Membrane Transporters: Fundamentals, Function and Their Role in ADME

JOHN KEOGH, BRUNO HAGENBUCH, CAROLINE RYNN, BRUNO STIEGER AND GLYNIS NICHOLLS

1.1 Introduction

In the middle of the 20th century, the absorption, distribution, metabolism and elimination (ADME) of pharmaceutical drugs was considered to be mediated primarily by simple diffusion and metabolism. The concept that membrane transporter proteins existed and could facilitate the flux of molecules across eukaryotic cell membranes was still in its infancy, but as knowledge and information expanded, it was recognised that transporter proteins could play an important role both in the movement of endogenous compounds within the body and in the ADME of drugs. Two superfamilies of transporters, the ATP binding cassette (ABC) and the solute carrier (SLC), comprising between them over 500 members, have now been identified in the human genome, although only a few transporters of specific interest to the pharmaceutical industry are described here.

This chapter summarises the discovery of transporters, their function in cellular processes, location and mechanism(s) of action, as well as outlining the key transporters currently considered to be clinically relevant. A description of how and why they are evaluated within drug discovery and development is included, outlining some of the key pharmacokinetic (PK) concepts useful to transporter scientists, and briefly discussing the methods and strategies used. While there are many different transporters within the body, this overview concentrates primarily on those transporters currently known to influence drug ADME and will not cover the transport of oligonucleotides or proteins. Links to current transporter databases and reviews are also included throughout the text for those wishing to pursue the area further.

1.2 The History of Transporter Science

1.2.1 The Discovery of Transport Processes

The basic functional unit of eukaryotic organisms is the cell, with each cell enclosed by a plasma membrane that forms an inherent physical barrier to the free transport of solutes. While small hydrophobic molecules are able to move freely across these phospholipid membranes by simple diffusion, the more water-soluble molecules require the presence of membrane proteins or channels embedded within the plasma membrane to gain access into and out of cells. This concept of transport via membrane proteins ('transporters' or 'drug transporters') and their involvement in the ADME of small drug molecules was first noted in the 1950's, although there were much earlier indications that transport processes may be present within the body.

Bile salts are highly amphipathic molecules that are synthesised and secreted by the liver and considered as model compounds for enterohepatic circulation. At the turn of the 18th century, Mauritius Reverhorst and Alfonso Borelli reported that the amount of bile recovered in faeces was much less than the amount of bile produced by the liver, and they coined the term "motus circularis bili" (or enterohepatic circulation, the cycling of compounds between the intestine and the liver). This insight into the selective preservation and recycling of bile can be viewed as an early but major step towards the appreciation of transport processes for solutes. Focusing on specific mechanisms, Tappeiner noted in the 19th century that bile salts are preferentially absorbed in the ileum of dogs, but not in the upper part of the small intestine, suggestive of a transporter-mediated process. In 1923, Eli Kennerly Marshal demonstrated the active secretion of phenolsulfonephthalein in the kidney, which may be considered the first demonstration of active (rather than passive) transport. Prior to this, phenoltetrachlorophthalein was used as a liver function marker. This work illustrated not only that both the liver and kidney are important drug clearance organs, but also that they can be selective, indicating the presence of specific, active mechanisms in these organs. However, it was not until 1958 that Crane published his observations on active transport, demonstrating that 6-deoxyglucose can be transported against a concentration gradient and subsequently that sugar absorption is dependent on the presence of sodium and ultimately that the process is electrogenic.

Applying the knowledge from these early findings, the very short half-life of penicillin in patients, concomitant with its rapid appearance in urine, was believed to be caused by active tubular secretion of the drug in the kidney. It was therefore reasoned that para-aminohippurate, also known to be secreted by the kidney, may interfere with the secretion of penicillin. The subsequent finding that co-administration of para-aminohippurate with penicillin resulted in a marked retention of penicillin in the blood of dogs may well be considered the first application of transporter research to ADME. These findings led to the development of probenecid, which inhibits penicillin secretion in the kidney and thereby prolongs its plasma half-life, although the specific transporter proteins involved were not identified until many decades later. At the same time, it was discovered that probenecid enhanced renal urate elimination, and probenecid is still used today as an uricosuric drug.

In the 1960s, following the development of the first chemotherapeutic drugs for the treatment of cancers, it was noted that some tumours developed resistance against these drugs. This was followed by descriptions of cross-resistant cell lines and the isolation and characterisation of several multidrug resistant cell lines, as reviewed by Gottesman and Ling. For example, a Danish group reported on active drug export from Ehrlich ascites tumour cells and Ling and Thompson, whilst isolating cells resistant to colchicine, observed that these cells were resistant to other chemotherapeutic drugs with different pharmacodynamic (PD) properties. The authors concluded that this cross-resistance was caused by a reduced permeability of the cell lines to drugs and identified a glycoprotein of about 170 kDa that was only expressed in colchicine resistant and not in revertant colchicine sensitive cells. They named this protein P-glycoprotein (P-gp), whereby "P" designated permeability. Sequencing of a mouse multidrug resistance protein (MDR) sequence demonstrated that P-gp is a member of the ABC superfamily of transporters, subfamily member B1 (ABCB1), which had previously been identified. Purification and reconstitution of human multidrug resistance protein 1 (MDR1) finally demonstrated that MDR1 displayed drug-stimulated ATP hydrolysis and drug transport. Hence, P-gp is also known as MDR1 and ABCB1. These discoveries led to a series of programmes over many decades testing clinical in vivo inhibitors of P-gp to overcome multidrug resistance in chemotherapy, with limited success.

1.2.2 The Development of Transporter Science in Industry

Drug transporters first came to the attention of drug discovery and development specialists in the drug formulation and drug metabolism and PK (DMPK) fields, as these are disciplines that characterise how the body handles pharmaceutical compounds. Arguably, the earliest appreciation of differential drug-tissue distributions (and later of drug transport mechanisms) in the DMPK field arose with the development of semi-quantitative radiolabelled drug–tissue distribution studies and later quantitative whole body autoradiography in rats. Distribution studies revealed that, for some drugs, the tissue concentrations of drug related material were much greater than blood concentrations, sometimes by many orders of magnitude, whereas in others, drug related material in tissues was virtually undetectable. These observations were presumed to be due to the physicochemical properties of the molecules (lipophilicity, charge at pH 7.4, solubility, etc.) and/or biological phenomena such as metabolism, and plasma and tissue protein binding. However, with the discovery of P-gp, these assumptions began to be re-evaluated. Over a period of time, a body of evidence emerged, demonstrating that many marketed drugs were in fact both in vitro substrates and inhibitors of P-gp. This raised concerns that P-gp could influence drug absorption, distribution and elimination (ADE), and therefore be a potential mechanism driving both systemic and target organ exposure, as well as drug–drug interactions (DDIs). Given its abundant localisation at the blood–brain barrier, P-gp was most often evaluated as a modulator of drug penetration of the central nervous system (CNS), becoming the first drug transporter routinely investigated during drug discovery and development in the pharmaceutical industry.

As the science, understanding and discovery of drug transporters and their function continued to develop, it became clear that a number of pharmaceutical products were substrates, inhibitors and inducers of multiple drug transporters of different types. These included widely prescribed drugs such as digoxin, transported by P-gp and the organic anion transporting polypeptide member 4C1 (OATP4C1); metformin, transported by organic cation transporter member 1 (OCT1) and multidrug and toxin extrusion proteins (MATEs); 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoA) inhibitors, transported by OATP1B1; fexofenadine, transported by OATPs and P-gp; and acyclovir, transported by organic anion transporters (OATs), to name just a few. One notable example is that of rosuvastatin, a HMG-CoA reductase inhibitor (also known as a 'statin') with a high distribution into the liver that was shown to be mediated via transporter dependent mechanisms. Subsequent work showed that rosuvastatin and other statins were substrates of some of the OATPs, members of the SLC organic anion transporter (SLCO) gene family. This and other work led to an explosion of interest in hepatic transporter systems, as it became clear that a number of DDIs between statins and other molecules could be readily explained by considering hepatic OATPs and other drug transporters, such as breast cancer resistance protein (BCRP; the protein product of the ABCG2 gene), multidrug resistance associated protein 2 (MRP2; the protein product of the ABCC2 gene), and the organic anion and cation transporters (OATs and OCTs; protein products of the SLC22 gene family). Even some molecules ultimately cleared by metabolism were discovered to be clinical substrates of drug transporters, which could result in the restriction or enhancement of systemic exposures and thereby influence the drugs' PK. For example, atorvastatin is a substrate of OATPs and P-gp, is primarily eliminated as metabolites, but is subject to DDIs when co-administered with transporter and/or metabolism inhibitors.

Several findings regarding other transporter-mediated processes were also noted, and there are now many examples of the PK of marketed drugs being influenced by the action of drug transporters. The clinical consequences of many of these interactions are still being investigated, but continue to result in changes and warnings on labels for drug use. This topic is discussed in more detail in Chapter 11. As knowledge of transporter tissue distribution and localisation grew, particularly for barrier tissues such as the gastrointestinal tract (GIT) and in the major clearance organs (kidney and liver), there was a growing appreciation that drug transporters could significantly influence drug PK. This was particularly true for molecules with low simple diffusion (see Figure 1.1a), as this limits their transfer across cellular membranes. In these cases, drug transporters were found to be an important mechanism for oral absorption, systemic clearance, and renal and faecal excretion. Furthermore, evidence that drug molecules were substrates, inhibitors or inducers of multiple transporters and drug metabolising enzymes (DMEs), challenged commonly held assumptions that associated clinical DDIs were due to a single mechanism, and led to the reevaluation of these DDIs. However, identifying the precise contributions of multiple mechanisms to overall clinical PK remains a remarkably challenging goal. This is due in part to limitations of the current preclinical toolkit, as well as the reliance of PK on measurement of drug concentrations in the blood compartment rather than within organs or tissues. While subtle changes in systemic drug concentrations can be measured, they may not always reflect much larger concentration changes in tissues and organs, which may result in unpredicted toxicology and/or pharmacology.

These findings raised many questions for drug discovery and development scientists, as they alerted the scientific, medical and regulatory communities that they could have relevance to drug safety, efficacy and toxicity. A good example of the depth and breadth of a typical investigation now is that of bosentan DDIs and drug induced liver injury (DILI). Bosentan is a nonpeptide dual endothelin receptor antagonist that is used to treat pulmonary arterial hypertension. It is metabolised in the liver to three major oxidative metabolites, undergoes biliary elimination and was shown to be a substrate for OATPs expressed in human hepatocytes. Studies in rats indicated that cyclosporine inhibited the uptake of bosentan into hepatocytes via members of the rat OATPs (rOATPs), and in vitro studies identified rifampicin as an inhibitor of rat and human OATPs expressed in hepatocytes. Several drugs were subsequently tested for PK interactions on co-administration with bosentan, including cyclosporine and rifampicin. Both of these compounds increased the minimum blood concentrations of repeat-dose bosentan, suggesting inhibition of hepatic uptake and/or metabolism of bosentan by these drugs. Additionally, long term treatment of patients with rifampicin led to a reduction in serum concentrations of bosentan, which could be explained by the induction of cytochrome P450 (CYP450) 3A4 (CYP3A4), which generated a metabolismdriven sink for bosentan in hepatocytes, stimulating its OATP mediated uptake.

In terms of DILI, elevated liver enzymes, preceded by an increase in serum bile salts, were observed in patients receiving bosentan, which resolved on withdrawal of the drug. Interference with bile salt elimination, possibly through modulation of the bile salt export pump (BSEP; protein product of the ABCB11 gene) was suspected. In addition, in a chronic heart failure trial, bosentan and glibenclamide were found to act synergistically on liver injury, elevating serum bile salt levels. This was also confirmed in rats. In vitro investigations using isolated rat canalicular plasma membrane vesicles and vesicles from the insect cell line Sf9 over-expressing the rat bile salt export pump (rBSEP) demonstrated that bosentan and its three metabolites competitively inhibited rBSEP and human BSEP mediated transport of the bile salt taurocholate. Glibenclamide also competitively inhibited rBSEP and, as the serum markers for liver injury in patients spontaneously resolved after discontinuation of bosentan, the molecular mechanism of liver injury was linked to drug-induced cholestasis (i.e. the slowing or blockage of bile flow). In vivo studies with bosentan in rats indicated no change in bile salt output, but there was stimulation of bile flow by bosentan mediated by rat MRP2 (rMRP2). This was confirmed in vitro using human MRP2 and BSEP expressed in insect cells, indicating that bosentan inhibited BSEP but activated MRP2 transport. Such a functional MRP2-BSEP interaction has also been observed for estradiol-17ß-glucuronide and gives an indication of the complexity of interactions that can occur for compounds that are substrates and/or inhibitors of membrane transporters.

Recently, it has been asserted that all solute transport across cell membranes is via transporter proteins, and that simple diffusion does not happen. It should however be kept in mind that cells are unable to control simple diffusion and hence are vulnerable to the entry of potentially toxic substances, unless they possess protective mechanisms. Efflux transporters may limit the entry of such toxins, and indeed MDR1 has been dubbed a "vacuum cleaner", because of its ability to efflux substrates from the cell membrane to the external environment, thus protecting the cell. Such considerations may support the concept that all solutes enter (or are expelled from) cells by transporter proteins. This is disputed by other workers in the field and remains a matter of debate.