Supporting the Improvement of Your Success Rate in Drug Development with Our Advanced In Vitro and In Vivo Pharmacokinetic Studies

Early-stage pharmacokinetic characterization during drug discovery is critical for pharmaceutical companies to improve the success rate of drug development. To support them, Sumika Chemical Analysis Service, Ltd. (SCAS) has extensive experience in conducting both in vitro and in vivo pharmacokinetic screening and offering services applicable to all stages of drug discovery.

• We respond to your wide range of requests from exploratory to nonclinical and clinical phases, and those related to translational research.
• Major pharmacokinetic studies listed below are available as our comprehensive package service or as individual services.
• We focus on rapid reporting to our clients by conducting studies in compliance with ISO9001. However, in the case of regulatory submissions, such as clinical trial applications, please contact us.
• Test conditions can be flexibly customized.

• Metabolic Stability Study:
  Evaluation is performed by incubating test compounds with liver microsomes or hepatocytes, followed by the calculation of the remaining compound ratio of metabolized to non-metabolized groups.
• CYP Isoform Identification Study:
  Expression system-based microsomal assay and inhibitor-based assay are conducted to identify CYP isoforms responsible for the metabolism of test compounds.
• Reactive Metabolite Study:
  The potential for formation of highly reactive metabolites is assessed using dGSH (dansylated glutathione) and CysGlu-Dan (N2-(L-Cysteinyl)-N5-(2-{[5-(dimethylamino)naphthalene-1-sulfonyl]amino}ethyl)-L-glutamine) as trapping agents, allowing semi-quantitative evaluation.
• Enzyme Inhibition Study:
  The inhibitory effect and mechanism of test compounds on seven major CYP isoforms in human liver microsomes are evaluated using cocktail substrates.
• Enzyme Induction Study:
  Induction potential is assessed by measuring changes in mRNA expression level and metabolic activity of three major CYP isoforms in cryopreserved human hepatocytes or HepaRG® cells.
• Protein Binding Study:
  The binding rate between test compounds and serum/plasma proteins from various animal species is assessed using equilibrium dialysis.
• Membrane Permeability Study:
  Apparent permeability is evaluated using Caco-2 cells on a Transwell®.
• Transporter Study:
  The affinity of test compounds for P-glycoprotein (P-gp) and BCRP, as well as P-gp inhibition, is assessed. The efflux ratio is calculated using bidirectional permeability coefficients in Caco-2 cells expressing the relevant transporters.

• Exploratory PK Studies and Endogenous Biomarker Quantification:
  The concentrations of test compounds in plasma, tissues and other biological matrices are quantified using LC-MS/MS or Ligand Binding Assay (LBA). Quantitative analysis of endogenous biomarkers (e.g., steroid hormones, lipids, nucleic acids, amino acids, peptides) is also available.

• Liver Microsome Assay:
  To exert sufficient pharmacological effect in vivo, the test compounds that are resistant to metabolism should be generally selected. Metabolic stability studies using liver microsomes are valuable for pharmacokinetic evaluation during early drug discovery. All processes—from sample dispensing to completion of metabolic reactions, MS analysis optimization, and analytical method development—are automated enhancing reproducibility and throughput. High-sensitivity LC-MS/MS is utilized for analysis.
  [Standard Protocol]
  Number of test compounds: Up to 24 compounds/batch
  Test concentration: 1 concentration
  Repeat number: n = 2
• Hepatocyte Assay:
  In order to predict in vivo metabolism, isolated hepatocytes are an excellent model enabling comprehensive metabolic assessment, compared with microsomal fractions or liver slices. We use cryopreserved human hepatocytes and evaluate hepatic intrinsic clearance levels with HPLC or LC-MS/MS.
  [Standard Protocol]
  Number of test compounds: Up to 12 conditions (species × number of compounds)/batch
  Test concentration: 1 concentration
  Repeat number: n = 2
  Positive control; buspirone

Identifying CYP isoforms involved in compound metabolism is essential for predicting and avoiding drug-drug interactions. If metabolism by a specific enzyme contributes to >25% of total clearance, a drug-drug interaction study is required per ICH M12 guidelines. We conduct both enzyme-expression-system-based microsomal assay and inhibitor-based assay using normal hepatic microsomes.

• Enzyme-expression-system-based microsome assay
  [Standard Protocol]
  Number of test compounds: Up to 4 compounds/batch
  Test concentration: 1 concentration
  Repeat number: n = 2
  CYP isoforms evaluated: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5 (total of eight isoforms)
• Inhibition-based assay
  [Standard Protocol]
  Number of test compounds: Up to 2 compounds/batch
  Test concentration: 2 concentrations
  Repeat number: n = 2
  CYP isoforms evaluated: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 (total of 7 isoforms)

Reactive metabolites can cause serious drug-induced toxicities. We perform a trapping assay using dansylated glutathione (dGSH), allowing semi-quantitative evaluation via fluorescence intensity to evaluate soft reactive metabolites (as defined by the HSAB principle). CysGlu-Dan is also used to simultaneously evaluate both soft and hard reactive metabolites.
[Standard Protocol]
Number of test compounds: Up to 45 compounds/batch
Test concentration: 1 concentration
Repeat number: n = 2
Positive control: troglitazone

Most pharmacokinetic interactions result from CYP inhibition, which can be reversible or time-dependent. Time-dependent inhibition is particularly associated with severe adverse effects.
IC50 shift assays are performed using cocktail substrates against the seven major CYP isoforms and human liver microsomes.
[Standard Protocol]
Number of test compounds: Up to 160 compounds/6 batches
Test concentration: 4 concentrations (common ratio 5)
Repeat number: n = 1
CYP isoforms evaluated: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6,
CYP3A4 (total of 7 isoforms)
Positive control using inhibitor cocktail

• By using Cryopreserved Human Hepatocytes:
  Compounds may induce drug-metabolizing enzymes such as CYPs, leading to reduced systemic exposure. Induction potential is evaluated by measuring changes in mRNA expression level and enzyme activity.
  [Standard Protocol]
  Number of test compounds: Up to 5 compounds/batch
  Test concentration: 3 concentrations
  Repeat number: n = 2
  CYP isoforms evaluated: 1A2, 2B6, 3A4 (total of 3 isoforms)
  Positive control: omeprazole (1A2), phenobarbital (2B6), rifampin (3A4)
  mRNA expression level (Real-time PCR), enzymatic activity (LC-MS/MS)
• By using HepaRG® Cells:
  HepaRG® cells, derived from human hepatoma, retain many features of primary hepatocyte. They are suitable for CYP induction studies, because of minimal batch variability and high availability.
  [Standard Protocol]
  Number of test compounds: Up to 13 compounds/batch
  Test concentration: 3 concentrations
  Repeat number: n = 2
  CYP isoforms evaluated: 1A2, 2B6, 3A4 (total of 3 isoforms)
  Positive control: omeprazole (1A2), phenobarbital (2B6), rifampin (3A4)
  mRNA expression level (Real-time PCR), enzymatic activity (LC-MS/MS)

Only unbound drugs to proteins in tissue and serum are pharmacologically active and can contribute to therapeutic and adverse effects. Therefore, the protein binding study is critical to accurately evaluate the efficacy and safety of the drugs. Protein binding is assessed using a high-throughput, well-plate-based equilibrium dialysis method, followed by LC-MS/MS analysis.
[Standard Protocol]
Number of test compounds: Up to 24 compounds/batch
Test concentration: 1 concentration
Repeat number: n = 2
Quality control sample: Warfarin

Oral drug development requires prediction of human intestinal absorption. Caco-2 cell-based assay is used to estimate permeability coefficients, which is suggested to correlate with in vivo absorption.
[Standard Protocol]
Number of test compounds: Up to 22 compounds/batch
Test concentration: 1 concentration
Number of repetitions: n = 3
Indicator compounds: propranolol, atenolol

P-glycoprotein (P-gp) and BCRP are key transporters influencing drug absorption and disposition. Per ICH M12, we evaluate substrate affinity and inhibition potential using Caco-2 cells which express these transporters.

Exploratory PK Study and Endogenous Biomarker Quantification
The concentrations of test compounds in plasma, tissues and other biological matrices are quantified using LC-MS/MS or ligand binding assays (LBA). Quantitative analysis of endogenous biomarkers (e.g., steroid hormones, lipids, nucleic acids, amino acids, peptides) is available.

• We also accept analytical method development and validation.
• Administration of test compounds to animals, blood sampling, and bioanalysis can be packaged (administration and blood sampling will be outsourced).

• Pharmaceuticals and Medical Devices Agency (PMDA), ICH M12 Drug Interaction Study: https://www.pmda.go.jp/int-activities/int-harmony/ich/0101.html (accessed 2025.11.28)