In Vitro Evaluation of In Vitro Drug Transporter: Exploring the Roles of ABC Vesicles and SLC Transporter Cells

Keywords: ATP-binding cassette(ABC), ABC Transporter, SLC Transporter, Membrane Vesicle, MDR1(P-gp), BSEP, BCRP, MATE1, MATE2-K, OAT1, OATP1B1, MDCK II, Caco-2, Transporter Inhibition, Transporter Substrate Identification, ICH M12 Draft Guidance on Drug Interaction Studies，HEK293 MOCK, MOCK SLC Transporter

IPHASE Products

Examples of substrates for transporters (In Vitro Studies) provided by ICH M12 Draft Guidance on Drug Interaction Studies

Examples of inhibitors for transporters (In Vitro Studies) provided by ICH M12 Draft Guidance on Drug Interaction Studies

Background

In clinical applications patients often use multiple drugs simultaneously, and these drugs may produce drug-drug interactions(DDI) that have the potential to cause serious adverse reactions or alter the effectiveness of treatment. DDI assessment usually begins with in vitro testing to identify factors that may affect drug disposition to elucidate potential DDI mechanisms and to obtain kinetic parameters for further study. Drug metabolizing enzymes have long been the focus of DDI research. In recent years, with the development of molecular biology technology, significant progress has been made in the study of in vitro drug transporters, and the importance of in vitro evaluation of transporter based DDIs has attracted increasing attention.

Transporters and Their Roles

Transporter refers to transmembrane proteins throughout the cell membrane of various tissues that mediate the entry and exit of endogenous or exogenous substances into and out of biological membranes. In vitro drug transporter is a general term for proteins that take drugs as substrates, exist on the cell membrane surface of tissues or organs, and take up the function of transmembrane drug transport, mainly including two families: the ATP-binding cassette(ABC) superfamily and solute carrier superfamily (SLC transport), which dominate the transmembrane drug transport process. 

ABC transporters are ATP-dependent transporters that actively move various substrates (e.g., ions, lipids, and drugs) across cell membranes, often against concentration gradients. They play key roles in drug resistance, metabolism, and cellular detoxification. SLC transporters are mostly secondary active or facilitated transporters that mediate the movement of solutes such as glucose, amino acids, and neurotransmitters. Unlike ABC transporters, they do not require ATP directly but rely on ion gradients. 

Transporters that mediate drug efflux mainly include P-glycoprotein (P-gp), or multi-drug resistance 1 protein (MDR1), breast cancer resistance protein (BCRP). They are members of the ATP-binding cassette(ABC) transporter family, which use the energy of hydrolyzed ATP for the transport of drugs and endogenous substances. Transporters that mediate the entry of drugs into the cell can take up substrates to the target site to exert pharmacological effects, and belong to the solute transporter family members, mainly including Organic anion transporting polypeptide (OATPs), Organic anion transporter (OATs), Multidrug and toxin extrusion proteins (MATEs), Organic cation transporter (OCTs), etc.

Key Transporters and Their Roles: 

• MDR1(P-gp): A major efflux transporter expressed in the intestine, liver, and blood-brain barrier, MDR1 limits drug absorption and enhances excretion, contributing to multidrug resistance in cancer therapy.

• BSEP (Bile Salt Export Pump): Critical for bile acid secretion in the liver, BSEP dysfunction is linked to cholestatic liver disease. Drug-induced BSEP inhibition can cause hepatotoxicity.

• BCRP (Breast Cancer Resistance Protein): BCRP affects the bioavailability of chemotherapeutics and antivirals, exporting substrates from cells and influencing placental and blood-brain barrier penetration.

• MATE1/MATE2-K (Multidrug and Toxin Extrusion Proteins): Located in renal and hepatic tissues, MATE1 and MATE2-K excrete cationic drugs, working synergistically with OCT2 to mediate renal secretion.

• OATP1B1 (SLCO1B1): A hepatic uptake transporter critical for statin clearance. OATP1B1 plays a crucial role in mediating the uptake of endogenous compounds such as bilirubic acid, bilirubin, steroid-coupled compounds, and thyroid hormones, as well as the hepatic clearance of a wide range of clinical drugs such as statins, antibiotics, antivirals, and anticancer drugs.

• OAT1 (SLC22A6): Mediates renal uptake of anions, including antivirals and NSAIDs, influencing nephrotoxicity risk.

Transporter Inhibition and DDI

Transporter inhibition is an essential aspect of drug development. Transporter inhibition plays a pivotal role in drug-drug interactions (DDI) by affecting the absorption, distribution, metabolism, and excretion of co-administered drugs. Inhibition of efflux transporters such as MDR1(P-gp) and BCRP can reduce the cellular extrusion of drugs, potentially leading to higher intracellular and systemic concentrations. Similarly, inhibiting uptake transporters like OATP1B1 may decrease hepatic drug clearance, while BSEP inhibition can interfere with bile acid transport, raising the risk of cholestasis. Additionally, transporters like MATE1, MATE2-K, and OAT1, which are crucial for renal drug elimination, when inhibited, can alter renal excretion and contribute to drug accumulation. In vitro models, including MDCK II and Caco-2 cell lines, are widely used to evaluate these transporter interactions, providing essential insights into DDI potential during drug development.

In Vitro Models for Transporter Studies

Understanding transporter function and predicting transporter-mediated DDIs rely on specialized cell models. Researchers use HEK293 MOCK cells as a negative control, ensuring that any observed transport is due to the specific transporter. HEK293 MOCK provides a consistent background, while HEK293 MOCK remains central to validating experimental specificity. One method employs membrane vesicles from cells overexpressing transporters, often isolated from HEK293 MOCK cells. Similarly, MOCK SLC Transporter models are critical for distinguishing specific transporter activity from nonspecific uptake. MOCK SLC Transporter assays confirm that observed substrate movement is transporter-dependent, and MOCK SLC Transporter experiments reinforce the reliability of the model. This dual approach using HEK293 MOCK and MOCK SLC Transporter enhances our ability to study ATP-dependent transport without interference from cellular metabolism.

In addition to vesicular systems, cell-based assays are widely employed. For example, the MDCK II cell line is frequently used because of its polarized nature, which mimics epithelial barriers. Notably, Human MDR1 Knockin MDCK II Cells have been engineered to express human MDR1, allowing a direct evaluation of MDR1(P-gp)–mediated transport and inhibition in a relevant in vitro model.

The Caco-2 cell line, derived from human colon carcinoma, is another standard model used to simulate intestinal drug absorption. Its ability to differentiate into enterocyte-like cells that express various transporters—such as P-gp and BCRP—makes it invaluable for assessing both drug permeability and potential interactions.

Moreover, transporter-specific models like Human BCRP Expressing cells enable the investigation of substrate specificity, the kinetic behavior of drug transport, and the extent to which inhibitors can modulate transporter activity. When combined with data from vesicular assays, these cellular systems provide comprehensive insights into the dynamics of transporter-mediated drug disposition.

Conclusion

In summary, understanding transporter interactions is vital for optimizing drug safety and efficacy. Transporters such as MDR1(P-gp), BCRP, and BSEP alongside uptake transporters like OATP 1B1 and OAT1 slc Transporter play critical roles in drug pharmacokinetics. The use of in vitro models including MDCK II, Caco-2, Human BCRP Expressing cells, and Human MDR1 Knockin MDCK II Cells provides a robust framework for understanding how transporter inhibition can affect drug–drug interactions and overall drug disposition. These insights are invaluable for optimizing drug efficacy and safety in clinical settings.

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