Construction and Functional Evaluation of a Three-Dimensional Blood-Brain Barrier Model Equipped With Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells.

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.

Design, Synthesis, and Evaluation of 11C-Labeled 3-Acetyl-Indole Derivatives as a Novel Positron Emission Tomography Imaging Agent for Diacylglycerol Kinase Gamma (DGKγ) in Brain.

Diacylglycerol kinase gamma (DGKγ) is a subtype of DGK enzyme, which catalyzes ATP-dependent conversion of diacylglycerol to phosphatidic acid. DGKγ, localized in the brain, plays an important role in the central nervous system. However, its function has not been widely investigated. Positron emission tomography (PET) imaging of DGKγ validates target engagement of therapeutic DGKγ inhibitors and investigates DGKγ levels under normal and disease conditions. In this study, we designed and synthesized a series of 3-acetyl indole derivatives as candidates for PET imaging agents for DGKγ. Among the synthesized compounds, 2-((3-acetyl-1-(6-methoxypyridin-3-yl)-2-methyl-1H-indol-5-yl)oxy)-N-methylacetamide (9) exhibited potent inhibitory activity (IC50 = 30 nM) against DGKγ and desirable physicochemical properties allowing efficient blood-brain barrier penetration and low levels of undesirable nonspecific binding. The radiolabeling of 9 followed by PET imaging of wild-type and DGKγ-deficient mice and rats indicated that [11C]9 ([11C]T-278) specifically binds to DGKγ and yields a high signal-to-noise ratio for DGKγ in rodent brains.

Identification of Novel, Potent, and Orally Available GCN2 Inhibitors with Type I Half Binding Mode.

General control nonderepressible 2 (GCN2) is a master regulator kinase of amino acid homeostasis and important for cancer survival in the tumor microenvironment under amino acid depletion. We initiated studies aiming at the discovery of novel GCN2 inhibitors as first-in-class antitumor agents and conducted modification of the substructure of sulfonamide derivatives with expected type I half binding on GCN2. Our synthetic strategy mainly corresponding to the αC-helix allosteric pocket of GCN2 led to significant enhancement in potency and a good pharmacokinetic profile in mice. In addition, compound 6d, which showed slow dissociation in binding on GCN2, demonstrated antiproliferative activity in combination with the asparagine-depleting agent asparaginase in an acute lymphoblastic leukemia (ALL) cell line, and it also displayed suppression of GCN2 pathway activation with asparaginase treatment in the ALL cell line and mouse xenograft model.

Design, synthesis, and biological activities of novel hexahydropyrazino[1,2-a]indole derivatives as potent inhibitors of apoptosis (IAP) proteins antagonists with improved membrane permeability across MDR1 expressing cells.

We previously reported octahydropyrrolo[1,2-a]pyrazine derivative 2 (T-3256336) as a potent antagonist for inhibitors of apoptosis (IAP) proteins. Because compound 2 was susceptible to MDR1 mediated efflux, we developed another scaffold, hexahydropyrazino[1,2-a]indole, using structure-based drug design. The fused benzene ring of this scaffold was aimed at increasing the lipophilicity and decreasing the basicity of the scaffold to improve the membrane permeability across MDR1 expressing cells. We established a chiral pool synthetic route to yield the desired tricyclic chiral isomers. Chemical modification of the core scaffold led to a representative compound 50, which showed strong inhibition of IAP binding (X chromosome-linked IAP [XIAP]: IC50 23 nM and cellular IAP [cIAP]: IC50 1.1 nM) and cell growth inhibition (MDA-MB-231 cells: GI50 2.8 nM) with high permeability and low potential of MDR1 substrate.

Expression and transport function of drug uptake transporters in differentiated HepaRG cells.

HepaRG cells have the ability to differentiate into hepatocyte-like cells. Many papers have shown that these hepatocyte-like cells share several functional properties with intact human hepatocytes. However, although previous studies have indicated the partial maintenance of mRNA expression of drug transporters, their expression and function have not been quantitatively characterized. In the present study, the mRNA and protein expression levels and transport activities of hepatic uptake transporters, organic anion transporting polypeptides (OATPs) and Na(+)-taurocholate cotransporting polypeptide (NTCP) in HepaRG cells were compared with those in cryopreserved human hepatocytes. The mRNA expression levels of OATP1B1, OATP1B3, OATP2B1, and NTCP in HepaRG cells were 22-38%, 2-15%, 82-113%, and 191-247% of those in human hepatocytes, respectively. The relative protein expression of these transporters was comparable with their mRNA expression. We observed saturable uptake of typical substrates of NTCP and OATPs except for cholecystokinin octapeptide (OATP1B3-selective substrate), and Na(+)-dependent uptake of taurocholate was confirmed. Their relative uptake clearances were well explained by their mRNA and protein expression levels. Additionally, inhibition potencies of 12 OATP1B1 inhibitors were investigated both in HepaRG cells and in OATP1B1-expressing HEK293 cells to demonstrate the usefulness of HepaRG cells for the characterization of OATP1B1-mediated drug-drug interactions. The Ki values in both cell lines were comparable and showed significant correlation. These results suggest that the hepatic uptake transport function of OATP and NTCP transporters was relatively well maintained in HepaRG, although OATP1B3 function was too low to be detected.

Hepatic uptake in the dog: comparison of uptake in hepatocytes and human embryonic kidney cells expressing dog organic anion-transporting polypeptide 1B4.

Although the dog is frequently used in pharmacological, pharmacokinetic, and drug safety studies, little is known about canine drug transporters. Dog organic anion-transporting polypeptide (Oatp1b4) has recently been cloned (Comp Biochem Physiol C Toxicol Pharmacol 151:393-399, 2010), but the contribution of Oatp1b4 to hepatic uptake has yet to be clarified. This study compares the transport characteristics of dog Oatp1b4 with those of human OATP1B1/1B3 and demonstrates the importance of Oatp1b4 in the uptake of anionic compounds in dog hepatocytes. Oatp1b4 is the predominant Oatp in dog liver with expression levels double and 30 times those of Oatp2b1 and Oatp1a2, respectively. Uptake of a range of typical OATP substrates by Oatp1b4-expressing HEK293 cells was compared with that in fresh dog hepatocytes. All compounds tested were transported by Oatp1b4 and uptake intrinsic clearance (CL(int, uptake)) in dog hepatocytes in sodium-free buffer was correlated significantly with CL(int, uptake) in Oatp1b4-expressing cells. Dog in vivo clearance for five substrates was predicted more accurately from CL(int, uptake) than from metabolic intrinsic clearance (CL(int, met)), indicating that uptake governs the overall in vivo hepatic clearance of these anionic compounds in dog. The substrate specificities of dog Oatp1b4 appear to be similar to those of human OATP1B1/OATP1B3, whereas the relative uptake clearance of substrates for Oatp1b4 correlate better with OATP1B3 than with the more abundant hepatic analog OATP1B1.

Prediction of the overall renal tubular secretion and hepatic clearance of anionic drugs and a renal drug-drug interaction involving organic anion transporter 3 in humans by in vitro uptake experiments.

The present study investigated prediction of the overall renal tubular secretion and hepatic clearances of anionic drugs based on in vitro transport studies. The saturable uptake of eight drugs, most of which were OAT3 substrates (rosuvastatin, pravastatin, pitavastatin, valsartan, olmesartan, trichlormethiazide, p-aminohippurate, and benzylpenicillin) by freshly prepared human kidney slices underestimated the overall intrinsic clearance of the tubular secretion; therefore, a scaling factor of 10 was required for in vitro-in vivo extrapolation. We examined the effect of gemfibrozil and its metabolites, gemfibrozil glucuronide and the carboxylic metabolite, gemfibrozil M3, on pravastatin uptake by human kidney slices. The inhibition study using human kidney slices suggests that OAT3 plays a predominant role in the renal uptake of pravastatin. Comparison of unbound concentrations and K(i) values (1.5, 9.1, and 4.0 µM, for gemfibrozil, gemfibrozil glucuronide, and gemfibrozil M3, respectively) suggests that the mechanism of the interaction is due mainly to inhibition by gemfibrozil and gemfibrozil glucuronide. Furthermore, extrapolation of saturable uptake by cryopreserved human hepatocytes predicts clearance comparable with the observed hepatic clearance although fluvastatin and rosuvastatin required a scaling factor of 11 and 6.9, respectively. This study suggests that in vitro uptake assays using human kidney slices and hepatocytes provide a good prediction of the overall tubular secretion and hepatic clearances of anionic drugs and renal drug-drug interactions. It is also recommended that in vitro-in vivo extrapolation be performed in animals to obtain more reliable prediction.

Involvement of multiple efflux transporters in hepatic disposition of fexofenadine.

Fexofenadine (FEX) is mainly eliminated from the liver into bile in unchanged form. We demonstrated previously that organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are involved in the hepatic uptake of FEX. However, little is known about the mechanisms controlling the hepatic efflux of FEX from the liver to bile and blood. In the present study, the involvement of hepatic efflux transporters in the pharmacokinetics of FEX was investigated in both in vitro and in vivo studies. Vectorial transport of FEX was observed in OATP1B3/human bile salt export pump (hBSEP) double transfectants but not in OATP1B3/human breast cancer resistance protein double transfectants, which indicates the possible contribution of hBSEP to the biliary excretion of FEX in humans. In multidrug resistance-associated protein 2 (Mrp2)(-/-) mice, the biliary excretion clearance based on the plasma concentration and the liver-to-plasma concentration ratio significantly decreased, whereas the biliary excretion clearance based on the liver concentration decreased only with 20%, suggesting the minimum contribution of Mrp2 to its biliary excretion. ATP-dependent transport of FEX was observed in hMRP3-enriched membrane vesicles but not hMRP4. In Mrp3(-/-) mice, the biliary excretion clearance based on both the plasma and liver concentration and the liver-to-plasma concentration ratio increased, suggesting the significant contribution of Mrp3 to its sinusoidal efflux and the up-regulation of its biliary excretion in Mrp3(-/-) mice. On the other hand, pharmacokinetics of FEX remained unchanged in Mrp4(-/-) mice. This information provides a novel insight into the transporters important for FEX disposition.

Synchronous evaluation of toxico- and pharmaco-dynamics of yttrium-90 by a novel autoradiographic procedure.

A synchronous evaluation was performed, using a quick in vivo [2-(14)C]thymidine labeling method, of the toxico- and pharmaco-dynamics of a given dose of yttrium-90 (90Y) at a given time after injection to nude BALB/c mice loaded with 10(7) HuO9 cells. Quantitative data were 14C-microautoradiographs of the liver lobule, intestinal crypts, epiphysial growth plate, secondary ossification center containing pluripotent stem cells, perifollicular zone containing unipotent stem cells in the spleen, and plasmacytoma cells in the osteogenic sarcoma in each mouse following a 10-min labeling with 14C at 0.5, 6, and 24 h after i.v. injection of 90Y. Results show that the cell proliferation rate of the stem cells in respective tissues was markedly suppressed, dependent on time after dosing and the dose of 90Y; 3.7, 37, 370, 3700, and 37,000 kBq per mouse (25 g). In addition to the above, the sensitivity of the proliferation rate was dependent on amitosis or mitosis and the AUC value of 90Y-concentration at specific locations of the cells in the mouse body. The most sensitive cells were the plasmacytoma cells, followed by the pluripotent and unipotent stem cells, the intestinal crypts, epiphysial growth plate, and liver cells.