Robust physiologically based pharmacokinetic model of rifampicin for predicting drug-drug interactions via P-glycoprotein induction and inhibition in the intestine, liver, and kidney.

P-glycoprotein (P-gp) is an efflux transporter that plays an important role in the pharmacokinetics of its substrate, and P-gp activities can be altered by induction and inhibition effects of rifampicin. This study aimed to establish a physiologically based pharmacokinetic (PBPK) model of rifampicin to predict the P-gp-mediated drug-drug interactions (DDIs) and assess the DDI impact in the intestine, liver, and kidney. The induction and inhibition parameters of rifampicin for P-gp were estimated using two of seven DDI cases of rifampicin and digoxin and incorporated into our previously constructed PBPK model of rifampicin. The constructed rifampicin model was verified using the remaining five DDI cases with digoxin and five DDI cases with other P-gp substrates (talinolol and quinidine). Based on the established PBPK model, following repeated dosing of 600 mg rifampicin, the deduced net effect was an approximately threefold induction in P-gp activities in the intestine, liver, and kidney. Furthermore, in all 12 cases the predicted area under the plasma concentration-time curve ratios of the P-gp substrates were within the predefined acceptance criteria with various dosing regimens. Intestinal effects of P-gp-mediated DDIs had their greatest impact on the pharmacokinetics of digoxin and talinolol, with a minimal impact on the liver and kidney. For quinidine, predicted intestinal P-gp/cytochrome P450 3A-mediated DDIs were slightly underestimated because of the complexity of nonlinearity and transporter-enzyme interplay. These findings demonstrate that our rifampicin model can be applicable to quantitatively predict the net impact of P-gp induction and/or inhibition on diverse P-gp substrates and investigate the magnitude of DDIs in each tissue.

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.

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.

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.

Evaluation of hepatic disposition of paroxetine using sandwich-cultured rat and human hepatocytes.

Paroxetine, a selective serotonin reuptake inhibitor, is metabolized in the liver and excreted into bile and urine as metabolites, but species differences have been observed in hepatic disposition between rats and humans. A major metabolite in rats is M1-glucuronide, whereas M1-glucuronide and M1-sulfate are found in humans. The primary excretion route of paroxetine-derived radioactivity in rats and humans is bile and urine, respectively. The aim of this study was to examine the usefulness of sandwich-cultured hepatocytes (SCH) to evaluate in vivo species differences of the hepatic disposition of paroxetine between rats and humans. The metabolite profile of [(3)H]paroxetine in SCH was similar to that in hepatocytes in suspension, and the in vitro metabolite profiles were similar to the published in vivo metabolic pathways for both species. Furthermore, the biliary excretion index (BEI) of formed M1-glucuronide in rat SCH (25.8-50.9%) was higher than that in human SCH (15.1-16.7%). The BEI of formed M1-sulfate (16.4-29.1%) was comparable to that of M1-glucuronide in human SCH, whereas the BEIs of paroxetine were negligible in SCH of both species. Moreover, M1-glucuronide was demonstrated to be a multidrug resistance-associated protein 2 substrate in both species, as determined by its uptake into ATP-binding cassette transporter-expressing membrane vesicles. SCH should prove to be useful to evaluate the processes of hepatic uptake and metabolism of parent drugs and the simultaneous examination of the biliary excretion of both parent drug and liver-derived metabolites.

In vitro metabolism and inhibitory effects of pranlukast in human liver microsomes.

We investigated the metabolism of pranlukast, a selective leukotriene agonist, and the potential for drug-drug interactions. Although cytochrome P450 (CYP) 3A4 appeared to be the major cytochrome P450 isoform involved in the metabolism of pranlukast, the results suggested that pranlukast metabolism was inhibited less than 50% by ketoconazole, a reversible CYP3A4 inhibitor, or by anti-CYP3A4 antibodies. Irreversible macrolide CYP3A4 inhibitors, clarithromycin, erythromycin and roxithromycin, exhibited little effect on pranlukast metabolism. On the other hand, pranlukast reversibly inhibited CYP2C8 and/or 2C9, and CYP3A4, with K(i) values of 3.9 and 4.1 micromol/l, respectively. The [I](in,max,u)/K(i) ratios were 0.004 and 0.003, respectively. The K(i) values were about 300-fold greater than the [I](in,max,u), therefore it is suggested that, at clinical doses, pranlukast will not affect the pharmacokinetics of concomitantly administered drugs that are primarily metabolized by CYP2C8 and/or 2C9 or CYP3A4.

Cloning and Characterization of the Murine and Rat mrp1 Promoter Regions.

The ATP-binding cassette transporter multidrug resistance protein 1 (MRP1) confers resistance to a number of clinically important chemotherapeutic agents. The proximal promoter region of MRP1 is GC-rich and contains binding sites for members of the Sp1 family of trans-acting factors that seem to be important for basal expression. As an approach to searching for other elements that may contribute to expression, we have sequenced and functionally compared the promoters of the murine and rat mrp1 genes with that of the human gene. All three promoters are GC-rich, TATA-less, and CAAT-less. Conservation of sequence between rodent and human promoters is limited to a proximal region of 100 nucleotides containing binding sites for members of the Sp1 family and a putative activator protein-1 element. The 5'-untranslated region (UTR) of human MRP1 contains an insertion of approximately 160 nucleotides comprising a GCC-triplet repeat and a GC-rich tandem repeat that is absent from the rodent sequences. Transient transfection analyses demonstrated that the conserved GC-boxes of all three genes are the major determinants of basal activity. Based on electrophoretic mobility shift assays, each GC-box can be bound by Sp1 or Sp3. Unlike the rodent genes, the human MRP1 5'UTR also binds Sp1 but not Sp3, and the human promoter retains substantial activity even in the absence of the conserved GC-boxes. Finally, we show that the tumor suppressor protein p53 can repress the human and rodent promoters by a mechanism that is independent of the Sp1 elements.