Transporters are ubiquitous membrane proteins that pump compounds into (uptake transporters) or out of (efflux transporters) cells throughout the body. Some of the efflux transporters are members of the ABC (ATP-binding cassette) protein superfamily, for which the hydrolysis of ATP provides the driving force for the transmembrane transport of substrates. Many, if not all, of the efflux transporters were initially discovered due to their upregulation in chemotherapy-resistant tumor cells. Subsequently, their roles in normal cell function have been recognized and characterized. Not all transporters pump drugs, but a fairly complete listing of the ones that do can be found on the FDA website at www.fda.gov/cder/drug/drugInteractions/default.htm.

The best understood of the drug transporters is ABCB1 (also known as MDR1 in humans or mdr1 in rodents), commonly referred to as P-glycoprotein or P-gp. In terms of drug development, P-gp is significant for several reasons:

• Upregulation of P-gp expression is one means by which cancer cells acquire resistance to chemotherapeutic drugs, so drugs that are not P-gp substrates should be able to circumvent this particular defense mechanism.
• Expression of P-gp at the apical surface of the endothelial cells lining cerebral capillaries limits the distribution of drugs into the brain. This is also the case in some other tissues. Thus, non-substrates of P-gp tend to penetrate the brain more readily, which could obviously be significant for a drug targeting a CNS target.
• P-gp is present at every major barrier that is significant in terms of the absorption and excretion of drugs: the apical surface of epithelial cells lining the lumen of the small intestine, the canalicular surface of hepatocytes, and the apical surface renal proximal tubule epithelial cells. As a result, P-gp non-substrates tend to be absorbed more readily and excreted into the urine and bile less readily, the consequence being higher oral bioavailability.
• Because P-gp is expressed at every barrier that determines absorption, distribution and excretion of drugs, the potential exists for its involvement in drug-drug interactions in cases where a P-gp substrate is co-administered with a P-gp inhibitor. This is not only a theoretical possibility, it is also a clinical reality (link to Jiunn Lin's 2003 review) and is the reason the FDA is very interested.

Since the release, in September 2006, of a draft Guidance for Industry entitled Drug Interaction Studies-Study Design, Data Analysis, and Implications for Dosing and Labeling (http://www.fda.gov/cder/guidance/6695dft.pdf), the FDA has required drug sponsors to include data on P-gp interactions as part of an NDA submission. Besides updating a 1997 guidance on drug metabolism and metabolic drug interactions, for the first time the agency required that sponsors provide data indicating whether or not an NCE is a substrate or an inhibitor of P-gp. One problem afflicting the field of drug transporters is that definitive models and reagents do not exist, particularly with regard to the bidirectional transport assay format that the FDA specified in the draft guidance.

• Other assays, such as measuring the ATPase activity associated with P-gp activity or the uptake of fluorogenic P-gp substrates such as calcein-AM, are amenable to high-throughput screening but are unsuitable for regulatory purposes because they cannot distinguish between substrates and inhibitors and because they fail to identify low-permeability substrates.
• All of the cell lines that are currently available for use in the bidirectional transport assay format are non-ideal for one reason or another:
  • MDR1-MDCK and MDR1-LLCPK1 cells express high levels of human P-gp (they have been transfected to overexpress the human MDR1 gene) but also contain canine and porcine P-gp, respectively. Thus, in order to prove that an observed result is due to interaction with human P-gp it is necessary to run the non-transfected (wild-type) cells in parallel.
  • Caco-2 cells express no non-human P-gp but do express at least two other efflux transporters that could confound the interpretation of experimental data, BCRP and MRP-2. Thus, Caco-2 is a good model to detect transporter-mediated efflux, but not a good one with which to identify which transporter is involved.
• If transporter-specific substrates and/or inhibitors were available, one could use a model such as Caco-2 in the absence and presence of one inhibitor at a time to identify which transporter a test compound interacts with. However, the currently available substrates and inhibitors of various transporters are notoriously non-specific, making this type of pharmacological approach unreliable.

That's why Absorption Systems has developed CellPort Technologies™, a suite of proprietary cell lines that can be used to determine, definitively and unambiguously, with which efflux transporter a test compound interacts.