Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates

Burak Ozgür; Lasse Saaby; Kristine Langthaler; Birger Brodin

doi:10.1016/j.ejps.2017.11.007

Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates

Burak Ozgür, Lasse Saaby, Kristine Langthaler, Birger Brodin

2 Citations (Scopus)

Abstract

Recently, we transfected the porcine intestinal cell line IPEC-J2, with human P-glycoprotein (P-gp, ABCB1). The resulting cell line, iP-gp, has a high expression of functional human P-gp in the apical membrane, and a low expression of nonhuman ATP-binding cassette (ABC) transporters. The aim of the present work was to investigate the usability of iP-gp cell line for determining transepithelial transport kinetics of the prototypical P-gp substrates digoxin and rhodamine 123. The cell line generated tight monolayers after 16 days of culture, reflected by high transepithelial electrical resistance values (TEER > 15,000 Ω·cm2 ), immunocytochemistry and low fluxes of the paracellular flux marker [ 14C]-mannitol. Monolayer integrity was not affected the common solvents dimethyl sulfoxide (DMSO), methanol and ethanol in concentrations up to 2% (v/v). Transepithelial fluxes of [3 H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. Km and Vmax of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332 ± 124 μM and 111 ± 16 pmol·cm−2·min−1 (n = 3, total N = 6), respectively. Vmax and Km of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC50) of the selective P-gp inhibitor, zosuquidar (LY335979), were estimated to 0.05 ± 0.01 μM (n = 3, total N = 6) and 0.04 ± 0.01 μM (n = 3, total N = 6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC50 values of inhibition, Ki values were only possible to obtain using rhodamine 123. The observed tightness, robustness towards solvents and the high efflux ratios confirmed that the iP-gp cell line may serve as a useful screening tool for investigations of substrate-P-gp interactions and modulation of P-gp function.

Original language	English
Journal	European Journal of Pharmaceutical Sciences
Volume	112
Pages (from-to)	112-121
Number of pages	10
ISSN	0928-0987
DOIs	https://doi.org/10.1016/j.ejps.2017.11.007
Publication status	Published - 15 Jan 2018

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10.1016/j.ejps.2017.11.007

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@article{eb78039e34534ce396abcdebac661fd5,

title = "Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates",

abstract = "Recently, we transfected the porcine intestinal cell line IPEC-J2, with human P-glycoprotein (P-gp, ABCB1). The resulting cell line, iP-gp, has a high expression of functional human P-gp in the apical membrane, and a low expression of nonhuman ATP-binding cassette (ABC) transporters. The aim of the present work was to investigate the usability of iP-gp cell line for determining transepithelial transport kinetics of the prototypical P-gp substrates digoxin and rhodamine 123. The cell line generated tight monolayers after 16 days of culture, reflected by high transepithelial electrical resistance values (TEER > 15,000 Ω·cm2 ), immunocytochemistry and low fluxes of the paracellular flux marker [ 14C]-mannitol. Monolayer integrity was not affected the common solvents dimethyl sulfoxide (DMSO), methanol and ethanol in concentrations up to 2% (v/v). Transepithelial fluxes of [3 H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. Km and Vmax of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332 ± 124 μM and 111 ± 16 pmol·cm−2·min−1 (n = 3, total N = 6), respectively. Vmax and Km of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC50) of the selective P-gp inhibitor, zosuquidar (LY335979), were estimated to 0.05 ± 0.01 μM (n = 3, total N = 6) and 0.04 ± 0.01 μM (n = 3, total N = 6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC50 values of inhibition, Ki values were only possible to obtain using rhodamine 123. The observed tightness, robustness towards solvents and the high efflux ratios confirmed that the iP-gp cell line may serve as a useful screening tool for investigations of substrate-P-gp interactions and modulation of P-gp function.",

author = "Burak Ozg{\"u}r and Lasse Saaby and Kristine Langthaler and Birger Brodin",

year = "2018",

month = jan,

day = "15",

doi = "10.1016/j.ejps.2017.11.007",

language = "English",

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journal = "Norvegica Pharmaceutica Acta",

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TY - JOUR

T1 - Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates

AU - Ozgür, Burak

AU - Saaby, Lasse

AU - Langthaler, Kristine

AU - Brodin, Birger

PY - 2018/1/15

Y1 - 2018/1/15

N2 - Recently, we transfected the porcine intestinal cell line IPEC-J2, with human P-glycoprotein (P-gp, ABCB1). The resulting cell line, iP-gp, has a high expression of functional human P-gp in the apical membrane, and a low expression of nonhuman ATP-binding cassette (ABC) transporters. The aim of the present work was to investigate the usability of iP-gp cell line for determining transepithelial transport kinetics of the prototypical P-gp substrates digoxin and rhodamine 123. The cell line generated tight monolayers after 16 days of culture, reflected by high transepithelial electrical resistance values (TEER > 15,000 Ω·cm2 ), immunocytochemistry and low fluxes of the paracellular flux marker [ 14C]-mannitol. Monolayer integrity was not affected the common solvents dimethyl sulfoxide (DMSO), methanol and ethanol in concentrations up to 2% (v/v). Transepithelial fluxes of [3 H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. Km and Vmax of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332 ± 124 μM and 111 ± 16 pmol·cm−2·min−1 (n = 3, total N = 6), respectively. Vmax and Km of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC50) of the selective P-gp inhibitor, zosuquidar (LY335979), were estimated to 0.05 ± 0.01 μM (n = 3, total N = 6) and 0.04 ± 0.01 μM (n = 3, total N = 6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC50 values of inhibition, Ki values were only possible to obtain using rhodamine 123. The observed tightness, robustness towards solvents and the high efflux ratios confirmed that the iP-gp cell line may serve as a useful screening tool for investigations of substrate-P-gp interactions and modulation of P-gp function.

AB - Recently, we transfected the porcine intestinal cell line IPEC-J2, with human P-glycoprotein (P-gp, ABCB1). The resulting cell line, iP-gp, has a high expression of functional human P-gp in the apical membrane, and a low expression of nonhuman ATP-binding cassette (ABC) transporters. The aim of the present work was to investigate the usability of iP-gp cell line for determining transepithelial transport kinetics of the prototypical P-gp substrates digoxin and rhodamine 123. The cell line generated tight monolayers after 16 days of culture, reflected by high transepithelial electrical resistance values (TEER > 15,000 Ω·cm2 ), immunocytochemistry and low fluxes of the paracellular flux marker [ 14C]-mannitol. Monolayer integrity was not affected the common solvents dimethyl sulfoxide (DMSO), methanol and ethanol in concentrations up to 2% (v/v). Transepithelial fluxes of [3 H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. Km and Vmax of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332 ± 124 μM and 111 ± 16 pmol·cm−2·min−1 (n = 3, total N = 6), respectively. Vmax and Km of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC50) of the selective P-gp inhibitor, zosuquidar (LY335979), were estimated to 0.05 ± 0.01 μM (n = 3, total N = 6) and 0.04 ± 0.01 μM (n = 3, total N = 6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC50 values of inhibition, Ki values were only possible to obtain using rhodamine 123. The observed tightness, robustness towards solvents and the high efflux ratios confirmed that the iP-gp cell line may serve as a useful screening tool for investigations of substrate-P-gp interactions and modulation of P-gp function.

U2 - 10.1016/j.ejps.2017.11.007

DO - 10.1016/j.ejps.2017.11.007

M3 - Journal article

C2 - 29146563

SN - 0928-0987

VL - 112

SP - 112

EP - 121

JO - Norvegica Pharmaceutica Acta

JF - Norvegica Pharmaceutica Acta

ER -