Inhibitory Effect Of Pharmaceutical Excipients On Organic Cation And Anion Uptake Transporters

Pharmaceutical excipients are materials added into pharmaceutical formulations in addition to active pharmaceutical ingredients（APIs）.They are extensively applied as the basic materials and essential components of pharmaceutical preparations.Drug transporters are proteins located in the cell membrane and responsible for the transportation of a broad range of endogenous and exogenous substrates into or out of cells.They are divided into two super families:ATP-binding cassette（ABC）transporter family and solute carrier（SLC）transporter family.Human organic cation transporter 1and 2（h OCT1 and h OCT2）,human organic anion transporter 1 and 3（h OAT1 and h OAT3）,as well as human organic anion transporting polypeptide 1B1 and 1B3（h OATP1B1 and h OATP1B3）are SLC uptake transporters.They play major roles in drug disposition and excretion in liver and kidney.Additionally,they have also been recognized as important sites for drug-drug interactions（DDIs）.Objective:Pharmaceutical excipients are conventionally considered inactive and safe materials.However,emerging data have suggested that some pharmaceutical excipients could alter the functions of membrane transporters such as P-glycoprotein（P-gp）and breast cancer resistance protein（BCRP）.Given the little information about the interactions between excipients and uptake transporters,the present study aimed to investigate the inhibitory effects and mechanisms of sixteen commonly used pharmaceutical excipients on six major uptake transporters in liver and kidney,namely h OCT1,h OCT2,h OAT1,h OAT3,h OATP1B1,and h OATP1B3.Methods:Human embryonic kidney（HEK293）cells overexpressing h OCT1（HEK-h OCT1）and h OCT2（HEK-h OCT2）were established using the Flp-In^TM System and validated by quantitative real-time PCR（q RT-PCR）and cell uptake assays.HEK293 cells overexpressing h OAT1（HEK-h OAT1）,h OAT3（HEK-h OAT3）,and h OATP1B3（HEK-h OATP1B3）were previously established in our lab.Chinese Hamster Ovary（CHO）cells overexpressing human OATP1B1（CHO-h OATP1B1）were kindly provided by Dr.Chunshan Gui from Soochow University（Suzhou,China）.Fluorescence-based high-throughput screening assays were used to evaluate the effects of excipients on the uptake of fluorescent substrates.4-（Dimethylamino）styryl)-N-methylpyridinium（ASP⁺）was used for h OCT1 and h OCT2.6-Carboxyfluorescein（6-CF）was used for h OAT1 and h OAT3.Dibromofluorescein（DBF）was used for h OATP1B1.Fluorescein-methotrexate（FMTX）was used for h OATP1B3.Three excipients（Triton X-100,Tween 20,and Tween 80）were selected to investigate their inhibitory potency and determine their half maximal inhibitory concentrations(IC₅₀)using concentration-dependent inhibition assays.The clinical relevance of their effects was evaluated by calculating the in vitro inhibition values([I]/IC₅₀).Their inhibitory mechanisms were further investigated utilizing Lineweaver-Burk plot method.Results:1.HEK-h OCT1 and HEK-h OCT2 cell lines were successfully established and validated.2.Triton X-100 showed inhibitory effects on all six transporters,whereas Tween 20 and Tween 80 inhibited the uptake functions of h OCT2,h OAT1,h OAT3,h OATP1B1,and h OATP1B3.3.Triton X-100 was a strong inhibitor of h OCT1,h OCT2,and h OAT3 with IC₅₀ values≤20.1μg/m L,whereas Tween 20 and Tween 80 were potent inhibitors of h OAT3 with IC₅₀ values≤31.0μg/m L.4.The inhibitory effects of Tween 80 were clinically relevant.5.Mechanism experiments revealed that Triton X-100 was a non-competitive inhibitor for all six transporters;Tween 20 inhibited h OCT2,h OAT1,h OAT3,and h OATP1B3 in a competitive and non-competitive mixed way,but competitively inhibited h OATP1B1;Tween 80 was a competitive inhibitor for h OCT2,a non-competitive inhibitor for h OATP1B1 and h OATP1B3,as well as a mixed inhibitor for h OAT1 and h OAT3.Conclusion:The present findings demonstrate that Triton X-100,Tween 20,and Tween 80 have inhibitory effects on the functions of uptake transporters located in liver and kidney.Excipient-drug interactions may occur when these excipients are used simultaneously with the substrates of the transporters.Therefore,the effects of excipients should be taken into account for a scientific and reasonable formulation design.The present study is significant in understanding the excipient-drug interactions and provides valuable information for excipient selection in drug development.