Publications by authors named "Kazuhisa Ozeki"

15 Publications

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- correlation of antitumor activity of heat shock protein 90 (HSP90) inhibitors with a pharmacokinetics/pharmacodynamics analysis using NCI-N87 xenograft mice.

Xenobiotica 2021 Jun 16:1-30. Epub 2021 Jun 16.

Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.

The antitumor activity (e.g., IC) of anticancer drugs is important for selecting candidate compounds for drug efficacy study in the early stage of drug discovery. In this study, we investigated the relationship between IC and EC using six heat shock protein 90 (HSP90) inhibitors.IC of each compound was calculated from cell proliferation assays using the NCI-N87 cancer cell line. Each compound was administered to NCI-N87 xenograft mice, and EC and the maximum tumor-killing rate constant were calculated from pharmacokinetics/pharmacodynamics analyses using plasma concentrations and tumor volumes.IC obtained was poorly correlated with EC obtained , while a good correlation ( = 0.856) was observed between them when corrected with the unbound fraction ratio.The results of this study using of HSP90 inhibitors as model compounds suggest importance of the consideration of an unbound fraction to evaluate the relationship between IC and EC. These results will contribute to improvement in the prediction accuracy of drug efficacy from activity and the efficiency of drug discovery research.
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http://dx.doi.org/10.1080/00498254.2021.1942588DOI Listing
June 2021

A cell based assay for evaluating binding and uptake of an antibody using hepatic nonparenchymal cells.

Sci Rep 2021 Apr 16;11(1):8383. Epub 2021 Apr 16.

Laboratory of DDS Design and Drug Disposition, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, Chiba, 260-0856, Japan.

Evaluation of the binding and uptake of an antibody in liver non-parenchymal cells (NPC), including liver sinusoidal endothelial cells, is important for revealing its pharmacokinetic (PK) behavior, since NPC has important roles in eliminating an antibody from the blood via the Fc fragment of IgG receptor IIB (FcγRIIB). However, there is currently no in vitro quantitative assay using NPC. This study reports on the development of a cell-based assay for evaluating the binding and uptake of such an antibody using liver NPC of mice and monkeys. In mice, the FcγRIIB-expressing cells were identified in the CD146-positive and CD45-negative fraction by flow cytometry. A titration assay was performed to determine the PK parameters, and the obtained parameter was comparable to that determined by the fitting of the in vivo PK. This approach was also extended to NPC from monkeys. The concentration-dependent binding and uptake was measured to determine the PK parameters using monkey NPC, the FcγRIIB-expressing fraction of which was identified by CD31 and CD45. The findings presented herein demonstrate that the in vitro liver NPC assay using flow cytometry is a useful tool to determine the binding and uptake of biologics and to predict the PK.
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http://dx.doi.org/10.1038/s41598-021-87912-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052349PMC
April 2021

Combination of cassette-dosing and microsampling for reduced animal usage for antibody pharmacokinetics in cynomolgus monkeys, wild-type mice, and human FcRn transgenic mice.

Pharm Res 2021 Apr 29;38(4):583-592. Epub 2021 Mar 29.

Research Division, Chugai Pharmaceutical Co. Ltd., 1-135 Komakado, Gotemba-shi, Shizuoka, 412-8513, Japan.

Purpose: The aim of this study was to develop a useful antibody PK evaluation tool using a combination of cassette-dosing and microsampling in mice and monkeys in order to reduce the number of animals used.

Methods: Cetuximab, denosumab, infliximab, and a mixture of the three antibodies, i.e., cassette-dosing, were administered intravenously to cynomolgus monkeys, C57BL/6J mice, and homozygous human neonatal Fc-receptor transgenic (Tg32) mice. Mouse blood was collected from one animal continuously via the jugular vein at nine points.

Results: In cynomolgus monkeys, infliximab showed faster elimination in the cassette-dosing group than in the single-dose group. Anti-drug antibody production was observed, but the PK parameters of the clearance and distribution volume were similar in both groups. In C57BL/6J and Tg32 mice, each of the plasma concentrations-time profiles after cassette-dosing were similar to those after single dosing. PK evaluation using a combination of cassette-dosing and microsampling in mice may reduce the number of mice used by approximately 90% compared with the conventional method.

Conclusions: The combination of antibody cassette-dosing and microsampling is a promising PK evaluation method as a high-throughput and reliable with reduced numbers of mice and cynomolgus monkeys.
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http://dx.doi.org/10.1007/s11095-021-03028-6DOI Listing
April 2021

Prediction of Human Pharmacokinetics Profile of Monoclonal Antibody Using hFcRn Transgenic Mouse Model.

Biol Pharm Bull 2021 ;44(3):389-395

Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama.

Human pharmacokinetics (PK) profiles of monoclonal antibodies (mAbs) are usually predicted using non-human primates (NHP), but this comes with drawbacks in terms of cost and throughput. Therefore, we established a human PK profile prediction method using human neonatal Fc receptor (hFcRn) transgenic mice (TgM). We administered launched 13 mAbs to hFcRn TgM and measured the concentration in plasma using electro-chemiluminescence immunoassay. This was then used to calculate PK parameters and predict human PK profiles. The mAbs showed a bi-phased elimination pattern, and clearance (CL) (mL/d/kg) and distribution volume at steady state (V) (mL/kg) ranges were 11.0 to 131 and 110 to 285, respectively. There was a correlation in half-life at elimination phase (t) between hFcRn TgM and humans for 10 mAbs showing CL of more than 80% in the elimination phase (R = 0.714). Human t was predicted using hFcRn TgM t; 9 out of 10 mAbs were within 2-fold the actual values, and all mAbs were within 3-fold. Regarding the predicted CL values, 7 out of 10 mAbs were within 2-fold the human values and all mAbs were within 3-fold. Furthermore, even on day 7 the predicted CL values of 8 out of 10 mAbs were within 2-fold the observed value, with all mAbs within 3-fold. These results suggest human PK profiles can be predicted using hFcRn TgM data. These methods can accelerate the development of antibody drugs while also reducing cost and improving throughput.
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http://dx.doi.org/10.1248/bpb.b20-00775DOI Listing
January 2021

Evaluation of Methods to Assess CYP3A Induction Risk in Clinical Practice Using in Vitro Induction Parameters.

Biol Pharm Bull 2021 ;44(3):338-349

Research division, Chugai Pharmaceutical Co., Ltd.

Established guidelines have recommended a number of methods based on in vitro data to assess the CYP3A induction risk of new chemical entities in clinical practice. In this study, we evaluated the predictability of various assessment methods. We collected in vitro parameters from a variety of literature that includes data on 19 batches of hepatocytes. Clinical CYP3A induction was predicted using 3 direct approaches-the fold-change, basic model, and mechanistic static models-as well as 5 correlation approaches, including the relative induction score (RIS) and the relative factor (RF) method. These predictions were then compared with data from 30 clinical inductions. Collected in vitro parameters varied greatly between hepatocyte batches. Direct assessment methods using fixed cut-off values provided a lot of false predictions due to hepatocyte variability, which can overlook induction risk or lead to needless clinical drug-drug interaction (DDI) studies. On the other hand, correlation methods with the cut-off values set for each batch of hepatocytes accurately predicted the induction risk. Among these, the AUC/inducer concentrations for half the maximum induction (EC) and the RF methods which use the area under the curve (AUC) of the unbound inducers for calculating induction potential showed an especially good correlation with clinical induction. Correlation methods were better at predicting clinical induction risk than the other methods, regardless of hepatocyte variability. The AUC/EC and the RF methods in particular had a small number of false predictions, and can therefore be used to assess induction risk along with the other correlation methods recommended in guidelines.
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http://dx.doi.org/10.1248/bpb.b20-00578DOI Listing
January 2021

Exploitation of Elevated Extracellular ATP to Specifically Direct Antibody to Tumor Microenvironment.

Cell Rep 2020 12;33(12):108542

Research Division, Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd., 200, Kajiwara, Kamakura, Kanagawa, 247-8530, Japan.

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.
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http://dx.doi.org/10.1016/j.celrep.2020.108542DOI Listing
December 2020

Pharmacokinetic prediction of an antibody in mice based on an in vitro cell-based approach using target receptor-expressing cells.

Sci Rep 2020 10 1;10(1):16268. Epub 2020 Oct 1.

Laboratory of DDS Design and Drug Disposition, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, Chiba, 260-0856, Japan.

In vivo pharmacokinetics (PK) studies using mice and monkeys are the main approaches for evaluating and predicting the PK of antibodies, and there is a strong demand for methods that do not require animal experiments. In this work, we focused on quantitatively predicting the nonlinear PK of an antibody based on cell-based assays. An anti-mouse Fc gamma receptor IIB antibody was used as a model antibody. To determine the PK parameters related to nonspecific elimination in vivo, the plasma concentration profile at 100 mg/kg, at which target-specific clearance is saturated, was analyzed by a 2-compartment model. To estimate the parameters related to target-specific elimination, the Michaelis-Menten constant (K) and the maximum elimination rate (V) were determined by an uptake assay using Chinese hamster ovary (CHO) cells expressing the target receptor. Finally, the integration of all of these parameters permitted the PK to be predicted at doses ranging from 1 to 100 mg/kg regardless of whether target-specific clearance was saturated or nonsaturated. The findings presented herein show that in vitro assays using target-expressing cells are useful tools for obtaining PK parameters and predicting PK profiles and, in some cases, eliminate the need for in vivo PK studies using experimental animals.
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http://dx.doi.org/10.1038/s41598-020-73255-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529773PMC
October 2020

Predicting Method for the Human Plasma Concentration-Time Profile of a Monoclonal Antibody from the Half-life of Non-human Primates.

Biol Pharm Bull 2020 ;43(5):823-830

Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama.

Efficiency (speed and cost) and animal welfare are important factors in the development of new drugs. A novel method (the half-life method) was developed to predict the human plasma concentration-time profile of a monoclonal antibody (mAb) after intravenous (i.v.) administration using less data compared to the conventional approach; moreover, predicted results were comparable to conventional method. This new method use human geometric means of pharmacokinetics (PK) parameters and the non-human primates (NHP) half-life of each mAb. PK data on mAbs in humans and NHPs were collected from literature focusing on linear elimination, and the two-compartment model was used for analysis. The following features were revealed in humans: 1) the coefficient of variation in the distribution volume of the central compartment and at steady state of mAbs was small (22.6 and 23.8%, respectively) and 2) half-life at the elimination phase (t1/2) was the main contributor to plasma clearance. Moreover, distribution volume showed no significant correlation between humans and NHPs, and human t1/2 showed a good correlation with allometrically scaled t1/2 of NHP. Based on the features revealed in this study, we propose a new method for predicting the human plasma concentration-time profile of mAbs after i.v. dosing. When tested, this half-life method showed reasonable human prediction compared with a conventional empirical approach. The half-life method only requires t1/2 to predict human PK, and is therefore able to improve animal welfare and potentially accelerate the drug development process.
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http://dx.doi.org/10.1248/bpb.b19-01042DOI Listing
January 2021

Quantitative evaluation of hepatic and intestinal induction of CYP3A in clinical practice.

Xenobiotica 2020 Aug 7;50(8):875-884. Epub 2020 Jan 7.

Research division, Chugai Pharmaceutical Co., Ltd, Gotemba, Shizuoka, Japan.

This is the first report quantitatively evaluating the clinical induction of CYP3A in the liver and the intestine.To evaluate hepatic induction, we collected literature data on endogenous biomarkers of hepatic CYP3A induction which we then used to calculate the fold-induction (inducer-mediated change in biomarker level). Literature data on decreases in the area under the curve (AUC) of alfentanil, a CYP3A substrate, caused by CYP3A inducers were also collected. We used the hepatic intrinsic clearance of alfentanil to calculate the hepatic induction ratio (inducer-mediated change in intrinsic clearance). For intestinal induction, the intestinal bioavailability (F) of alfentanil was used to calculate the intestinal induction ratio. We determined in vivo maximum induction (E) and the average unbound plasma concentration (C) required for half the maximum induction (EC) for inducers using an E model analysis.In our results, fold-induction was comparable to the induction ratio at several inducer concentrations, and almost the maximum induction was achieved by a therapeutic dose. Induction ratios in the intestine were similar to the liver.Our findings suggest that, by knowing only hepatic induction ratios for common inducers, we can quantitatively predict the decreases in the AUC of substrates by CYP3A induction.
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http://dx.doi.org/10.1080/00498254.2019.1710620DOI Listing
August 2020

Simplified Method to Determine the Efflux Ratio on P-Glycoprotein Substrates Using Three-Compartment Model Analysis for Caco-2 Cell Assay Data.

Pharm Res 2019 Dec 23;37(1):13. Epub 2019 Dec 23.

Laboratory of Biopharmacy, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka, 422-8256, Japan.

Purpose: Multiple time-point sampling is required in transcellular transport studies to accurately calculate the appropriate efflux ratio (ER). Our study sought to develop a simplified method to determine the ER in Caco-2 cells.

Methods: The equation for the ER was derived from a three-compartment model of apical to basal and basal to apical transport. Transcellular transport studies were conducted with 10 non-P-glycoprotein (P-gp) and 6 P-gp substrates in Caco-2 cells, and the ER was calculated using this equation.

Results: The equation for the ER used the concentration ratio in the receiver compartment at the same time-point; therefore, the ER can theoretically be calculated using only a single point. The ER of all non-P-gp substrates tested was close to 1 at all sampling times. The ERs of cyclosporine A calculated from the concentration ratio at 30, 60, 90, and 120 min incubation were 2.93, 6.43, 7.12, and 9.57, respectively, and the ER at 120 min was almost identical to the theoretical value (9.62) calculated using three-compartment model analysis. The other 5 P-gp substrates showed a similar tendency. Single-point sampling can be used to accurately calculate ER at 120 min.

Conclusions: Single-point sampling is a promising approach for calculating appropriate ERs in the drug discovery stage.
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http://dx.doi.org/10.1007/s11095-019-2729-xDOI Listing
December 2019

Three-Compartment Model Analysis with Minimal Sampling Points in the Caco-2 Permeability Assay.

Biol Pharm Bull 2019 ;42(9):1600-1604

Laboratory of Biopharmacy, School of Pharmaceutical Sciences, University of Shizuoka.

The purpose of this study was to establish a modified method of three-compartment model analysis that minimized the sampling frequency. A Caco-2 permeability assay was performed on ten structurally diverse compounds with passive diffusion. A three-compartment model was analyzed by a conventional method and a method with fewer sampling points, called the simplified method, using concentration-time profiles in the donor, intracellular, and receiver compartments. The concentration-time profiles in all compartments were well described by the conventional method. The calculated unbound fraction of intracellular (fu) and apparent permeability coefficient (P) were 0.0107-1.22 and 0.886-146 × 10 cm/s, respectively. The simplified method also described the concentration profiles in the compartments of all ten compounds except one, ibuprofen. The difference in values calculated by the simplified method compared to the conventional method was between -7 and 7% for fu and between -6 and 42% for P. These results suggested that the parameter values from the simplified method were comparable with those from the conventional method. The simplified method may be a promising approach to improve the throughput of three-compartment model analyses of Caco-2 permeability assays in the early stages of drug discovery.
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http://dx.doi.org/10.1248/bpb.b19-00221DOI Listing
January 2020

Pharmacokinetics of an intracerebroventricularly administered antibody in rats.

MAbs 2017 10 6;9(7):1210-1215. Epub 2017 Jul 6.

a Research Division , Chugai Pharmaceutical Co., Ltd. , Shizuoka , Japan.

The pharmacokinetics (PK) of an antibody in the brain and the spinal cord is insufficiently understood, which is an obstacle to the discovery of antibody drugs that target diseases in the central nervous system. In this study, we focused on the elimination of IgG from cerebrospinal fluid (CSF) circulating in the brain and the spinal cord in rats, and, to evaluate the influence of CSF bulk flow on the clearance of IgG, also examined the PK of inulin in CSF. To monitor their concentrations in CSF, IgG and inulin were co-administered into the lateral ventricle via a catheter, and CSF was collected from the cisterna magna via another catheter time-sequentially. Blood was also obtained from the same individuals, and the concentrations of IgG and inulin in CSF and plasma were measured. The results revealed that PK parameters of IgG were similar to those of inulin; half-life and clearance of IgG were 47.0 ± 6.49 min and 29.0 ± 15.2 mL/day/kg, and those of inulin were 52.8 ± 25.4 min and 29.0 ± 13.3 mL/day/kg. Moreover, deconvolution analysis indicated that all of the IgG administered in the lateral ventricle was transferred to plasma from CSF within 24 hours. This study demonstrated that IgG in CSF was eliminated by bulk flow and transferred totally to blood circulation.
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http://dx.doi.org/10.1080/19420862.2017.1345834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627584PMC
October 2017

PK/PD analysis of a novel pH-dependent antigen-binding antibody using a dynamic antibody-antigen binding model.

Drug Metab Pharmacokinet 2016 Apr 6;31(2):123-32. Epub 2016 Jan 6.

Research Division, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka, Japan.

Previously, we have reported novel engineered antibody with pH-dependent antigen-binding (recycling antibody), and with both pH-dependent antigen-binding and increased FcRn-binding at neutral pH (sweeping antibody). The purpose of this study is to perform PK/PD predictions to better understand the potential applications of the antibodies as therapeutics. To demonstrate the applicability of recycling and sweeping antibodies over conventional antibodies, PK/PD analyses were performed. PK/PD parameters for antibody and antigen dynamics were estimated from the results of a pharmacokinetic study in human FcRn transgenic mice. A simulation study was performed using the estimated PK/PD parameters with various target antigen profiles. In comparison to conventional antibody, recycling antibody enhanced antibody-antigen complex clearance by 3 folds, while sweeping antibody accelerated antigen clearance by 10 folds in a pharmacokinetic study. Simulation results showed that recycling and sweeping antibodies can improve dosage frequency and reduce the required dose for target antigens with various clearances, plasma concentrations or binding kinetics. Moreover, importance of the association rate constant to enhance the beneficial effect of antibodies was shown. These results support the conclusion that recycling and sweeping antibodies can be applied to various target antigens with different profiles, and expand the number of antigens that antibodies can target.
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http://dx.doi.org/10.1016/j.dmpk.2015.12.007DOI Listing
April 2016

Evaluation of the appropriate time range for estimating the apparent permeability coefficient (P(app)) in a transcellular transport study.

Int J Pharm 2015 Nov 24;495(2):963-71. Epub 2015 Sep 24.

Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan. Electronic address:

In a transcellular transport study, the apparent permeability coefficient (Papp) of a compound is evaluated using the range by which the amount of compound accumulated on the receiver side is assumed to be proportional to time. However, the time profile of the concentration of the compound in receiver (C3) often shows a lag time before reaching the linear range and later changes from linear to steady state. In this study, the linear range needed to calculate Papp in the C3-time profile was evaluated by a 3-compartment model. C3 was described by an equation with two steady states (C3=A3(1-e(-αt))+B3(1-e(-βt)), α>β), and by a simple approximate line (C3=A3-A3×αt) in the time range of 3/α
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http://dx.doi.org/10.1016/j.ijpharm.2015.09.035DOI Listing
November 2015

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

PLoS Pathog 2012 16;8(8):e1002860. Epub 2012 Aug 16.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.
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http://dx.doi.org/10.1371/journal.ppat.1002860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420934PMC
December 2012
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