes in Tc and C isf seen in LPS-injected Slco2a1 +/+ mice were partially attenuated in monocyte-/macrophage-specific Slco2a1 -/- ( Slco2a1 Fl/Fl / LysM Cre/+ ) mice. Thus, OATP2A1 facilitates the LPS-induced febrile response by maintaining a high level of C isf , possibly by regulating PGE 2 secretion from F4/80-positive glial cells and/or facilitating PGE 2 transport across the blood-brain barrier. These findings suggest that OATP2A1 is a useful therapeutic target for neuroinflammation. SIGNIFICANCE STATEMENT Fever is a physiological response caused by pyrogen-induced release of prostaglandin E 2 (PGE 2 ) in the hypothalamus, which plays a central role in regulating the set-point of body temperature. However, it is unclear whether the prostaglandin transporter OATP2A1/ SLCO2A1 is involved in this response. We show here that LPS-induced fever is associated with increased PGE 2 concentration in hypothalamus interstitial fluid ( C isf ), but not in CSF ( C csf ), by means of a microdialysis study in global Slco2a1 -knock-out mice and monocyte-/macrophage-specific Slco2a1 -knock-out mice. The results suggest that OATP2A1 serves as a regulator of C isf in F4/80-positive glial cells. OATP2A1 was detected immunohistochemically in brain capillary endothelial cells and, therefore, may also play a role in PGE 2 transport across the blood-brain barrier., (Copyright © 2018 the authors 0270-6474/18/385584-12$15.00/0.)

Brain microdialysis is a powerful tool to estimate brain-to-plasma unbound concentration ratio at the steady state (Kp,uu) of compounds by direct measurement of the unbound concentration in brain interstitial fluid. Here, we evaluated a method to estimate Kp,uu values of multiple organic-cationic drugs simultaneously, by means of brain microdialysis combined with cocktail dosing. Five cationic drugs (diphenhydramine, memantine, oxycodone, pyrilamine, and tramadol), substrates of the proton-coupled organic cation antiport system, were selected as model drugs, and compared under single-dosing and cocktail-dosing conditions. We selected doses of the drugs at which no significant drug-drug interaction occurs at the proton-coupled organic cation antiport system in the blood-brain barrier (BBB). This was confirmed by uptake studies in hCMEC/D3 cells, an in vitro BBB model. The Kp,uu values after cocktail administration were in the range of 1.8-5.2, and were in good agreement with those after single administration. These results suggest that the microdialysis method with cocktail dosing is suitable to estimate Kp,uu values of several cationic drugs simultaneously, if there is no drug-drug interaction during BBB transport. The method could be useful for evaluating drug candidates with high Kp,uu values at an early stage in the development of central nervous system-acting drugs., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Memantine is clinically used for the treatment of patients with Alzheimer's disease and is highly distributed to the brain. The aim of this study is to characterize memantine transport at the blood-brain barrier (BBB) using hCMEC/D3 cells, a human BBB model. The initial uptake velocity of memantine in hCMEC/D3 cells was concentration-dependent, and was reduced by metabolic inhibitors, but was independent of extracellular sodium ion and membrane potential. Intracellular alkalization and intracellular acidification markedly reduced and enhanced the uptake, respectively. The uptake was strongly inhibited by quinidine, pyrilamine and verapamil, and was moderately inhibited by TEA (substrate of OCTs and OCTNs) and l-carnitine (substrate of OCTN2), but was not inhibited by MPP(+) (substrate of OCTs and PMAT) or ergothioneine (substrate of OCTN1). Although relatively abundant expression of OCTN2 gene has been observed in hCMEC/D3 cells, knockdown of OCTN2 with siRNA did not decrease memantine uptake. Memantine and diphenhydramine each showed inhibition of the other's uptake in a competitive manner. Thus, proton-coupled organic cation antiporter(s) appears to be involved in the transport of memantine in hCMEC/D3 cells, at least in part. Our results indicate that the in vivo BBB permeability of memantine in humans can be predicted from the in vitro uptake clearance in hCMEC/D3 cells., (Copyright © 2014 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The present study aimed to characterize bladder endothelin-1 (ET-1) receptor binding of clinically used ET-1 receptor antagonists by using [(125)I]ET-1. The inhibition of specific [(125)I]ET-1 binding was measured in the presence of ET-1 and its receptor antagonists. Specific binding of [(125)I]ET-1 in rat bladder was saturable and of high affinity, which characterized selective labeling of bladder ET-1 receptors. ET-1, bosentan, ambrisentan, and CI-1020 inhibited specific [(125)I]ET-1 binding in a concentration-dependent manner at nanomolar ranges of IC50. Nonlinear least squares regression analysis revealed the presence of high- and low-affinity ET-1 receptor sites for ambrisentan and CI-1020. Bosentan and ambrisentan significantly increased the dissociation constant for bladder [(125)I]ET-1 binding without affecting maximal number of binding sites (Bmax). Thus, bosentan and ambrisentan seem to bind to bladder ET-1 receptor in a competitive and reversible manner. Oral administration of bosentan caused a dose-dependent decrease in Bmax for bladder [(125)I]ET-1 binding, suggesting significant binding of bladder ET-1 receptors in vivo. A significant amount of pharmacologically relevant ET-1 receptors may exist in the bladder. These receptors may be implicated in the pathogenesis of lower urinary tract symptoms and may also be promising targets for the development of therapeutic agents.

Aim: To compare the effectiveness of the different parameters for predicting alveolar macrophage (AM) uptake, interactions between quinolones, including two amphipathic bases, and phospholipid membrane were evaluated by three different membrane-like systems., Methods: AM cells were isolated, cultured as confluent monolayers, then incubated with drug solution at 37 degrees C. At designated time points, uptake was terminated by aspirating solution, followed by rinsing, cell lysis, a nd analysis of drug and protein concentrations. Immobilized artificial membrane (IAM) chromatography and liposome/buffer system were used to determine interactions with phospholipid membrane, expressed as lipophilicity indices, lg k(IAM) and lg D(L/B,7.4), respectively. An n-octano l/buffer system was also employed as the reference hydrophobicity, lg D(O/B,7.4)4., Results: For the tested set, lg k(IAM) correlated more significantly with lg D(L/B,7.4) (r2 = 0.93) than with lgD(O/B,7.4) (r2 = 0.65). There were better correlations between either lg k(IAM) or lg D(L/B,7.4) and the extent of accumulation in AM than did lg DO/B,7.4 (r2 = 0.89, 0.92, and 0.67, respectively). Correlations obtained using lg k(IAM), lg D(L/B,7.4), and lg D(O/B,7.4) were comparable when regressed against the logarithm of influx rate into AM f or a set consisting of five amphoteric quinolones and quinidine., Conclusion: Liposome/buffer system and IAM chromatography could provide nearly similar scale of lipophilicity measurement, both distinct from n-octanol/buffer system. Accumulation by AM was better described by lg k(IAM) or lg D(L/B,7.4) than lg D(O/B,7.4), and the passive diffusion was principal form during drugs transported across AM membrane.