Scientific background

Since the publication of the human genome in 2001 the significance of the membrane transporters has been acknowledged even further (26 % of the entire genome are transporter molecules). The ABC (efflux) transporters (49 different molecules) and the SLC (uptake) transporters (hitherto 328 are known) actively pump substrates through the cell membranes against a concentration gradient. ATP is the source of energy for ABC transporters, whereas uptake transporters use other molecules to bring about transport of substrates. Uptake and efflux transporters are abundantly expressed in all pharmacologically significant barriers (intestinal cells, blood-brain barrier (BBB), hepatocytes, kidney etc.). Transporters influence the absorption, distribution, metabolism, excretion, and toxicology (ADME-Tox), bioavailability and pharmacokinetic properties of drugs, nutrients, endobiotics and xenobiotics. Transporter substrate/inhibitor interactions directly or indirectly have an impact on the molecules reaching their target site of action as well as inducing adverse side-effects e.g. drug-drug interactions, or entering the brain. Multidrug resistance (MDR) caused by overexpression of efflux transporters on tumor cells has been acknowledged for a long time. Recent findings suggest that transporter induced MDR can also develop during the treatment of other diseases, like in the case of Rheumatoid arthritis (Kis et al. 2008).

One of the most important functions of the mammalian BBB is to restrict the free movement of different substances between blood and neural tissue meanwhile sustain the homeostasis of the central nervous system (CNS). Yet, BBB can also be a major impediment for the treatment of diseases of the CNS (CNS tumors, therapy resistant epilepsy, stroke etc.): due to the relative impermeability of the barrier many drugs are unable to reach the brain at therapeutic concentrations. Therefore it is of pivotal importance to study the permeability of the BBB for potential new drugs designed for the therapy of CNS disorders and for non-CNS drugs to prevent neurotoxic side-effects. Many efflux (MDR1, MRP1, MRP2, MXR) and uptake transporters (OATP-A, OAT1, OAT3, OCT3, OCT2) have been identified in the BBB and blood-cerebrospinal fluid barrier (BCSFB) (for review see Kushuhara and Sugiyama, 2005, Bollók et al. 2008). Several models exist with different complexities to study BBB-transporter interactions, ranging from membrane assays to in vivo microdialyses (Erdő et al., 2008).

Hepatocytes are highly polarized cells that depend on the maintenance of two distinct plasma membrane domains for normal hepatic functions. The canalicular (MDR1, MDR3, MRP2, BSEP, OCT1) and sinusoidal domains (MRP1, MRP3, MRP5, MRP6, OATP-B, OATP-C, OATP-8, NTCP, OAT2, OCT2) each possess specialized transport processes related to xenobiotic transport. MRP2 is the major efflux transporter that clears endogenous compounds, including LTC4 as well as toxic compounds, phase II metabolites, such as glucuronides (e.g. bilirubin conjugates) and glutathione conjugates into the bile. Blockade of this transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. SOLVO has developed a vesicular transport assay to enable the determination of drug-drug and drug-endogenous compound interactions of ABC transporters (see Heredi-Szabó, 2008). Sandwich cultured hepatocytes are considered as one of the best cell culture models of liver function, as they express transporters and enzymes close to physiological levels (Lengyel et al. 2008). BSEP is mainly responsible for transport of bile salts into the bile; and inhibition of its transport activity may lead to chemical cholestasis. Rodents are commonly used in the pharmaceutical industry as model species for pre-clinical in vivo experiments. However, a key difference between rodents and humans is their sensitivity to Bsep activity impairment. The consequences of hampering BSEP function are far less severe in mice than in humans and therefore the hepatic cholestasis induction potential of drugs may considerably differ. For this reason SOLVO has developed, characterized and validated a range of BSEP assays, including human and mouse Bsep ATPase (Kis et al, JBS in press) and VT. The above example also supports the notion that species specific differences may be an important factor to consider and that human liver toxicity can not always be reliably predicted by preclinical animal species alone. For fully understanding and characterizing the behavior of new chemical entities and improving the prediction of their hepatotoxic potential complex studies are needed, starting from high throughput membrane assays to preclinical animal studies.

SOLVO provides various assay systems to investigate xenobiotic transporter interactions and in vitro - in vivo correlations (Heredi-Szabo et al, submitted). The IVIVC results are invaluable tools to understand and improve the determination of transporter-xenobiotic interactions and to assist data interpretation.

High-through-put screening (HTS) technologies are suitable for testing several thousands of molecules in the early phase of drug development (screening, lead selection and optimization), thus allowing fast and more cost-efficient delivery of revolutionary, bio-pharmacological drugs to clinical applications. Furthermore, the risk of unpredicted and unwanted drug interactions can be reduced. SOLVO has developed and validated various membrane (ATPase, VT, nucleotide trapping Glavinas, H., et al., EOMT 2008) and cellular (e.g. dye extrusion like calcein assay or uptake assays) and monolayer assays (e.g. Caco-2 monolayer efflux assay) for several transporters (Glavinas, H., et al., Current Drug Delivery, 2004;1:27-42.). Insect cells, like Sf9 are commonly used to express mammalian transporters. SOLVO's proprietary membrane technology is based on the combination of high throughput assay set up and optimized membranes. During assay development differences in membrane properties e.g. cholesterol content (Pal et al. 2007) and species differences (Glavinas et al.2007, Mehn et al. in press) are taken into account. SOLVO also works extensively on developing screening strategies to assist pharmaceutical companies' drug development (von Richter, 2008).

Beside transporter based drug-drug and drug-transporter interactions, nutrients and toxic substances also exhibit extensive interactions with a wide range of transporters. For this reason considerable research has been carried out regarding nutrient transport (Williamson et al. 2007; Zhang et al. 2007), especially uptake as well as on the potential role of transporters related to toxic compounds (Lespine et al. 2006; Oosterhuis et al. 2008)