Abstract

The body is continuously exposed to a variety of toxins and metabolic waste products but is able to rid itself of these by using various detoxification mechanisms such as enzymes and transmembrane transporters. A large number of the transporters which play an essential role in the detoxification mechanisms are found in the liver, kidney and intestines. The largest family of transporters is the Solute Carrier (SLC) Superfamily with 255 members in humans. While most SLC transporters are highly specialized, a number of these transporters are polyspecific, generalized transporters that play a major role in the elimination process. Accordingly, the aim of this research programme was to develop novel methodology to enable the study of the interaction of drugs and related compounds with these transporters, in a rapid and facile manner.
In the initial studies the application of affinity chromatography was carried out with the use of α1-acid glycoprotein (AGP) column to achieve enantioselective separation of the drug ketamine and its metabolites. The enantioselective separation of ketamine and norketamine from plasma samples was achieved and the assay conducted was sensitive and reproducible. Building upon the experience gained in this work on affinity chromatography and LC-MS, these useful tools for the determination of chiral drugs in biological fluids were also used in the study of drug-protein interactions. With the nicotinic acetylcholine receptors it was possible to explore the use of immobilized liquid chromatographic stationary phases containing drug transporters in on-line high throughput screening (HTS).
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Thus having demonstrated that a stationary phase containing immobilized membranes can be used to identify substrate/inhibitors of an expressed receptor/transporter the next and principal phase of the programme was to adapt the methodology to other target biopolymers such as P-glycoprotein which over the last decade has been focused upon for its role in drug resistance in cancer treatments. In particular liquid chromatographic columns containing the drug transporters, P-glycoprotein (Pgp) and human organic cation transporter 1 (hOCT1) were prepared, evaluated and exploited.
Initial studies were conducted to confirm the functionality of a stably transfected cell line expressing Pgp (Pgp-(+), LCC6/MDR1 cell line) through a comparison with the non-transfected cell line (Pgp-(-), LCC6 cell line).
Initially membranes from the Pgp(+) and Pgp(-) cell lines were then immobilized on immobilized artificial membranes. However, although the resulting column could actively bind the Pgp substrates, strong non-specific interactions with the IAM backbone led to large retention times and peak tailing. A more successful approach was to immobilize on the surface of open tubular capillaries. Such columns were used to sort compounds with or without affinity for Pgp, by comparing the differential retention time on the Pgp (+) and Pgp (-) columns. In this way the non-specific interactions with the constituents of the cellular membrane were compensated for. The results from the sorting by differential chromatography were compared with the behavior of the same compounds in Caco-2 monolayers cultured in 96-well transwell plates, the standard method for the determination of substrates for Pgp. A group of 14 compounds
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previously characterized as substrates or non-substrates of Pgp were studied using the chromatographic and Caco-2 methods. In the parallel chromatographic screen, the value of