P-glycoprotein Substrate Assessment in Drug Discovery: Application of Modeling to Bridge Differential Protein Expression Across In Vitro Tools

• Published in: Journal of pharmaceutical sciences Vol. 110; no. 1; pp. 325 - 337
• Main Authors: Li, Na, Kulkarni, Priyanka, Badrinarayanan, Akshay, Kefelegn, Adey, Manoukian, Raffi, Li, Xingwen, Prasad, Bhagwat, Karasu, Matthew, McCarty, William J., Knutson, Charles G., Gupta, Anshul
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2021
• Subjects: Blood brain barrier; Drug transporter; Efflux; In vitro models; Kinetics; MDCK cells; MDR1; P-glycoprotein; P-gp expression; In vitro models; Efflux; A; B; BBB; P-gp; Kpuu; IVIVE; Drug transporter; 3-C; CNS; NIH; Blood brain barrier; ER; P-glycoprotein; BSA; LC-MS/MS; NCE; MDR1; MDCK cells; Kinetics; HBSS; MDCK; P-gp expression
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1016/j.xphs.2020.09.017

P-glycoprotein (P-gp) efflux assay is an integral part of discovery screening, especially for drugs requiring brain penetration as P-gp efflux ratio (ER) inversely correlates with brain exposure. However, significant variability in P-gp ER generated across cell lines can lead to misclassification of a P-gp substrate and subsequently disconnect with brain exposure data. We hypothesized that the ER depends on P-gp protein expression level in the in vitro assay. Quantitative proteomics and immunofluorescence staining were utilized to characterize P-gp protein expression and localization in four recombinant cell lines, over-expressing human or mouse P-gp isoforms, followed by functional evaluation. Efflux data generated in each cell line was compared against available rodent brain distribution data. The results suggested that the cell line with highest P-gp expression (hMDCK-MDR1 sourced from NIH) led to greatest dynamic range for efflux; thus, proving to be the most sensitive model to predict brain penetration. Cell lines with lower P-gp expression exhibited the greatest tendency for compound-dependent in vitro efflux saturation leading to false negative results. Ultimately, P-gp kinetics were characterized using a compartmental model to generate system-independent parameters to resolve such discrepancy. This study highlights the need for careful choice of well characterized P-gp in vitro tools and utility of modeling techniques to enable appropriate interpretation of the data.