P-glycoprotein (P-gp) belongs to the ATP Binding Cassette (ABC) protein family and is an ATP dependent transmembrane efflux transporter. It is mainly encoded by the multidrug resistance gene (MDR1), also known as ABCB1 gene, in the human body. P-gp is related to multidrug resistance and drug drug interactions (DDIs). Drug regulatory authorities in the United States, Europe, and China have successively issued guidance principles, listing transporter mediated DDI research as an important indicator for evaluating the safety and efficacy of new drugs. It is also explicitly stated that in vitro studies should be conducted to evaluate whether the investigational drug is a substrate, inhibitor, or inducer of P-gp, providing reference for in vivo studies.

Based on the above requirements, IPHASE/Huizhi and Yuanji have successfully constructed SLC transporter cells, including OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1, and MATE2-K. In addition, they have also successfully constructed the first P-gp transporter membrane vesicle in China to assist in the development of new drugs.

Introduction to P-gp

P-gp is the most well-known efflux transporter in drug discovery, capable of excreting compounds from various cells. It was first discovered by researchers in 1976 on the plasma membrane of a Chinese hamster ovary (CHO) cell line resistant to colchicine, and was the first cloned ABC transporter protein.

In the human body, P-gp was initially thought to be an efflux transporter causing multi drug resistance (MDR) in acute myeloid leukemia. Later, it was gradually discovered to exist in various tissue cells, such as various epithelial cells, liver cells, kidney cells, and also expressed in blood cells and immune cells. P-gp mainly actively transports various hydrophobic compounds, including small molecule compounds, alkaloids, and various natural or artificially synthesized toxic substances.

P-gp is composed of 1280 amino acid residues, with a molecular weight ranging from 130 to 190 kDa due to differences in glycosylation degree. The P-gp molecule consists of two homologous fragments at the N-terminus and C-terminus, each with six hydrophobic transmembrane domains (TMDs) and one hydrophilic ATP binding domain (NBD). These transmembrane domains bind to neutral or positively charged lipophilic drugs, activating the ATPase activity of P-gp. The energy released by the hydrolysis of two ATP molecules is used to transport one drug molecule directly from the lipid bilayer to the extracellular space, reducing the intracellular concentration of the drug and leading to multidrug resistance.Research Model of P-gp Transport

At present, in vitro evaluation of DDI models caused by ABC transporters mainly includes membrane vesicle systems, polarized cell-based bidirectional transport systems, and sandwich cultured liver cells.

2.1 Membrane vesicle system

The membrane vesicle system is the most commonly used model for studying whether investigational drugs are substrates and inhibitors of P-gp. Membrane vesicles can be prepared by infecting insect cells (such as Sf9 or Sf21) or transfecting mammalian cells (such as HEK293, HeLa, V79, or MDCK). After the vesicle is inverted, the transporter originally expressed inside the cell will be inverted outside the vesicle. When incubated with drugs, the drug directly acts on the transporter protein on the inverted membrane. The transporter protein can transport the drug into the vesicle and detect drug absorption rather than efflux. That is, measuring the drug content inside the vesicle can reflect the effect of the transporter on the drug. This model not only has the advantages of easy operation and no need for cell culture, but more importantly, if the molecular weight of the compound is large and difficult to penetrate through the cell membrane, the cell model will not be able to study the drug transport mechanism, while the outer membrane vesicle model is not affected by the permeability of the compound.2.2 Bipolar cell based bidirectional transport system

The bidirectional transport system based on polarized cells refers to cell lines that can spontaneously polarize and form complete cell monolayers, such as Caco-2, MDCK, LLC-PK1, etc. Inoculate the cells onto a Transwell chamber and culture until a complete polarized cell monolayer is formed. Add the test drug to the apical (AP) or basal (BL) side of the monolayer cells, measure the amount of drug that permeates into the receiving chamber, and calculate the efflux ratio (ER) based on the apparent permeability coefficient (Papp) in the AP → BL (absorption) and BL → AP (efflux) directions to evaluate whether the compound is a substrate for the ABC transporter. In addition, by measuring the net flow rate or ER value changes of probe substrates in the BL → AP direction, the inhibitory effect of compounds on specific transporters can also be evaluated.

2.3 Sandwich culture of liver cells

In addition to abundant drug metabolizing enzymes, liver cells also express various important transporters. Sandwich culture is the process of culturing liver cells between two layers of collagen. The bottom layer is made of mouse tail glue to allow the liver cells to adhere to the wall, while the top layer is made of Matrigen glue to form a liver plate like structure. After a few days of cultivation, liver cells form a complete bile duct network while maintaining tight connections, and liver transporters are expressed normally and localized in appropriate membrane regions, which can be used for transporter function research.

IPHASE transporter products

As a leader in in vitro research of biological reagents, IPHASE successfully constructed transfected recombinant P-gp transporters using HEK293 as a carrier, and prepared them into inverted membrane vesicles through special methods to assist drug transporter mediated DDI research.

IPHASE technicians used Talinolol with a final concentration of 1 μ M as a substrate and incubated it with P-gp vesicles under two different conditions, ATP and AMP. LC-MS/MS was used to detect the transport of Talinolol, and the results showed that the transport activity of P-gp transporter membrane vesicles in the ATP group was 5.8 times higher than that in the AMP group, which was twice as high as the "Drug Interaction Guidelines 2020", indicating successful construction of P-gp transporter membrane vesicles!

In addition to ABC transporter vesicle P-gp, IPHASE has successfully launched SLC transporter cells and supporting products that fully match the guiding principle requirements for customers to choose from. Meanwhile, ABC transporter vesicle BCRP is also being optimized, please stay tuned