Pharmacological importance of ATP binding cassette

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The active drug efflux transporters of the ATP binding cassette (ABC)-containing protein family greatly affect the pharmacological behaviour of numerous clinically relevant drugs. One pharmacological property affected by ABC transporters is the oral bioavailability while the distribution of drugs into various important pharmacological sanctuaries, such as the brain, testis, and fetus, can be extensively limited by ABC transporters. This essays aims to provide an overview of these properties of the ABC transporters in order to display their effect on the clinical usefulness and toxicity risks of drugs.

Although the family of mammalian ABC transporters is highly extensive and functionally diverse, focus is placed on the transporters that have been demonstrated to have a well-defined role in drug transport. These are the P-glycoproteins (P-gp), the multidrug resistance proteins (MRPs) 1-5, and breast cancer resistance protein (BCRP). These drug efflux transporters share extensive sequence homology and domain organisation. All contain a highly conserved ATP-binding cassette (200-250 aa) which consists of two nucleotide-binding domains (NBDs). Based on their predicted structure and amino acid sequence homology, the transporters discussed can be divided into four classes (Figure 1). Discovered first, P-gp consists of two very similar halves and the same overall basic architecture is found in the MRP family. However BCRP, the most recently discovered ABC drug transporter, is a member of the ABC White subfamily and contains 'half-transporters'. Evidence supports that BCRP most likely functions as a homo-dimer.

Fig. 1. Secondary structures of ABC transporters. Grey TM, transmembrane domain; NBD, nucleotide binding domains; Red branch, N-glycosylation; N, amino-terminal; C, carboxy-terminal; in, Intracellular; out, extracellular. a. contains an intracellular ATP binding site. b. has an N-terminal extension of 5 putative TM segments and the N-terminus is located extracellularly. c. lacks the N-terminal extension. d. NBD is at the N-terminal end.

Mainly located in the plasma membrane, ABC transporters transport a variety of structurally diverse drugs from the cell and have very broad yet distinct substrate specificities. The putative binding sites which interact with the drugs are located within the TM domains (TMDs). At least 12 transmembrane helices are required in order for the complex reaction with the transported ligand to occur. The current 'hydrophobic vacuum cleaner' model describes an alternating access and release mechanism, whereby ATP-binding promotes the outward-facing conformation of the TMDs while dissociation of the hydrolysis products promotes the inward-facing conformation. This basic mechanism can, thus, explain drug export by ABC transporters (Figure 2).

Direct evidence for this mechanism has been provided by studies on the substrate-induced changes in the TMDs. Cyclosporin A was shown to induce cross-linking between TM6 and TM11 of P-gp while colchicine induced cross-linking between TM6 and TM12. On the other hand, vinblastine, verapamil, and flupenthixol, did not promote cross-linking. This ability of substrates to induce changes in cross-linkages supports the ability of TM segments to change shape in order to accommodate the structurally diverse substrates. The vast number of permutations of residues that can contribute to the drug-binding site may be due to minute rotational and lateral movements in TM segments. Each substrate can cause specific shifts in the different TM segments, enabling common residues to be able to bind diverse substrates.

Fig. 2. Molecular mechanism of ABC transporters. The 12 TM helices form a central cavity. Left; the NBD are open, no ATP is bound, and the TM helices form an inward-facing conformation of the TMDs. ATP-driven tight dimerization of the NBD results in the outward-facing conformation of the TMDs, and dissociation of the transported ligand.

Following its absorption or systemic administration, a drug distributes into the interstitial and intracellular fluids. This tissue distribution depends on the partitioning of the drug between the blood and the specific tissue. As the kidney and liver are major organs for overall drug excretion from the body and ABC transporters are vital for drug elimination into urine or bile, they are highly expressed in the polarized tissues of kidney and liver. Moreover, ABC transports are all expressed in the apical side of the intestinal epithelium, where they export various xenobiotics, including many clinically relevant drugs.

Many ABC transporters are expressed on the blood side of the endothelial and epithelial cells that limit the penetration of xenobiotics into naive tissues. For example, at the blood brain barrier (BBB; Figure 2), the blood-cerebrospinal fluid (CSF) barrier (MRP1, MRP4 on the basolateral blood side of choroid plexus epithelia), the blood-testis barrier (MRP1 on the basolateral membrane of mouse Sertoli cells, P-gp in human testicular cells), and the blood-placenta barrier (P-gp, MRP2, BCRP on the luminal maternal side, MRP1 on the antiluminal fetal side of placental trophoblasts). While these transporters protect the vulnerable areas, they also restrict drug accessibility in pathological situations, such as in central nervous diseases (neurodegenerative diseases, intracranial tumors, dementia and epilepsy).

Studies using P-gp-knockout mice have been used to establish the implication of P-gp in limiting drug entry into the brain. The knock-out mice were shown to be almost 100 times more sensitive to the neurotoxic effects of ivermectin, an antiparasitic compound than the wild-type mice (WT). The effect of many other P-gp substrates, such as digoxin, ciclosporin A, loperamide, domperidone and ondansetron, HIV protease inhibitors (indinavir, saquinavir, nelfinavir), or paclitaxel was also shown. These accumulated in the brains of knockout mice up to 35 or 40 times more than in WT mice, thus, clearly demonstrating the role of P-gp as a gatekeeper at the luminal side of the BBB.

The transporter BCRP appears to have a more moderate function in transport of drugs, such as imatinib and mitoxantrone. Other studies have, however, showed the BCRP acts synergistically with P-gp and limits the brain penetration of topotecan and lapatinib. In P-gp deficiency, BCRP expression at the BBB increases, resulting in a greater export of its substrates, like mitoxantrone or prazosin. Moreover, the dual localization of MRP4 - expressed at both the apical membrane of BBB endothelial cells and at the basolateral membrane of epithelial cells at the blood-CSF barrier - enables clearance of MRP4 substrates from both the CSF and the brain, as shown for topotecan.

In the placenta, the P-gp expressed protects the fetus from potential teratogenic compounds, and from many drugs including digoxin, saquinavir and paclitaxel by exporting them into the maternal blood. Similarly, BCRP, expressed in placental syncytiotrophoblasts, limits the foetal absorption of topotecan.

Fig. 3. Tissue distribution of the ABC transporters P-gp, BCRP, and MRP1-3. Blue circles represent tight junctions. Although, BCRP and Pgp are the main ABC transporters expressed at the human BBB, MRPs are also detected but with a lower expression and their functional role at the BBB still needs to be clearly determined.

Bioavailability defines the fractional extent to which a dose of a drug reaches its target site. All three ABC-transporters are present in both the intestine and the liver and can, therefore, decrease oral bioavailability through two mechanisms: first, by direct inhibition of the uptake from the gut, and second, by rapid elimination of the xenobiotics and their metabolites in the bile. Absence of intestinal ABC transporters results in an increased xenobiotic uptake into the portal vein increasing the levels in the systemic blood and organs. If hepatic elimination fails the hepatic tissue levels increase or, when basolateral resecretion from the hepatocyte into the systemic circulation occurs, the systemic blood and organ levels increase.

More specifically, the abundance of P-gp on the intestinal epithelium restricts the absorption of substrates from the intestinal lumen into the bloodstream. Many drugs, such as the anticancer drug paclitaxel, are P-gp substrates and the bioavailability of many orally administered drugs is restricted by P-gp. In one particular experiment, knockout mice deficient in P-gp developed an inflammation of the large intestine which was similar to the inflammatory bowel disease seen in humans. This inflammation depended on the presence of intestinal bacteria and it seemed likely that the inflammation was caused from toxins produced by the intestinal bacteria, which would have normally been kept out of the intestines by the P-gp activity.

Fig. 4. Determinants of oral bioavailability. Although ABC transporters are only present in the intestine and liver, they influence almost all parameters of bioavailability. The orange arrows indicate changes in the absence of transporters.

Oral bioavailability is very important for pharmacotherapy purposes as oral drug administration is highly preferred due to its cheapness, relative safety, and administration ease. If a drug has low oral bioavailability, the plasma level of the drug may not reach sufficient levels to have a therapeutic effect. Moreover, low drug bioavailability is usually connected to variable drug uptake, and this is unfavourable if a drug has a narrow therapeutic concentration window. By limiting drugs in achieving therapeutic levels, the ABC transporters complicate drug discovery.

A tremendous amount of knowledge on the pharmacological impact of ABC transporters has been gained. These transporters were once considered to have relevance only in making cancer cells resistant to anticancer drugs. It is now obvious that the ability to interfere with the activity of the ABC transporters can have some very important pharmaco-therapeutic benefits and the challenge for the future lies in forming the complete picture of the impact of all these transporters and their cross-interactions.

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