Hydrolase Activity of the Genetic Variants of Human Alpha-1-Acid Glycoprotein.

In human plasma, the main agent of hydrolysis of the ester-type prodrug of levodopa, designated ONO-2160, is alpha-1-acid glycoprotein (AGP), which is a mixture of the F1*S and A variants at molar ratios of 3:1 to 2:1. In this study, the mechanism of AGP esterase-like activity was investigated by evaluating the contribution of the F1*S and A variants to ONO-2160 hydrolysis and identifying the AGP hydrolase active site. We found that although both variants hydrolyzed ONO-2160, their hydrolase activities were different. The intrinsic plasma clearance of the F1*S variant (0.441 mL/h/mg protein) was approximately 30 times higher than that of the A variant (0.0148 mL/h/mg protein), indicating that the F1*S variant contributed the most to AGP esterase-like activity. To identify the hydrolase active site of AGP, we performed inhibition studies of ONO-2160 hydrolysis using 12 AGP-binding drugs with various ligand-binding constants and binding selectivities to the two AGP variants. Inhibition of activity was positively correlated with the constant of ligand binding to the F1*S variant. In addition, compounds with high affinity to the F1*S variant inhibited ONO-2160 hydrolysis the most. Together, our data indicate that ONO-2160 is predominantly hydrolyzed by the F1*S variant at its ligand-binding site.

Species Difference in Hydrolysis of an Ester-type Prodrug of Levodopa in Human and Animal Plasma: Different Contributions of Alpha-1 Acid Glycoprotein.

Previously, we found that ONO-2160, an ester-type prodrug of levodopa (3-hydroxy-l-tyrosine), was mainly hydrolyzed in human plasma by α1-acid glycoprotein (AGP) with a partial contribution of albumin. In this study, we investigated whether ONO-2160 was hydrolyzed in the plasma of preclinical species (dog, rabbit, rat, and mouse) and humans and whether AGP and albumin are involved in its hydrolysis. ONO-2160 was hydrolyzed to some extent in the plasma of all tested species with the order of magnitude mouse > human > rabbit > rat > dog. Except for dogs, ONO-2160 was partially hydrolyzed by animal AGP and albumin. This indicated that, similar to albumin, AGP possesses esterase-like activity in mice, rats, and rabbits, as well as humans. A comparison of the values of intrinsic clearance per milliliter of plasma demonstrated that AGP was the major contributor to the hydrolysis of ONO-2160 in rabbit plasma, whereas albumin was primarily responsible for the hydrolysis of ONO-2160 in mouse plasma. This was confirmed by experiments using AGP-knockout mouse plasma. This study reports the first evidence for the existence of species differences in the hydrolysis of ONO-2160 in plasma related to the different contributions of AGP and albumin.

Predicting successful/unsuccessful extrapolation for in vivo total clearance of model compounds with a variety of hepatic intrinsic metabolism and protein bindings in humans from pharmacokinetic data using chimeric mice with humanised liver.

1. Immunodeficient chimeric mice with humanised liver have been useful in predicting total clearance values of drugs in humans. However, their usefulness may currently be limited for specific compounds with interspecies differences.2. In vivo total clearance and in vitro hepatic intrinsic clearance values of 16 model compounds were determined in control/humanised-liver mice and in mouse and human hepatocytes, respectively, for extrapolating the total clearance values of compounds in humans.3. The predictability of in vivo total clearance values of 11 model compounds in humans was adequate using pharmacokinetic data from humanised-liver mice. The predictability of total clearance values using humanised-liver mice was better than conventional allometric scaling for compounds with large interspecies differences in in vitro hepatic intrinsic clearance or plasma unbound fractions.4. There were trends that total clearance values in control and humanised-liver mice were similar to or higher than reported hepatic blood flow rates in normal mice among four compounds with poor predictability. Diazepam, with the poorest predictability, showed 38-fold-higher hepatic intrinsic clearance in mice than in humans.5. These results could lead to guidelines describing that compounds may be suited or unsuited to extrapolating total clearance values in humans from pharmacokinetics in humanised-liver mice.

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.

Unique Hydrolysis of an Ester-Type Prodrug of Levodopa in Human Plasma: Relay-Type Role Sharing between Alpha-1 Acid Glycoprotein and Human Serum Albumin.

ONO-2160 is a newly developed oral ester-type prodrug of levodopa for removing the problems in use of levodopa. It has a structure in which two of the same substituents are bound to levodopa. It is important to understand the pharmacokinetics and metabolic pathway for new drug candidate compounds. The aim of this study was to identify the major enzymes that contribute to the metabolism of ONO-2160 in human plasma. ONO-2160 was hydrolyzed by human serum albumin (HSA) and α1-acid glycoprotein (AGP) in human plasma, although the hydrolysis was not inhibited by various reported esterase inhibitors. The value of the intrinsic clearance per milliliter of plasma of ONO-2160 in AGP solution was greater than that in HSA solution and was comparable to that in human plasma. Therefore, AGP is responsible for the hydrolysis of ONO-2160 in human plasma. ONO-M, which is an intermediate metabolite of ONO-2160, has a structure in which one substituent is removed from ONO-2160 and was mainly generated in AGP solution, but not in human plasma or HSA solution. The hydrolysis of ONO-M by HSA was much greater than by AGP. These results indicate that ONO-M, which is mainly generated from ONO-2160 by AGP, is rapidly hydrolyzed by HSA, and that ONO-2160 generates levodopa via ONO-M in a relay-type reaction through AGP and HSA in human plasma. It has not been reported that AGP has esterase-like activity. These findings could be useful information for drug development of the ester-type prodrug.

Expanded Physiologically-Based Pharmacokinetic Model of Rifampicin for Predicting Interactions With Drugs and an Endogenous Biomarker via Complex Mechanisms Including Organic Anion Transporting Polypeptide 1B Induction.

As rifampicin can cause the induction and inhibition of multiple metabolizing enzymes and transporters, it has been challenging to accurately predict the complex drug-drug interactions (DDIs). We previously constructed a physiologically-based pharmacokinetic (PBPK) model of rifampicin accounting for the components for the induction of cytochrome P450 (CYP) 3A/CYP2C9 and the inhibition of organic anion transporting polypeptide 1B (OATP1B). This study aimed to expand and verify the PBPK model for rifampicin by incorporating additional components for the induction of OATP1B and CYP2C8 and the inhibition of multidrug resistance protein 2. The established PBPK model was capable of accurately predicting complex rifampicin-induced alterations in the profiles of glibenclamide, repaglinide, and coproporphyrin I (an endogenous biomarker of OATP1B activities) with various dosing regimens. Our comprehensive rifampicin PBPK model may enable quantitative prediction of DDIs across diverse potential victim drugs and endogenous biomarkers handled by multiple metabolizing enzymes and transporters.

Comprehensive PBPK Model of Rifampicin for Quantitative Prediction of Complex Drug-Drug Interactions: CYP3A/2C9 Induction and OATP Inhibition Effects.

This study aimed to construct a physiologically based pharmacokinetic (PBPK) model of rifampicin that can accurately and quantitatively predict complex drug-drug interactions (DDIs) involving its saturable hepatic uptake and auto-induction. Using in silico and in vitro parameters, and reported clinical pharmacokinetic data, rifampicin PBPK model was built and relevant parameters for saturable hepatic uptake and UDP-glucuronosyltransferase (UGT) auto-induction were optimized by fitting. The parameters for cytochrome P450 (CYP) 3A and CYP2C9 induction by rifampicin were similarly optimized using clinical DDI data with midazolam and tolbutamide as probe substrates, respectively. For validation, our current PBPK model was applied to simulate complex DDIs with glibenclamide (a substrate of CYP3A/2C9 and hepatic organic anion transporting polypeptides (OATPs)). Simulated results were in quite good accordance with the observed data. Altogether, our constructed PBPK model of rifampicin demonstrates the robustness and utility in quantitatively predicting CYP3A/2C9 induction-mediated and/or OATP inhibition-mediated DDIs with victim drugs.

Sandwich-Cultured Hepatocytes for Mechanistic Understanding of Hepatic Disposition of Parent Drugs and Metabolites by Transporter-Enzyme Interplay.

Functional interplay between transporters and drug-metabolizing enzymes is currently one of the hottest topics in the field of drug metabolism and pharmacokinetics. Uptake transporter-enzyme interplay is important to determine intrinsic hepatic clearance based on the extended clearance concept. Enzyme and efflux transporter interplay, which includes both sinusoidal (basolateral) and canalicular efflux transporters, determines the fate of metabolites formed in the liver. As sandwich-cultured hepatocytes (SCHs) maintain metabolic activities and form a canalicular network, the whole interplay between uptake and efflux transporters and drug-metabolizing enzymes can be investigated simultaneously. In this article, we review the utility and applicability of SCHs for mechanistic understanding of hepatic disposition of both parent drugs and metabolites. In addition, the utility of SCHs for mimicking species-specific disposition of parent drugs and metabolites in vivo is described. We also review application of SCHs for clinically relevant prediction of drug-drug interactions caused by drugs and metabolites. The usefulness of mathematical modeling of hepatic disposition of parent drugs and metabolites in SCHs is described to allow a quantitative understanding of an event in vitro and to develop a more advanced model to predict in vivo disposition.

Analysis of Nonlinear Pharmacokinetics of a Highly Albumin-Bound Compound: Contribution of Albumin-Mediated Hepatic Uptake Mechanism.

The cause of nonlinear pharmacokinetics (PK) (more than dose-proportional increase in exposure) of a urea derivative under development (compound A: anionic compound [pKa: 4.4]; LogP: 6.5; and plasma protein binding: 99.95%) observed in a clinical trial was investigated. Compound A was metabolized by CYP3A4, UGT1A1, and UGT1A3 with unbound Km of 3.3-17.8 µmol/L. OATP1B3-mediated uptake of compound A determined in the presence of human serum albumin (HSA) showed that unbound Km and Vmax decreased with increased HSA concentration. A greater decrease in unbound Km than in Vmax resulted in increased uptake clearance (Vmax/unbound Km) with increased HSA concentration, the so-called albumin-mediated uptake. At 2% HSA concentration, unbound Km was 0.00657 µmol/L. A physiologically based PK model assuming saturable hepatic uptake nearly replicated clinical PK of compound A. Unbound Km for hepatic uptake estimated from the model was 0.000767 µmol/L, lower than the in vitro unbound Km at 2% HSA concentration, whereas decreased Km with increased concentration of HSA in vitro indicated lower Km at physiological HSA concentration (4%-5%). In addition, unbound Km values for metabolizing enzymes were much higher than unbound Km for OATP1B3, indicating that the nonlinear PK of compound A is primarily attributed to saturated OATP1B3-mediated hepatic uptake of compound A.

Analysis of the Metabolic Pathway of Bosentan and of the Cytotoxicity of Bosentan Metabolites Based on a Quantitative Modeling of Metabolism and Transport in Sandwich-Cultured Human Hepatocytes.

To quantitatively understand the events in the human liver, we modeled a hepatic disposition of bosentan and its three known metabolites (Ro 48-5033, Ro 47-8634, and Ro 64-1056) in sandwich-cultured human hepatocytes based on the known metabolic pathway. In addition, the hepatotoxicity of Ro 47-8634 and Ro 64-1056 was investigated because bosentan is well known as a hepatotoxic drug. A model illustrating the hepatic disposition of bosentan and its three metabolites suggested the presence of a novel metabolic pathway(s) from the three metabolites. By performing in vitro metabolism studies on human liver microsomes, a novel metabolite (M4) was identified in Ro 47-8634 metabolism, and its structure was determined. Moreover, by incorporating the metabolic pathway of Ro 47-8634 to M4 into the model, the hepatic disposition of bosentan and its three metabolites was successfully estimated. In hepatocyte toxicity studies, the cell viability of human hepatocytes decreased after exposure to Ro 47-8634, and the observed hepatotoxicity was diminished by pretreatment with tienilic acid (CYP2C9-specific inactivator). Pretreatment with 1-aminobenzotriazole (broad cytochrome P450 inactivator) also tended to maintain the cell viability. Furthermore, Ro 64-1056 showed hepatotoxicity in a concentration-dependent manner. These results suggest that Ro 64-1056 is directly involved in bosentan-induced liver injury partly because CYP2C9 specifically mediates hydroxylation of the t-butyl group of Ro 47-8634. Our findings demonstrate the usefulness of a quantitative modeling of hepatic disposition of drugs and metabolites in sandwich-cultured hepatocytes. In addition, the newly identified metabolic pathway may be an alternative route that can avoid Ro 64-1056-induced liver injury.

Modeling approach for multiple transporters-mediated drug-drug interactions in sandwich-cultured human hepatocytes: effect of cyclosporin A on hepatic disposition of mycophenolic acid phenyl-glucuronide.

A lower exposure of mycophenolic acid (MPA) in patients receiving MPA-mofetil in combination with cyclosporin A (CsA) is thought to be due to the inhibition of enterohepatic circulation of phenyl-glucuronide of MPA (MPAG). This study aimed to evaluate the interaction of CsA with hepatic disposition of MPA and MPAG in sandwich-cultured human hepatocytes (SCHH) by a mathematical modeling approach. In addition, the inhibition of CsA for glucuronidation of MPA to MPAG was examined in human liver microsomes. Inhibitory parameters of CsA for hepatic disposition of MPAG were estimated using a non-linear mixed effect model program, NONMEM. As a result, CsA did not influence the conversion of MPA to MPAG in either SCHH or human liver microsomes. In contrast, CsA inhibited the basolateral uptake of MPAG with an estimated maximum inhibitory effect (Imax) of 32.4%. CsA also inhibited basolateral efflux and biliary excretion of MPAG formed in SCHH, and the concentration producing 50% of Imax (IC50) for biliary excretion was lower than that for basolateral efflux. Our modeling approach suggests that CsA inhibits both basolateral uptake and biliary excretion of MPAG and leads to changes in systemic exposure of MPA and MPAG in humans.

Inclusion of human intravenous study data in new drug applications-impact of the Japanese guidance.

Human pharmacokinetic study after intravenous (i.v.) administration is inevitable in calculating basic pharmacokinetic parameters such as bioavailability, clearance, and distribution volume. However, i.v. data are not always included in the dossier, although the regulatory guidance recommends the inclusion of i.v. studies for Japanese new drug applications. We investigated the adherence to this guidance on the inclusion of i.v. data along with various factors. Logistic regression analyses were carried out on 117 new oral drugs approved in Japan between 1999 and 2009. Inclusion of i.v. data conspicuously increased after the issuance of the guidance for self-originated drugs developed by Japanese firms [odds ratio (OR) = 30-41). Moreover, drugs exhibiting large interindividual variations in pharmacokinetics showed a positive association (OR = 4.1-8.2), whereas solubility did not show notable association (OR = 1.0) with inclusion of i.v. data. Inclusion of i.v. data was not associated with physicochemical feasibility, but the developer's willingness to comply with the guidance seems to be involved.

Prediction of human bioavailability from human oral administration data and animal pharmacokinetic data without data from intravenous administration of drugs in humans.

PURPOSE: To predict the absolute oral bioavailabilities (BAs) of drugs in humans without using pharmacokinetic data from intravenous administration in humans. METHODS: The distribution volume of the terminal phase (Vd(beta)) in humans was predicted by three methods using animal pharmacokinetic data. Then, total body clearance (CL(tot)) was calculated by multiplying the elimination rate constant and Vd(beta), and the BA was calculated as a ratio between CL(tot) and oral clearance. The predicted and observed values were compared for 67 drugs for which pharmacokinetic data after intravenous administration in humans were available. RESULTS: For Vd(beta), predicted values within twice the observed value were obtained for 72.1% of drugs by both methods Ia and Ib, respectively, in which only rat pharmacokinetic data were used. The corresponding percentage was 75.0% for method II, in which pharmacokinetic data from animals other than rats were used. For BA, predicted values within 1.3 times the observed values were obtained for 66.7% and 57.4% of drugs by methods Ia and Ib, respectively, and 75.0% by method II. CONCLUSIONS: Using the present methods, it is possible to predict BA from human oral administration data combined with animal pharmacokinetic data to a certain level without using intravenous injection data.

The effect of continuous arterial infusion of gabexate mesilate (FOY-007) on experimental acute pancreatitis.

The effectiveness of continuous arterial infusion of Gabexate Mesilate (FOY-007) on experimental acute pancreatitis was investigated. Acute necrotizing pancreatitis was induced by an injection of 10% Na-taurocholate (1ml/kg) into the main pancreatic duct of mongrel dogs. Animals were divided into three groups; Group A: non-treated control, Group B: after the induction of pancreatitis, injected with FOY-007 intravenously (5mg/kg/hr), Group C: after the induction of pancreatitis, injected with FOY-007 via the celiac artery. The changes in the values of amylase and lipase in serum and ascites etc. were examined. A histological examination was done and the FOY-007 concentration of the pancreas was measured. In both groups B and C, the serum levels of amylase and lipase reached significantly to low levels compared with those in group A. The extents of pancreatic parenchyma necrosis in each group were 36.1, 25.3 and 19.5%, respectively, and were significantly improved in group C. In addition, the FOY-007 levels in pancreas specimens in the intraarterial infusion group exceeded those in the intravenous infusion group by 32 times. The results suggest that continuous FOY-007 arterial infusion therapy is useful as a local treatment for severe acute pancreatitis.