Pre-Plated Cell Lines for ADMETox Applications in the Pharmaceutical Industry.

Membrane transporters significantly modulate membrane permeability of endobiotics and xenobiotics, such as bile acids and drugs, respectively. Various in vitro methods have been established for both ATP-binding cassette (ABC) transporters to examine cellular efflux and uptake, and for solute carriers (SLC) to examine cellular uptake of substrates. Cell-based systems are the models of choice to test drug-transporter interactions as well as drug-drug interactions for research and regulatory purposes, albeit, for low passive permeability substrates of ABC transporters, vesicular uptake assays are also recommended. Commercially available pre-plated cells (e.g., immortalized or transfected) offer a useful alternative to in-house cell culture. Three main methods are known to manufacture pre-plated cultures: regular culture medium with vacuum seal, cryopreserved delivery, and the solid shipping media technology. The regular culture medium and the solid shipping media technologies provide ready-to-use models for end users. Models expressing a broad selection of transporters are available in pre-plated formats for absorption, distribution, metabolism, excretion, and toxicity (ADMETox) studies. Conversely, the application and utility of pre-plated cultures coupled with personal experiences have not been extensively covered in published research papers or reviews, despite availability and significant use of pre-plated products in the pharmaceutical industry. In this overview, we will briefly describe: 1) in vitro tools commonly used for ADMETox testing; 2) methods employed in manufacturing, shipment and preparation of pre-plated cell lines; 3) cell-membrane barrier models currently available in pre-plated format to reproduce passage restriction of physiological barriers to certain compounds; and 4) recommended pre-plated cell lines overexpressing uptake transporters for ADMETox applications.

In vitro methods in drug transporter interaction assessment.

Drug transporter proteins recruit to pharmacological barrier tissues and profoundly affect the ADME properties of a large number of drugs. In vitro assays optimized for drug transporters have grown into routine tools in the determination of molecular level interactions as well as prediction of barrier penetration and system level pharmacokinetics. Regulatory position mandates increasing interest in the application of these assays during drug development.

Mouse Bsep ATPase assay: a nonradioactive tool for assessment of the cholestatic potential of drugs.

The mouse ortholog of the human bile salt export pump (BSEP) transporter was expressed in a baculovirus-infected insect cell (Sf9) system to study the effect of membrane cholesterol content on the transporter function. The transport activity of cholesterol-loaded mouse Bsep-HAM-Sf9 vesicles was determined in a vesicular transport assay with taurochenodeoxycholate (TCDC), a known BSEP substrate. Mouse Bsep transports TCDC at a high rate that can be sensitively detected in the ATPase assay. Cholesterol upload of the Sf9 membrane potentiates both TCDC transport and TCDC-stimulated ATPase activities. Inhibitory effect of BSEP interactors on probe substrate transport was tested in both vesicular transport and ATPase assays using cholesterol-loaded membrane vesicles. A good rank order correlation was found between IC(50) values measured in TCDC-stimulated mBsep ATPase assay and in the human BSEP vesicular transport assay utilizing taurocholate (TC) as probe substrate. This upgraded form of the mouse Bsep-HAM ATPase assay is a user friendly, sensitive, nonradioactive method for early high-throughput screening of drugs with BSEP-related cholestatic potential. It may complement the human BSEP-mediated taurocholate vesicular transport inhibition assay.