Abstract
Two different cellular assay models were assessed as in vitro systems for P-glycoprotein (P-gp) substrate identification: cellular accumulation studies with KB-V1, a human MDR1P-gp-overexpressing multidrug-resistant human epidermoid carcinoma cell line; and transcellular transport studies with L-MDR1 (or L-mdr1a), a human MDR1 (or mouse mdr1a)-transfected porcine renal epithelial cell line. The in vitro-in vivo correlation for P-gp-mediated transport activity was also examined by comparing in vitro data obtained from L-mdr1a cell studies and in vivo data frommdr1a (−/−)/(+/+) CF-1 mice studies for several compounds. The results are summarized as follows: 1) two in vitro assay systems routinely identified the substrate for humanMDR1 P-gp-mediated transport with similar quantitative results; 2) in vitro studies with L-MDR1 and L-mdr1a cells demonstrated that the P-gp substrate susceptibility is different between human and mouse for certain compounds (species difference); and 3) in vivo brain concentration ratios of mdr1a (−/−) to (+/+) CF-1 mice, either at a certain time point or up to 60 min, correlated well with the in vitro transcellular transport ratios from L-mdr1a cells (r2 = 0.968 and 0.926, respectively). This indicates that, at least in mice, the in vitro data are valid predictors of the in vivo contribution of P-gp: the contribution of P-gp to the distribution of the compound to the brain up to 60 min post i.v. administration. These results provide a rationale for predicting in vivo relevance of P-gp in human from in vitro data using human P-gp-expressing cells.
Footnotes
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Send reprint requests to: Masayo Yamazaki, Ph.D., WP75-200, Merck Research Laboratories, West Point, PA 19486. E-mail:Masayo_Yamazaki{at}Merck.com
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↵1 Current address: Department of Drug Metabolism, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., 3 Okubo, Tsukuba, 300-2611, Japan.
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↵2 We have confirmed that several compounds show mouse ≫ human (one compound exhibited ∼10-fold difference in the ratio between L-mdr1a versus L-MDR1) and/or human ≫ mouse, and we have found that the results were consistent irrespective of the B-to-A/A-to-B ratio or the absolute difference in directional transport. Due to the restriction of the disclosure of their chemical structures, we cannot include the data for these compounds in this manuscript.
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We have chosen transport amount ratios at 3 h as a “practical” measure for P-gp activity for the following reason. We would like to compare the in vitro transport results in terms of P-gp for a series of compounds with diverse physicochemical properties. Some of the test compounds such as MK-0826 and MK-0991 showed extremely low membrane permeability. Furthermore, we observed a very small difference in dpm values between A-to-B samples of compound A and D and background (approximately <100 dpm for samples versus ∼25 dpm for background) at the first time point (1 h). This is presumably due to relatively low specific activity of 3H/14C labels we used and extensive efflux by P-gp. At a longer time period (at 3 h), dpm counts of the samples for the A-to-B direction were more consistent for both compounds. To increase the reliability of the data in A-to-B direction, however, we may have underestimated the intrinsic permeability for B-to-A direction significantly. However, it was likely not the case for the present study: we calculated the permeability coefficient, percentage of transport, and the ratio between B-to-A and A-to B at 1 and 3 h for several compounds that were most likely to be affected by nonlinearity and by “not adhering to sink condition” among the compounds tested. As a result, differences in any of the parameters calculated at 1 h and at 3 h were at most <25%, demonstrating that there were no significant differences in using data evaluated at 1 or 3 h for the present study.
- Abbreviations:
- P-gp
- P-glycoprotein
- MDR
- multidrug resistance
- DG
- digoxin
- VBL
- vinblastine
- FCS
- fetal calf serum
- B-to-A
- basal-to-apical
- A-to-B
- apical-to-basal
- AUC
- area under the curve
- Kp,brain
- brain-to-plasma concentration ratio
- LC-MS
- liquid chromatography-mass spectrometry
- Received August 3, 2000.
- Accepted November 24, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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