- Email: [email protected]
Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates
Accepted Manuscript 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 PII: DOI: Reference:
S0928-0987(17)30625-5 doi:10.1016/j.ejps.2017.11.007 PHASCI 4296
To appear in:
European Journal of Pharmaceutical Sciences
Received date: Revised date: Accepted date:
11 September 2017 1 November 2017 8 November 2017
Please cite this article as: Burak Ozgür, Lasse Saaby, Kristine Langthaler, Birger Brodin , Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of Pgp substrates. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), doi:10.1016/j.ejps.2017.11.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Data article Title: Data demonstrating the challenges of determining the kinetic parameters of P-gp mediated
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transport of low-water soluble substrates
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Authors:
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Burak Ozgür1, Lasse Saaby1,2, Kristine Langthaler3 & Birger Brodin1*
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Affiliations:
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1. Section of Pharmaceutical Design and Drug Delivery, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, Denmark 2. Bioneer-FARMA, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, Denmark 3. Lundbeck Pharma A/S, Ottiliavej 9, 2500 Valby, Denmark Contact email:
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*Corresponding author: Birger Brodin, Email; [email protected] Voice: +453533616 Abstract
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The presented data are related to the research article entitled “Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates” (Ozgur et al., 2017). This
AC
data report describes the challenges of investigating the concentration-dependent transport for Pglycoprotein (P-gp) substrates with relatively low aqueous solubility. Thus, we provide solubility data on two prototypical P-gp substrates, digoxin and rhodamine 123, and present a simulated Michaelis-Menten curve of the P-gp mediated transport of digoxin. Furthermore, we present data from bidirectional transport of digoxin and rhodamine 123 across cell monolayers of the MDCK II MDR1 and iP-pg cell lines in the presence of the selective P-gp inhibitor, zosuquidar (LY335979).
ACCEPTED MANUSCRIPT
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Specifications Table [please fill in right-hand column of the table below] Subject area Pharmaceutic More specific subject area Efflux transporter, drug delivery Type of data Table, Figures How data was acquired HPLC-system consisting of an UltiMate 3000 pump (Thermo Fisher Scientific, Copenhagen, Denmark), a Kinetex column XB-C18 particle size 5 μm, 4.6 x 100 mm (Phenomenex, Torrance, CA, USA) and an UVdetector (Waltham, MA, USA). A fluorescence microplate reader (NOVOstar Microplate Reader, BMG Labtech, Offenburg, Germany). A liquid scintillation analyzer (Packard Tri-Carb 2100 TR; Canberra, Dreich, Germany) Data format Raw and analyzed Experimental factors N/A Experimental features The apparent solubility of both digoxin and rhodamine 123 was estimated thermodynamically by adding the experimental buffer systems directly to solid crystalline digoxin or rhodamine 123. Transport data were generated by measuring fluxes of digoxin and rhodamine 123 across cell monolayers of the MDCK II MDR1 and iPgp cell lines, as described previously [1]. Data source location University of Copenhagen, Denmark Data accessibility Data are presented in this article
Data present the aqueous solubility of two prototypical P-gp substrates, digoxin and rhodamine 123
Describes a key challenge for determining kinetic parameters of P-gp mediated transport
CE
PT
ED
Value of the data
of substrates with low aqueous solubility Data provide a comparison of the passive permeability of the two P-gp substrates, [3H]-
AC
digoxin and rhodamine 123, across cell monolayers of P-gp expressing cell lines
1. Data
The data described here show the challenges of determining the kinetic parameters of the P-gp mediated transport of digoxin, which has a relatively low aqueous solubility. This article provides solubility data on two prototypical P-gp substrates, digoxin and rhodamine 123, in
ACCEPTED MANUSCRIPT aqueous buffer systems supplemented with 2 % (v/v) DMSO and with or without 0.05 % (w/v) bovine serum albumin (BSA), respectively (Table 1). Solubility data in transport buffer supplemented with 0.5 % (v/v) DMSO can be found in ref: (Ozgur et al. 2017). A simulated Michaelis-Menten saturation curve of the P-gp mediated transport of digoxin was drawn to
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demonstrate that the maximal donor concentration, which could be reached in the experimental
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transport buffer, was too low to yield transport data in the saturable range of the Michaelis-
CR
Menten relationship (Figure 2). Figure 3 shows data on bidirectional transport of [3H]-digoxin and rhodamine 123 across cell monolayers of the MDCK II MDR1 and iP-gp cell lines in the
AN
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presence of the selective P-gp inhibitor, zosuquidar.
2. Experimental Design, Materials and Methods
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2.1 Solubility measurements of digoxin and rhodamine 123 in transport buffer
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The apparent aqueous solubility (Sapp) of digoxin and rhodamine 123 was estimated in the experimental transport buffer as described previously, ref: (Ozgur et al., 2017). Briefly, the
PT
buffer systems were Hank’s balanced salt solution (HBSS) supplemented with 10 mM HEPES
CE
(pH 7.4), 0.375 % (v/v) sodium carbonate, 2 % (v/v) DMSO, and finally with or without 0.05 % BSA. The buffer systems with and without 0.05 % BSA are abbreviated HBSS+ and HBSS-,
AC
respectively. The Sapp of the compounds was estimated thermodynamically by adding the experimental buffer systems directly to solid crystalline digoxin and rhodamine 123, respectively. These saturated solutions were incubated at 37 ℃ for 24 hours, where equilibrium between the dissolved and solid material (and between the dissolved and binding to BSA in HBSS+) were assumed to be reached. The saturated digoxin solutions were filtered using syringe filters (13 mm diameter, 0.45 µm pore size, material: 0.22 µM cellulose acetate, from Knebel,
ACCEPTED MANUSCRIPT Denmark), and mixed with methanol in a volume ratio of 1:1. Digoxin concentrations were quantified using HPLC combined with a UV-detector. The UV detection wavelength was 218 nm. The separation of digoxin was performed with a Kinetex column XB-C18 (particle size 5 µm, 4.6 x 100 mm) at 45 ⁰C. Water and acetonitrile with a volume ratio of 72:28 was used as the
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isocratic mobile phase, and the flow rate was fixed at 1.0 ml·min-1, which resulted in a retention
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time of 7.0 min (Figure 1). The saturated rhodamine 123 solutions were centrifuged, and the
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concentration of rhodamine 123 was quantified as emitted fluorescence using microplate reader.
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The excitation and emission wavelengths were 485 nm and 520 nm, respectively.
Table 1. The solubility of digoxin and rhodamine 123 in HBSS+ and HBSS-, supplemented with 2 %
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DMSO. Solubility data are shown as means ± SD of one individual experiments of triplicates (n=1, total
M
N=3).
Sapp in HBSS- + 2 % DMSO (µM)
Digoxin
55.0±5.7
42.9±5.4
1088±13
1053±20
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Compound
Sapp in HBSS+ + 2 % DMSO (µM)
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Rhodamine 123
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Figure 1. A representative HPLC-UV chromatogram of digoxin.
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2.2 The concentration dependent transport of digoxin
In the referred article, the apparent transport of digoxin was determined at a concentration range
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of 0.0251-13 µM across the iP-gp cell monolayers cultured on the permeable supports (1.12 cm2)
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in the basolateral to apical direction. Experiments were performed in the absence and presence of 2 µM zosuquidar in order to estimate the total and the passive transport, respectively. (Ozgur et
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al., 2017) The P-gp mediated transport was obtained by subtracting the passive transport from
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the total transport at the respective substrate concentrations. By plotting the fluxes from the P-gp mediated transport as function of donor concentration of digoxin, a linear relationship was observed. Using GraphPad Prism software (version 7.0, Inc. San Diego, CA, USA), simulated kinetic parameters were estimated. The simulated Km and Vmax values were determined to 253 µM and 136 nmol·cm-2·min-1, respectively. A hypothetical Michaelis-Menten relationship of the P-gp mediated transport of digoxin was drawn based on the simulation (Figure 2).
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Figure 2. Illustration of a hypothetical Michaelis-Menten curve of the P-gp mediated transport of digoxin.
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The box in the right figure shows the region, where experiments in the referred article were carried out,
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indicating the proposed Km were much higher than the apparent solubility limit of digoxin in the transport
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buffer (HBSS+).
2.3 Bidirectional transport of [3H]-digoxin and rhodamine 123
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The bidirectional transport experiments were performed as described previously, ref: (Ozgur et al., 2017). Briefly, the fluxes of [3H]-digoxin and rhodamine 123 were investigated across cell
AC
monolayers of the MDCK II MDR1 and iP-gp cell lines in the presence of 2 µM of zosuquidar (Figure 3). Transport was investigated in the apical to basolateral direction (A-B) and in the basolateral to apical direction (B-A), respectively. The concentration (C0) of [3H]-digoxin in the donor compartment was 0.0251 µM, while it was 3.12 µM for rhodamine 123. HBSS+ supplemented with 0.5 % DMSO was used as the experimental transport buffer. The fluxes were determined over a time course of 120 minutes. The apparent permeability coefficients, Papp, of
ACCEPTED MANUSCRIPT [3H]-digoxin and rhodamine 123 were calculated from the steady state fluxes, Jss, using following equation (1): 𝐽𝑠𝑠 𝐶0
(Equation 1)
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𝑃𝑎𝑝𝑝 =
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Figure 3. Bidirectional transport of [3H]-digoxin and rhodamine 123 across MDCK II MDR1 and iP-gp
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cell monolayers in the presence of 2 µM zosuquidar (ZSQ). The Papp values were calculated from the steady state fluxes. The filled bars illustrate Papp,A-B values, whereas open bars illustrate Papp,B-A. Values are
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presented as mean ± SEM of three individual passages of three individual permeable supports (n=3, total
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N=9).
Acknowledgements
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The authors wish to acknowledge Mette Frandsen and Lene Grønne Pedersen for expert technical assistance, and the Lundbeck Foundation for funding via the Lundbeck Foundation Research Network on Brain Barriers and Drug Delivery (RIBBDD).
References
Ozgur, B., Saaby, L., Langthaler, K., Brodin, B., 2017. Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates. J. Pharm. Sci.
ACCEPTED MANUSCRIPT
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Graphical abstract
Burak Ozgür, Lasse Saaby, Kristine Langthaler, Birger Brodin PII: DOI: Reference:
S0928-0987(17)30625-5 doi:10.1016/j.ejps.2017.11.007 PHASCI 4296
To appear in:
European Journal of Pharmaceutical Sciences
Received date: Revised date: Accepted date:
11 September 2017 1 November 2017 8 November 2017
Please cite this article as: Burak Ozgür, Lasse Saaby, Kristine Langthaler, Birger Brodin , Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of Pgp substrates. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), doi:10.1016/j.ejps.2017.11.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Data article Title: Data demonstrating the challenges of determining the kinetic parameters of P-gp mediated
T
transport of low-water soluble substrates
IP
Authors:
CR
Burak Ozgür1, Lasse Saaby1,2, Kristine Langthaler3 & Birger Brodin1*
US
Affiliations:
M
AN
1. Section of Pharmaceutical Design and Drug Delivery, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, Denmark 2. Bioneer-FARMA, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, Denmark 3. Lundbeck Pharma A/S, Ottiliavej 9, 2500 Valby, Denmark Contact email:
PT
ED
*Corresponding author: Birger Brodin, Email; [email protected] Voice: +453533616 Abstract
CE
The presented data are related to the research article entitled “Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates” (Ozgur et al., 2017). This
AC
data report describes the challenges of investigating the concentration-dependent transport for Pglycoprotein (P-gp) substrates with relatively low aqueous solubility. Thus, we provide solubility data on two prototypical P-gp substrates, digoxin and rhodamine 123, and present a simulated Michaelis-Menten curve of the P-gp mediated transport of digoxin. Furthermore, we present data from bidirectional transport of digoxin and rhodamine 123 across cell monolayers of the MDCK II MDR1 and iP-pg cell lines in the presence of the selective P-gp inhibitor, zosuquidar (LY335979).
ACCEPTED MANUSCRIPT
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AN
US
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Specifications Table [please fill in right-hand column of the table below] Subject area Pharmaceutic More specific subject area Efflux transporter, drug delivery Type of data Table, Figures How data was acquired HPLC-system consisting of an UltiMate 3000 pump (Thermo Fisher Scientific, Copenhagen, Denmark), a Kinetex column XB-C18 particle size 5 μm, 4.6 x 100 mm (Phenomenex, Torrance, CA, USA) and an UVdetector (Waltham, MA, USA). A fluorescence microplate reader (NOVOstar Microplate Reader, BMG Labtech, Offenburg, Germany). A liquid scintillation analyzer (Packard Tri-Carb 2100 TR; Canberra, Dreich, Germany) Data format Raw and analyzed Experimental factors N/A Experimental features The apparent solubility of both digoxin and rhodamine 123 was estimated thermodynamically by adding the experimental buffer systems directly to solid crystalline digoxin or rhodamine 123. Transport data were generated by measuring fluxes of digoxin and rhodamine 123 across cell monolayers of the MDCK II MDR1 and iPgp cell lines, as described previously [1]. Data source location University of Copenhagen, Denmark Data accessibility Data are presented in this article
Data present the aqueous solubility of two prototypical P-gp substrates, digoxin and rhodamine 123
Describes a key challenge for determining kinetic parameters of P-gp mediated transport
CE
PT
ED
Value of the data
of substrates with low aqueous solubility Data provide a comparison of the passive permeability of the two P-gp substrates, [3H]-
AC
digoxin and rhodamine 123, across cell monolayers of P-gp expressing cell lines
1. Data
The data described here show the challenges of determining the kinetic parameters of the P-gp mediated transport of digoxin, which has a relatively low aqueous solubility. This article provides solubility data on two prototypical P-gp substrates, digoxin and rhodamine 123, in
ACCEPTED MANUSCRIPT aqueous buffer systems supplemented with 2 % (v/v) DMSO and with or without 0.05 % (w/v) bovine serum albumin (BSA), respectively (Table 1). Solubility data in transport buffer supplemented with 0.5 % (v/v) DMSO can be found in ref: (Ozgur et al. 2017). A simulated Michaelis-Menten saturation curve of the P-gp mediated transport of digoxin was drawn to
T
demonstrate that the maximal donor concentration, which could be reached in the experimental
IP
transport buffer, was too low to yield transport data in the saturable range of the Michaelis-
CR
Menten relationship (Figure 2). Figure 3 shows data on bidirectional transport of [3H]-digoxin and rhodamine 123 across cell monolayers of the MDCK II MDR1 and iP-gp cell lines in the
AN
US
presence of the selective P-gp inhibitor, zosuquidar.
2. Experimental Design, Materials and Methods
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2.1 Solubility measurements of digoxin and rhodamine 123 in transport buffer
ED
The apparent aqueous solubility (Sapp) of digoxin and rhodamine 123 was estimated in the experimental transport buffer as described previously, ref: (Ozgur et al., 2017). Briefly, the
PT
buffer systems were Hank’s balanced salt solution (HBSS) supplemented with 10 mM HEPES
CE
(pH 7.4), 0.375 % (v/v) sodium carbonate, 2 % (v/v) DMSO, and finally with or without 0.05 % BSA. The buffer systems with and without 0.05 % BSA are abbreviated HBSS+ and HBSS-,
AC
respectively. The Sapp of the compounds was estimated thermodynamically by adding the experimental buffer systems directly to solid crystalline digoxin and rhodamine 123, respectively. These saturated solutions were incubated at 37 ℃ for 24 hours, where equilibrium between the dissolved and solid material (and between the dissolved and binding to BSA in HBSS+) were assumed to be reached. The saturated digoxin solutions were filtered using syringe filters (13 mm diameter, 0.45 µm pore size, material: 0.22 µM cellulose acetate, from Knebel,
ACCEPTED MANUSCRIPT Denmark), and mixed with methanol in a volume ratio of 1:1. Digoxin concentrations were quantified using HPLC combined with a UV-detector. The UV detection wavelength was 218 nm. The separation of digoxin was performed with a Kinetex column XB-C18 (particle size 5 µm, 4.6 x 100 mm) at 45 ⁰C. Water and acetonitrile with a volume ratio of 72:28 was used as the
T
isocratic mobile phase, and the flow rate was fixed at 1.0 ml·min-1, which resulted in a retention
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time of 7.0 min (Figure 1). The saturated rhodamine 123 solutions were centrifuged, and the
CR
concentration of rhodamine 123 was quantified as emitted fluorescence using microplate reader.
US
The excitation and emission wavelengths were 485 nm and 520 nm, respectively.
Table 1. The solubility of digoxin and rhodamine 123 in HBSS+ and HBSS-, supplemented with 2 %
AN
DMSO. Solubility data are shown as means ± SD of one individual experiments of triplicates (n=1, total
M
N=3).
Sapp in HBSS- + 2 % DMSO (µM)
Digoxin
55.0±5.7
42.9±5.4
1088±13
1053±20
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ED
Compound
Sapp in HBSS+ + 2 % DMSO (µM)
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Rhodamine 123
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ACCEPTED MANUSCRIPT
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Figure 1. A representative HPLC-UV chromatogram of digoxin.
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2.2 The concentration dependent transport of digoxin
In the referred article, the apparent transport of digoxin was determined at a concentration range
ED
of 0.0251-13 µM across the iP-gp cell monolayers cultured on the permeable supports (1.12 cm2)
PT
in the basolateral to apical direction. Experiments were performed in the absence and presence of 2 µM zosuquidar in order to estimate the total and the passive transport, respectively. (Ozgur et
CE
al., 2017) The P-gp mediated transport was obtained by subtracting the passive transport from
AC
the total transport at the respective substrate concentrations. By plotting the fluxes from the P-gp mediated transport as function of donor concentration of digoxin, a linear relationship was observed. Using GraphPad Prism software (version 7.0, Inc. San Diego, CA, USA), simulated kinetic parameters were estimated. The simulated Km and Vmax values were determined to 253 µM and 136 nmol·cm-2·min-1, respectively. A hypothetical Michaelis-Menten relationship of the P-gp mediated transport of digoxin was drawn based on the simulation (Figure 2).
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Figure 2. Illustration of a hypothetical Michaelis-Menten curve of the P-gp mediated transport of digoxin.
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The box in the right figure shows the region, where experiments in the referred article were carried out,
ED
indicating the proposed Km were much higher than the apparent solubility limit of digoxin in the transport
PT
buffer (HBSS+).
2.3 Bidirectional transport of [3H]-digoxin and rhodamine 123
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The bidirectional transport experiments were performed as described previously, ref: (Ozgur et al., 2017). Briefly, the fluxes of [3H]-digoxin and rhodamine 123 were investigated across cell
AC
monolayers of the MDCK II MDR1 and iP-gp cell lines in the presence of 2 µM of zosuquidar (Figure 3). Transport was investigated in the apical to basolateral direction (A-B) and in the basolateral to apical direction (B-A), respectively. The concentration (C0) of [3H]-digoxin in the donor compartment was 0.0251 µM, while it was 3.12 µM for rhodamine 123. HBSS+ supplemented with 0.5 % DMSO was used as the experimental transport buffer. The fluxes were determined over a time course of 120 minutes. The apparent permeability coefficients, Papp, of
ACCEPTED MANUSCRIPT [3H]-digoxin and rhodamine 123 were calculated from the steady state fluxes, Jss, using following equation (1): 𝐽𝑠𝑠 𝐶0
(Equation 1)
AN
US
CR
IP
T
𝑃𝑎𝑝𝑝 =
M
Figure 3. Bidirectional transport of [3H]-digoxin and rhodamine 123 across MDCK II MDR1 and iP-gp
ED
cell monolayers in the presence of 2 µM zosuquidar (ZSQ). The Papp values were calculated from the steady state fluxes. The filled bars illustrate Papp,A-B values, whereas open bars illustrate Papp,B-A. Values are
PT
presented as mean ± SEM of three individual passages of three individual permeable supports (n=3, total
CE
N=9).
Acknowledgements
AC
The authors wish to acknowledge Mette Frandsen and Lene Grønne Pedersen for expert technical assistance, and the Lundbeck Foundation for funding via the Lundbeck Foundation Research Network on Brain Barriers and Drug Delivery (RIBBDD).
References
Ozgur, B., Saaby, L., Langthaler, K., Brodin, B., 2017. Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates. J. Pharm. Sci.
ACCEPTED MANUSCRIPT
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Graphical abstract