Fluorosome®–trans-pgp Principles

The ability of a drug to reach and penetrate its intended target within the body is critical to its success in treating disease. However, active multidrug efflux transporters such as P-glycoprotein (Pgp) may pump drugs out of target tissues, leading to a reduction in efficacy. The presence of Pgp results in low oral absorption, poor brain penetration and resistance to chemotherapy treatment. Interaction of drugs with efflux transporters may also cause an increase in toxicity of co-administered compounds. Interaction of drugs with active transporters is of increasing interest to the pharmaceutical industry based, in large part, on new draft FDA guidelines requiring knowledge of whether a drug candidate is a substrate/inhibitor of Pgp. Current cell-based assays for identification of drugs that interact with Pgp are slow, cumbersome, and expensive, but the Fluorosome®–trans-pgp technique promises to revolutionize the testing process.

Fluorosome®–trans-pgp: Principles of Operation

tfcbio-trans-pgp diagram

View Fluorosome®–trans-pgp Animation

Fluorosome®–trans-pgp consists of purified Pgp reconstituted into the bilayers of Fluorosomes®, unilamellar liposomes that contain drug-binding fluorescent sensor molecules in their aqueous interior (see Figure).

  1. The Pgp probe substrate is introduced into a Fluorosome®–trans-pgp suspension.
  2. The probe substrate passively diffuses through the Fluorosome-trans-pgp membrane (this occurs in both the absence and presence of ATP).
  3. When it enters the aqueous interior of the Fluorosome®–trans-pgp particle, the probe substrate instantly binds to the fluorescent sensor, resulting in an alteration of the fluorescence signal intensity.
  4. On addition of ATP, more probe substrate is pumped against its concentration gradient into the interior of the Fluorosome®–trans-pgp particle by the Pgp molecules embedded in the membrane, resulting in further alteration of the fluorescence intensity (see Data)
  5. The test drug is added to this system at various concentrations, before the addition of ATP. If the test drug interacts with Pgp, it will inhibit transport of the probe substrate in a concentration-dependent manner. The IC50 for the test drug is estimated by fitting the inhibition data.