In Vitro Metabolism of 2-Methoxy-N-[3-[4-[3-methyl-4-[(6-methyl- 3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) by Aldehyde Oxidase and Predicting Its Percent Contribution Relative to CYP-Mediated Metabolism.

2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) is an anticancer drug that was discontinued due to hepatotoxicity found in clinical studies. Metabolite analysis of CP-724,714 was conducted using human hepatocytes, in which twelve oxidative metabolites and one hydrolyzed metabolite were formed. Among the three mono-oxidative metabolites, the formation of two was inhibited by adding 1-aminobenzotriazole, a pan-CYP inhibitor. In contrast, the remaining one was not affected by this inhibitor but partially inhibited by hydralazine, indicating that aldehyde oxidase (AO) was involved in metabolizing CP-724,714, which contains a quinazoline substructure, a heterocyclic aromatic quinazoline ring, known to be preferably metabolized by AO. One of the oxidative metabolites of CP-724,714 observed in human hepatocytes was also generated in recombinant human AO. Although CP-724,714 is metabolized by both CYPs and AO in human hepatocytes, the contribution level of AO could not be evaluated using its specific inhibitors because of low AO activity in in vitro human materials. Here, we present a metabolic pathway for CP-724,714 in human hepatocytes and the involvement of AO in CP-724,714 metabolism. We showed here a plausible workflow for predicting AO contribution to the metabolism of CP-724,714 based on DMPK screening data. SIGNIFICANCE STATEMENT: 2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) was identified as a substrate of aldehyde oxidase (AO) rather than xanthine oxidase. Since CP-724,714 is also metabolized by cytochrome P450s (CYPs), the contribution levels of AO and CYPs in the metabolism of CP-724,714 were estimated simultaneously based on in vitro drug metabolism screening data.

Human Immortalized Cell-Based Blood-Brain Barrier Spheroid Models Offer an Evaluation Tool for the Brain Penetration Properties of Macromolecules.

Blood-brain barrier (BBB)-permeable middle- or macromolecules (middle/macromolecules) have recently attracted significant attention as new drug delivery carriers into the human brain via receptor-mediated transcytosis (RMT). During the development process of such carriers, it is necessary to thoroughly evaluate their human BBB permeability levels. In such evaluations, our recently established human immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models) have shown high potential. However, the specifics of those capabilities have yet to be elucidated. Therefore, in this study, we characterize the ability of the hiMCS-BBB models to evaluate RMT-mediated BBB penetration properties of middle/macromolecules. More specifically, we began by validating transferrin receptor (TfR)-mediated RMT functionalities using transferrin in the hiMCS-BBB models and then examined the BBB permeability levels of MEM189 antibodies (known BBB-permeable anti-TfR antibodies). The obtained results showed that, as with the case of transferrin, temperature-dependent uptake of MEM189 antibodies was observed in the hiMCS-BBB models, and the extent of that uptake increased in a time-dependent manner until reaching a plateau after around 2 h. To further expand the evaluation applicability of the models, we also examined the BBB permeability levels of the recently developed SLS cyclic peptide and observed that peptide uptake was also temperature-dependent. To summarize, our results show that the hiMCS-BBB models possess the ability to evaluate the RMT-mediated BBB-permeable properties of antibodies and peptides and thus have the potential to provide valuable tools for use in the exploration and identification of middle/macromolecules showing excellent BBB permeability levels, thereby contributing powerfully to the development of new drug delivery carriers for transporting drugs into the human brain.

In Vitro-In Vivo Correlation of Blood-Brain Barrier Permeability of Drugs: A Feasibility Study Towards Development of Prediction Methods for Brain Drug Concentration in Humans.

PURPOSE: In vitro human blood-brain barrier (BBB) models in combination with central nervous system-physiologically based pharmacokinetic (CNS-PBPK) modeling, hereafter referred to as the "BBB/PBPK" method, are expected to contribute to prediction of brain drug concentration profiles in humans. As part of our ongoing effort to develop a BBB/PBPK method, we tried to clarify the relationship of in vivo BBB permeability data to those in vitro obtained from a human immortalized cell-based tri-culture BBB model (hiBBB), which we have recently created. METHODS: The hiBBB models were developed and functionally characterized as previously described. The in vitro BBB permeabilities (Pe, × 10-6 cm/s) of seventeen compounds were determined by permeability assays, and in vivo BBB permeabilities (QECF) for eight drugs were estimated by CNS-PBPK modeling. The correlation of the Pe values with the QECF values was analyzed by linear regression analysis. RESULTS: The hiBBB models showed intercellular barrier properties and several BBB transporter functions, which were enough to provide a wide dynamic range of Pe values from 5.7 ± 0.7 (rhodamine 123) to 2580.4 ± 781.9 (rivastigmine). Furthermore, the in vitro Pe values of the eight drugs showed a good correlation (R2 = 0.96) with their in vivo QECF values estimated from human clinical data. CONCLUSION: We show that in vitro human BBB models provide clinically relevant BBB permeability that can be used as input for CNS-PBPK modeling. Therefore, our findings will encourage the development of a BBB/PBPK method as a promising approach for predicting brain drug concentration profiles in humans.

Development, Characterization and Potential Applications of a Multicellular Spheroidal Human Blood-Brain Barrier Model Integrating Three Conditionally Immortalized Cell Lines.

In vitro blood-brain barrier (BBB) models are essential research tools for use in developing brain-targeted drugs and understanding the physiological and pathophysiological functions of the BBB. To develop BBB models with better functionalities, three-dimensional (3D) culture methods have gained significant attention as a promising approach. In this study, we report on the development of a human conditionally immortalized cell-based multicellular spheroidal BBB (hiMCS-BBB) model. After being seeded into non-attachment culture wells, HASTR/ci35 (astrocytes) and HBPC/ci37 cells (brain pericytes) self-assemble to form a spheroid core that is then covered with an outer monolayer of HBMEC/ci18 cells (brain microvascular endothelial cells). The results of immunocytochemistry showed the protein expression of several cellular junction and BBB-enriched transporter genes in HBMEC/ci18 cells of the spheroid model. The permeability assays showed that the hiMCS-BBB model exhibited barrier functions against the penetration of dextran (5 and 70 kDa) and rhodamine123 (a P-glycoprotein substrate) into the core. On the other hand, facilitation of 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG; a fluorescent glucose analog) uptake was observed in the hiMCS-BBB model. Furthermore, tumor necrosis factor-alpha treatment elicited an inflammatory response in HBMEC/ci18 cells, thereby suggesting that BBB inflammation can be recapitulated in the hiMCS-BBB model. To summarize, we have developed an hiMCS-BBB model that possesses fundamental BBB properties, which can be expected to provide a useful and highly accessible experimental platform for accelerating various BBB studies.

Bioactivation of diclofenac in human hepatocytes and the proposed human hepatic proteins modified by reactive metabolites.

To reveal putative bioactivation pathways of diclofenac, in vitro human liver materials such as microsomal fractions and hepatocytes were used to confirm metabolic activation of diclofenac by 35S-cysteine trapping assay and covalent binding assay. Candidate human liver proteins possibly targeted by 14C-diclofenac via bioactivation were investigated using two-dimensional gel electrophoresis followed by detection of remaining radioactivity on the modified proteins with bio-imaging analyzer.In the 35S-cysteine trapping assay, three and two adducts with 35S-cysteine were observed in NADPH-fortified and UDPGA-fortified human liver microsomes, respectively. In the covalent binding assay using 14C-diclofenac in human hepatocytes, the extent of covalent binding of diclofenac to human hepatic proteins increased time-dependently. Addition of glutathione attenuated the extent of covalent binding of 14C-diclofenac to human liver microsomal proteins.Fifty-nine proteins from human hepatocytes were proposed as the candidate proteins targeted by reactive metabolites of diclofenac. Proteins modified by cytochrome P450-mediated reactive metabolites were identified by using a cytochrome P450 inhibitor, 1-aminobenzyltriazole and seven of the nine radioactive protein spots were removed by 1-aminobenzyltriazole treatment.In contrast, the remaining two radioactive protein spots, mainly containing human serum albumin and heat shock proteins, were not affected by the addition of 1-aminobenzyltriazole, which suggested the involvement of the acyl glucuronide of diclofenac, formed via uridine diphosphate-glucuronosyl transferases, in the covalent modifications induced by diclofenac.

A Human Immortalized Cell-Based Blood-Brain Barrier Triculture Model: Development and Characterization as a Promising Tool for Drug-Brain Permeability Studies.

Brain microvascular endothelial cells (BMEC), together with astrocytes and pericytes, form the blood-brain barrier (BBB) that strictly restricts drug penetration into the brain. Therefore, in central nervous system drug development, the establishment of an in vitro human BBB model for use in studies estimating the in vivo human BBB permeability of drug candidates has long been awaited. The current study developed and characterized a human immortalized cell-based BBB triculture model, termed the "hiBBB" model. To set up the hiBBB model, human immortalized BMEC (HBMEC/ci18) were cocultured with human immortalized astrocytes (HASTR/ci35) and brain pericytes (HBPC/ci37) in a transwell system. The trans-endothelial electrical resistance of the hiBBB model was 134.4 ± 5.5 (Ω × cm2), and the efflux ratios of rhodamine123 and dantrolene were 1.72 ± 0.11 and 1.72 ± 0.45, respectively, suggesting that the hiBBB model possesses essential cellular junction and efflux transporter functions. In BBB permeability assays, the mean value of the permeability coefficients (Pe) of BBB permeable compounds (propranolol, pyrilamine, memantine, and diphenhydramine) was 960 × 10-6 cm/s, which was clearly distinguishable from that of BBB nonpermeable compounds (sodium fluorescein and Lucifer yellow, 18 × 10-6 cm/s). Collectively, this study successfully developed the hiBBB model, which exhibits essential BBB functionality. Taking into consideration the high availability of the immortalized cells used in the hiBBB model, our results are expected to become an initial step toward the establishment of a useful human BBB model to investigate drug penetration into the human brain.

Relative Activity Factor (RAF)-Based Scaling of Uptake Clearance Mediated by Organic Anion Transporting Polypeptide (OATP) 1B1 and OATP1B3 in Human Hepatocytes.

In vitro-in vivo extrapolation based on uptake clearance determined in human hepatocytes has been used to predict in vivo hepatic clearance of organic anion transporting polypeptide (OATP) substrates. This study evaluated the relative activity factor (RAF) approach to extrapolate active uptake clearance in transporter-transfected cell systems (CLuptake) to that in human hepatocyte suspensions (PSinf,act). RAF values for OATP1B1 and OATP1B3 were determined in two batches of cryopreserved human hepatocytes using estrone-3-sulfate and cholecystokinin octapeptide as reference substrates, respectively. Fourteen OATP1B substrate drugs selected (atorvastatin, bosentan, cerivastatin, fexofenadine, fluvastatin, glibenclamide, irbesartan, nateglinide, pitavastatin, pravastatin, rosuvastatin, telmisartan, torasemide, and valsartan) showed temperature-dependent uptake in human hepatocytes. In transporter-transfected cells, OATP1B1- and OATP1B3-mediated uptake was observed in all compounds except for telmisartan. RAF-based net CLuptake was mainly accounted for by OATP1B1 (72.3-99.7%) and fell within the 3-fold of PSinf,act observed in human hepatocytes in 11 out of 13 compounds (excluding telmisartan). This study demonstrated that the RAF approach provides a quantitative index of OATP1B1- and OATP1B3-mediated PSinf,act in human hepatocytes, which will facilitate the optimization of the pharmacokinetic properties of OATP1B substrates at nonclinical stages of drug development.

Synergism of perampanel and zonisamide in the rat amygdala kindling model of temporal lobe epilepsy.

OBJECTIVE: Anticonvulsive monotherapy fails to be effective in one third of patients with epilepsy resulting in the need for polytherapy regimens. However, with the still limited knowledge, drug choices for polytherapy remain empirical. Here we report experimental data from a chronic epilepsy model for the combination of perampanel and zonisamide, which can render guidance for clinical studies and individual drug choices. METHODS: The anticonvulsant effects of the combination of perampanel and zonisamide were evaluated in a rat amygdala kindling model. Furthermore, the potential for motor impairment was evaluated. The type of interaction was quantitatively assessed based on isobolographic analysis. RESULTS: When administered alone, zonisamide dose-dependently increased the afterdischarge threshold in fully kindled rats. Moreover, data confirmed efficacy of perampanel to inhibit seizure initiation and progression with an impact on propagation of activity from the focus. Pronounced threshold increases were observed following administration of a constant zonisamide dosage combined with different doses of perampanel. Isobolographic analysis of drug responses, which is based on individual drug dose-effect data, revealed a synergistic interaction substantiating the high efficacy of the combination. Furthermore, rotarod data indicated that the combination has a favorable tolerability profile when zonisamide is coadministered with low doses of perampanel. Plasma concentration analysis argued against a pharmacokinetic interaction as a basis for the synergism. SIGNIFICANCE: The findings clearly indicate a pronounced synergistic anticonvulsant effect for the combination of the noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist perampanel with zonisamide, which modulates voltage-sensitive sodium channels and T-type calcium currents. Consequently, polytherapy using these two antiepileptic drugs might be efficacious for clinical management of partial-onset seizures. The findings indicate that the impact of dose ratios on tolerability needs be taken into account. With regard to conclusions about the extent of the synergism and its implications further antiepileptic drug combinations need to be evaluated allowing direct comparison.

Investigation of Fluorescein Derivatives as Substrates of Organic Anion Transporting Polypeptide (OATP) 1B1 To Develop Sensitive Fluorescence-Based OATP1B1 Inhibition Assays.

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of various drugs. Because OATP1B1 is a site of drug-drug interactions (DDIs), evaluating the inhibitory potential of drug candidates on OATP1B1 is required during drug development. For establishing a highly sensitive, high-throughput fluorescence-based OATP1B1 inhibition assay system, the present study focused on fluorescein (FL) and its derivatives and evaluated their uptake via OATP1B1 as well as OATP1B3 and OATP2B1 using the transporter-expressing human embryonic kidney 293 cells. We identified 2',7'-dichlorofluorescein (DCF), 4',5'-dibromofluorescein (DBF), and Oregon green (OG) as good OATP1B1 substrates with Km values of 5.29, 4.16, and 54.1 µM and Vmax values of 87.9, 48.1, and 187 pmol/min/mg protein, respectively. In addition to FL, fluo-3, and 8-fluorescein-cAMP, OG, and DBF were identified as OATP1B3 substrates. FL, OG, DCF, and DBF were identified as OATP2B1 substrates. Among the FL derivatives, DCF displayed the highest OATP1B1-mediated uptake. The Ki values of 14 compounds on OATP1B1 determined with DCF as a probe exhibited good agreement with those obtained using [(3)H]estradiol-17ß-glucuronide (E2G) as a substrate, whereas [(3)H]estrone-3-sulfate and [(3)H]sulfobromophthalein yielded higher Ki values for all inhibitors than DCF. Mutually competitive inhibition observed between DCF and E2G suggested that they share the same binding site on OATP1B1. Therefore, DCF as well as E2G can be used as sensitive probes for in vitro OATP1B1 inhibition assays, which will help mitigate the risk of false-negative DDI predictions potentially caused by substrate-dependent Ki variations.

Investigation of the impact of substrate selection on in vitro organic anion transporting polypeptide 1B1 inhibition profiles for the prediction of drug-drug interactions.

The risk assessment of organic anion transporting polypeptide (OATP) 1B1-mediated drug-drug interactions (DDIs) is an indispensable part of drug development. We previously reported that in vitro inhibitory potencies of several inhibitors on OATP1B1 depend on the substrates when prototypical substrates, estradiol-17ß-glucuronide (E2G), estrone-3-sulfate, and sulfobromophthalein were used as test substrates. The purpose of this study was to comprehensively investigate this substrate-dependent inhibition of OATP1B1 using clinically relevant OATP1B1 inhibitors and substrate drugs. Effects of cyclosporine A (CsA), rifampin, and gemfibrozil on OATP1B1-mediated uptake of 12 substrate drugs were examined in OATP1B1-expressing human embryonic kidney 293 cells. The Ki values (µM) for CsA varied from 0.0771 to 0.486 (6.3-fold), for rifampin from 0.358 to 1.23 (3.4-fold), and for gemfibrozil from 9.65 to 252 (26-fold). Except for the inhibition of torasemide uptake by CsA and that of nateglinide uptake by gemfibrozil, the Ki values were within 2.8-fold of those obtained using E2G as a substrate. Preincubation potentiated the inhibitory effect of CsA on OATP1B1 with similar magnitude regardless of the substrates. R values calculated based on a static model showed some variation depending on the Ki values determined with various substrates, and such variability could have an impact on the DDI predictions particularly for a weak-to-moderate inhibitor (gemfibrozil). OATP1B1 substrate drugs except for torasemide and nateglinide, or E2G as a surrogate, is recommended as an in vitro probe in the inhibition experiments, which will help mitigate the risk of false-negative DDI predictions potentially caused by substrate-dependent Ki variation.