Validation Of In-vitro ABC Transporter Assays And Its Application In The Herbal-drug Interaction Study

Objective: Drug interaction is one of the major factors that affect the drug safety and efficacy. The joint use of Chinese medicines and chemical drugs is the main opportunities and challenges lying ahead of medical researchers(especially in China). The aim of this paper is to establish and validate in-vitro MDR1(P-glycoprotein) transporter assays and study its applications in evaluating the interaction of cholesterol-reducing Traditional Chinese Medicine(Fang-2) and Atorvastatin.Methods: The human MDR1 gene was expressed in the cultured Sf9 insect cells via recombinant baculovirus and membrane vesicle was prepared with nitrogen breaking Sf9 cells generating a high membrane ATPase activity. Fang-2 and its 6 simple prescriptions were prepared using a standardised method. Membrane-based ATPase assay and Caco-2 cell monolayer model were utilized to evaluate MDR1-mediated efflux of Fang-2 and its 6 simple prescriptions and the interaction of Fang-2 and Atorvastatin.Results: In this paper, the Sf9 cell membrane obtained provide stable expression of the MDR1 transporter with transporter activity of 50.75 nmol Pi/min/mg protein(ATPase assay).Atorvastatin was verified to be the substrate of P-gp, with Km, Vmax values of 40.58μM, 11.12 nmol Pi/min/mg protein respectively.The MDR1 transporter activity at 1 mg·mL-1, 10 mg·mL-1of Fang-2 was 27.2, 40.0 nmol Pi/min/mg protein respectively and showed a concentration-dependent manner.In the 6 simple prescriptions, the MDR1 transporter activity of aqueous extract Alisma orientalis, Mangnolia officinalis and Prunella vulgaris was 50.6, 42.6, 40.0 nmol Pi/min/mg protein.The Michaeli-Menten kinetics of MDR1 transporter activity was determined for Alisol B 23-acetate and Alisol A 24-acetate, two active components of Alisma orientalis. The Km values were 0.79±0.28μM, 2.01±0.67μM respectively and Vmax values were 50.51±3.72,56.28±4.6 nmol Pi/min/mg protein respectively, indicating the two components are probably the substrates of P-gp.Caco-2 cell monolayers were established with TEER value 100~800 ?·cm-2 and Papp of Lucifer Yellow is 0.1~0.7×10-6 cm·s-1. In this model system, the B-to-A permeability(10.02±1.20 ×10-6 cm·s-1) of Atorvastatin, significantly higher than A-to-B permeability(2.62±0.43 ×10-6 cm·s-1, P<0.01) and the Pratio value was 3.82±0.46. The Pratio value was reduced to 1.52±0.13 by adding the Fang-2, indicating that the Fang-2 may have inhibitory action on the MDR1 transport of Atorvastatin.Conclusions: The in-vitro MDR1 transporter assay(ATPase assay and Caco-2 cell monolayer efflux assay) was established and validate.There exsits MDR1-mediated interaction between this Fang-2 and Atorvastatin. Among the 6 aqueous extract, Alisma orientalis were mainly transported by MDR1. The two main active components Alisol B 23-acetate and Alisol A 24-acetate of Alisma orientalis were transported by MDR1.Since Fang-2 may probably be the substrate of MDR1 transporter, and Fang-2 was shown to have inhibitory effect on the MDR1 transport of Atorvastatin. Further research for mechanism of transporter mediated interaction of Fang-2 and Atorvastatin is necessary and provide clinical guidance for personalized medication.