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

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Abstract

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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Abbreviations

AtoB:

Apical to basal

BCRP:

Breast cancer resistance protein

BSA:

Bovine serum albumin

BtoA:

Basal to apical

DMEM:

Dulbecco’s modified Eagle’s medium

DMSO:

Dimethyl sulfoxide

EMA:

European Medicines Agency

ER:

Efflux ratio

FaSSIF:

Fasted state simulated intestinal fluid

FBS:

Fetal bovine serum

FDA:

U.S. Food and Drug Administration

HBSS:

Hank’s balanced salt solution

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Neaa:

MEM Non-essential amino acids solution

Papp:

Apparent permeability

P-gp:

P-glycoprotein

RMSD:

Root-mean square deviation

TEER:

Transepithelial electrical resistance

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Author information

Authors and Affiliations

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

    Miho Nagayasu, Kazuhisa Ozeki, Yuuji Sakurai & Haruka Tsutsui

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

    Miho Nagayasu & Satomi Onoue

Authors
  1. Miho Nagayasu
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  2. Kazuhisa Ozeki
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  4. Haruka Tsutsui
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  5. Satomi Onoue
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Contributions

Miho Nagayasu: Formal Analysis, Writing-Original draft preparation, Visualization. Kazuhisa Ozeki: Conceptualization, Methodology, Investigation, Project Administration. Yuuji Sakurai: Investigation, Data Curation. Haruka Tsutsui: Investigation. Satomi Onoue: Writing – Review & Editing, Supervision.

Corresponding author

Correspondence to Kazuhisa Ozeki.

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Conflict of Interest

Miho Nagayasu, Kazuhisa Ozeki, Yuuji Sakurai, and Hruka Tsutsui are employee of Chugai Pharmaceutical Co.,Ltd. Some authors have owns stock in Chugai.

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Nagayasu, M., Ozeki, K., Sakurai, Y. et al. Simplified Method to Determine the Efflux Ratio on P-Glycoprotein Substrates Using Three-Compartment Model Analysis for Caco-2 Cell Assay Data. Pharm Res 37, 13 (2020). https://doi.org/10.1007/s11095-019-2729-x

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  • DOI: https://doi.org/10.1007/s11095-019-2729-x

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