Abstract
Background: The efflux transporter P-glycoprotein (P-gp, product of the MDR1/ABCB1 gene) hinders
uptake of drug compounds to the brain, limits intestinal uptake, is a cause of resistance to chemoterapeutics and a potential "site" for drug-drug interaction. Regulatory agencies therefore recommend that new API's are evaluated with respect to P-gp interactions. Aim: The aim of the present work was to validate the suitability of the newly developed iP-gp cell line for investigating P-gp interactions with human P-gp.Methods: IPEC-J2 MDR1 (iP-gp) cells were cultured on permeable supports for 17-20 days in conventional growth media. Results: The iP-gp cell line generated tight monolayers after 16 days of culture (TEER > 15000 ohm·cm2). Efflux ratios (Papp, B-A / Papp, A-B) of the P-gp substrate digoxin were in the range of 80-100. P-gp was demonstrated to be responsible for the efflux transport by substrate profiling, combined with application of P-gp and BCRP inhibitors. Furthermore, the compounds atenolol, citalopram, and mitoxantrone were identified as P-gp substrates. Functional P-gp expression was shown to be stable through at least 10 cell passages.Transport kinetic analysis of rhodamine and digoxin B-A fluxes revealed that rhodamine 123 had a Km and Vmax of 332
µM +/- 124 µM and 111 +/- 16 pmol·cm-2·min-1, respectively. Vmax and Km of digoxin transport could not be estimated due to the low solubility of digoxin. The IC50 of the inhibitor, zosuquidar, were estimated to 0.05 +/- 0.01 µM and 0.04 +/- 0.01 µM in transport experiments including digoxin and rhodamine 123, respectively. Summary/Conclusion: The iP-gp cell line may become a useful screening tool for interactions between drug compounds and human P-gp.
uptake of drug compounds to the brain, limits intestinal uptake, is a cause of resistance to chemoterapeutics and a potential "site" for drug-drug interaction. Regulatory agencies therefore recommend that new API's are evaluated with respect to P-gp interactions. Aim: The aim of the present work was to validate the suitability of the newly developed iP-gp cell line for investigating P-gp interactions with human P-gp.Methods: IPEC-J2 MDR1 (iP-gp) cells were cultured on permeable supports for 17-20 days in conventional growth media. Results: The iP-gp cell line generated tight monolayers after 16 days of culture (TEER > 15000 ohm·cm2). Efflux ratios (Papp, B-A / Papp, A-B) of the P-gp substrate digoxin were in the range of 80-100. P-gp was demonstrated to be responsible for the efflux transport by substrate profiling, combined with application of P-gp and BCRP inhibitors. Furthermore, the compounds atenolol, citalopram, and mitoxantrone were identified as P-gp substrates. Functional P-gp expression was shown to be stable through at least 10 cell passages.Transport kinetic analysis of rhodamine and digoxin B-A fluxes revealed that rhodamine 123 had a Km and Vmax of 332
µM +/- 124 µM and 111 +/- 16 pmol·cm-2·min-1, respectively. Vmax and Km of digoxin transport could not be estimated due to the low solubility of digoxin. The IC50 of the inhibitor, zosuquidar, were estimated to 0.05 +/- 0.01 µM and 0.04 +/- 0.01 µM in transport experiments including digoxin and rhodamine 123, respectively. Summary/Conclusion: The iP-gp cell line may become a useful screening tool for interactions between drug compounds and human P-gp.
| Original language | English |
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| Publication date | 23 May 2017 |
| Publication status | Published - 23 May 2017 |
| Event | 6th FIP Pharmaceutical Sciences World Congress 2017. - Stockholm, Sweden Duration: 21 May 2017 → 24 May 2017 |
Conference
| Conference | 6th FIP Pharmaceutical Sciences World Congress 2017. |
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| Country/Territory | Sweden |
| City | Stockholm |
| Period | 21/05/2017 → 24/05/2017 |