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Drug Transporters Volume 2
Recent Advances and Emerging Technologies
By Glynis Nicholls, Kuresh Youdim The Royal Society of Chemistry
Copyright © 2016 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-78262-866-8
CHAPTER 1
Emerging Transporter Science and Challenges for the Future
GLYNIS NICHOLLS AND KURESH YOUDIM
Introduction
The role of membrane transporters in drug pharmacokinetics (PK) is now well established, even though in many respects it is still an emerging science, with our knowledge base continuing to expand. To date, the primary focus has been on the development of methods and models that identify the specific transporters involved in clinically relevant drug-drug interactions (DDIs), to elucidate their role in the disposition of a specific drug compound, and to find specific substrates and inhibitors that can be used as transporter probes. Due to their importance in absorption, distribution, metabolism and excretion (ADME), such work has concentrated on the key ADME organs of the liver, kidney, gastrointestinal tract, blood-brain barrier (BBB) and latterly the lung, as described in Volume 1 of this book. More recently, research has expanded into other, related areas to further understand the factors that may affect transporter-mediated interactions, including the impact of age or disease on transporter expression, their regulation (Chapter 2) and how enzymes (Chapter 4) and pharmacogenomics (Chapter 5) can impact the activity of specific transporters. Additionally, other organs and tissues are now being considered, to further understand how transporters in other parts of the body may influence the PK of administered drugs. Efforts are also being made to improve and expand the technology used for measuring and quantitating transporter-mediated interactions. This chapter is intended to give a brief overview of some of these areas, although it will no doubt soon be superseded by further advances as the science continues to evolve.
1.2 Membrane Transporters of Emerging Importance
Currently, the major focus for transporter based interaction studies within pharmaceutical companies is concerned with those transporters cited in the US Food and Drug Administration (FDA) 2012 DDI guidance document, i.e. P-glycoprotein [P-gp; multidrug resistance protein 1 (MDR1), ABCB1 gene family], breast cancer resistance protein (BCRP; ABCG2), the organic anion transporting polypeptides (OATPs) OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3), the organic anion transporters (OATs) OAT1 (SLC22A6) and OAT3 (SLC22A8), and organic cation transporter 2 (OCT2; SLC22A2). Consideration may also be given to OCT1 (SLC22A1) and the bile salt export pump (BSEP; ABCB11), as stated in the European 2013 guidance and latterly also to the multidrug and toxin extrusion (MATE) pumps MATE1/MATE2-K (SLC47A1/A2), as indicated in the Japanese 2014 guidance. Increasingly, however, as science progresses and new types of drugs and drug targets are being considered, the potential clinical importance of other transporters and gene families is becoming recognised. Recent commentaries from the International Transporter Consortium (ITC) give an excellent overview of this area and readers are referred to these papers for more detailed information. Its recommendations on the emerging transporters that should or could be considered during development of new molecular entities (NMEs) include:
• MATEs — due to their involvement in organic cation efflux, particularly in the kidney where renal DDIs can occur. Investigations are recommended for all NMEs where active renal secretion is an important route of elimination.
• Multidrug resistance associated proteins (MRPs; ABCC), which have a role as both drug and conjugate efflux pumps, with specific mention of investigation of MRP2 (ABCC2) inhibition if drug-induced hyperbilirubinaemia is observed in patients or preclinical species. MRP2, MRP3 (ABCC3) and MRP4 (ABCC4) should also be considered if glucuronate or glutathione drug conjugates are formed.
• BSEP — since impairment of function (by inhibition or deficiency) can cause cholestasis and liver injury, the investigation of BSEP inhibition should be considered if cholestasis is observed in clinical trials or safety studies.
In addition, the importance of considering other transporters for specific drug classes has been highlighted in the ITC commentaries, with mention of nucleoside drugs that may be transported by concentrative nucleoside transporters (CNTs; SLC28A) or equilibrative nucleoside transporters (ENTs; SLC29A), together with amino acid-type drugs and some antibiotics that may be transported by the peptide transporter 1 (PepT1; SLC15A1), as well as peptides via PepT2 (SLC15A2).
Equally, as knowledge expands, other transporters may also be found to be important in the disposition and/or DDIs of specific drugs or drug classes (for example in organs where membrane transporter expression is still only partly known, as described in Section 1.3). Potential transporters of interest include OATP1A2 (SLCO1A2), urate transporter 1 (URAT1; SLC22A12) or glucose transporter 9 (GLUT9; SLC2A6), and the organic solute transporters α and β (OSTα/β; SLC51A/B), amongst others.
1.3 Membrane Transporters in Less-studied Organs and Tissues
This section outlines the current status of transporter knowledge in organs and tissues that are not routinely considered in drug development, although the list is not exhaustive. For example, other organs and tissues that have received some attention in recent years include those of the muscle, breast and testis.
1.3.1 Placenta
The placenta is one of the less well-understood organs of the body, despite its importance to the growing foetus. During pregnancy, it supplies the foetus with nutrients, hormones and other endogenous factors, provides gaseous exchange, removes waste products, and also acts as a barrier to limit exposure to toxins and xenobiotics present within the maternal circulation. Its structure and gene expression change during pregnancy, being regulated by several nuclear receptors and ligand-activated transcription factors. From a drug development perspective, it is important to understand the mechanisms that regulate drug penetration, both to prevent damage to the foetus if a drug is present in the maternal circulation or alternatively to provide treatment to the developing embryo.
There is now growing evidence that the placental barrier function may be dependent, to some extent, on the presence of transporters. Several ATP binding cassette (ABC) efflux transporters are known to be expressed, including P-gp, BCRP and some MRPs. Several members of the solute carrier (SLC) gene super family are also known to be present, although their clinical significance in the placenta has not yet been evaluated. Transporter expression, controlled by maternal hormones as well as other factors, can vary with gestational age, as demonstrated for P-gp and MRP2, and in certain conditions such as inflammation or cholestasis. In addition, gene expression is subject to intra- and inter-individual variability due to transcriptional regulation and/or transporter polymorphisms. It can therefore be difficult to model the human placenta, although various in vitro, in vivo and in situ models have become available. However, the potential for species differences in transporter expression and function needs to be considered when extrapolating findings in animal models to humans.
As drug therapy (and polypharmacy) during pregnancy may be unavoidable, it is important to understand the transport mechanisms of drugs across the placental barrier. The first studies in this area focused on P-gp, with a study on pregnant control and CF-1 mice (naturally deficient in Mdr1a) exposed to the pesticide avermectin showing a protective role for P-gp. A more recent study using positron emission tomography (PET) in non-human primates was able to demonstrate that co-administration of the P-gp inhibitor cyclosporine A with the P-gp substrate [11C]-verapamil led to a modest increase in drug transport across the placental barrier. Dually-perfused human placenta models have also been used to demonstrate the barrier properties of P-gp, for example using the P-gp substrate saquinavir in the presence and absence of the inhibitor valspodar.
Relatively few studies have been performed for other transporters, apart from BCRP. The anti-diabetic drug glyburide, routinely used for gestational diabetes, has been used to study the barrier function of BCRP in the placenta, with a study in Bcrp-/- knockout mice suggesting that the transporter limits its entry into the placenta. A more recent study using in vitro and rat in situ methods determined the transport characteristics of tenofovir disoproxil fumarate (the prodrug of the HIV drug tenofovir) across the placenta, suggesting that it was effluxed back into the maternal circulation by P-gp and BCRP, but not MRP2. This area is covered more extensively in several comprehensive reviews.
1.3.2 Retina
The treatment of retinal disorders, which can result in severe vision loss in some instances, is challenging because of the presence of the blood-ocular barrier, which regulates entry into the retina. Similar to the BBB, it has tight junctions to restrict paracellular transport, helps to maintain neuroretinal homeostasis, and protects the intraocular environment from toxins and xenobiotics. The blood–ocular barrier consists of the blood–retinal barrier (BRB) and the blood–aqueous barrier (BAB), which maintains aqueous humour conditions. The BRB, which has inner and outer parts, separates the retina from the systemic blood circulation and has various transporters within the luminal (blood side) and abluminal (retinal side) membranes to both efficiently supply nutrients to the cells, and restrict or remove other compounds.
There are a variety of reported influx transporters capable of transporting essential nutrients into the retina, including GLUT1 (SLC2A1), the amino acid transporter LAT1 (SLC7A5), ENT2 (SLC29A2) and monocarboxylate transporter 1 (MCT1; SLC16A1), with ENT2 being suggested as a potential route for nucleoside drug delivery to the eye. Expression of SLC and SLCO transporters also occurs at the BRB and BAB, although information is more limited and appears to be mostly from the rat. However, a recent study using whole human eyes (orbs) found mRNA expression of several SLC transporters in the retina, including OCT2, OCT3 (SLC22A3), organic cation/ carnitine transporter 2 (OCTN2; SLC22A5), OATP1A2 (SLCO1A2) and OATP1B3. Expression varied between different regions of the eye. The same study also found expression of the efflux transporters P-gp, BCRP, MRP1 (ABCC1), MRP4 (ABCC4), MRP5 (ABCC5) and BSEP, with a separate study demonstrating protein expression of P-gp, MRP1, MRP2 and MRP6 (ABCC6). The presence and functionality of P-gp in the BRB has been confirmed using animal models, although some species differences were observed. However, information on BCRP is more limited, with a recent study in mice suggesting that its importance in the BRB was less than in the BBB.
The use of conventional treatments such as eye drops, ointments, etc. to treat retinal disorders remains limited because of poor penetration and bioavailability, and other approaches for systemic delivery of drugs are being considered. These target the transporters in the blood-ocular barrier by, for example, inhibiting efflux transporters, e.g. inhibition of MRPs with probenecid, or using drugs that are substrates of amino acid and peptide uptake transporters. A recent study in rats of pravastatin, a compound used to treat human diabetic retinopathy, suggested that multiple transporters may be involved in its retinal transport, with sufficient influx of pravastatin by rodent OATPs to allow a pharmacological effect prior to its efflux by ABC transporters.
However, this type of drug delivery faces the same issues as drug entry into the brain in terms of being able to penetrate the blood-ocular barrier and gain access. Consideration of the wider impact of this approach also needs to be assessed, since drugs able to enter the BRB will most likely also gain access into the central nervous system (CNS) via the BBB.
1.3.3 Heart
Although several transporters are known to be present in heart tissue, there is only limited information available in the literature in terms of their importance for DDIs. Accumulation of digoxin in the presence of verapamil has been observed in perfused rat hearts, likely caused by inhibition of P-gp. P-gp is also known to be important in the development and progression of cardiovascular disease, with the presence of one particular polymorphism (ABCB1 C3435T) being correlated with an increased risk of myocardial infarction and coronary heart disease. BCRP has been found in the endothelial cells of heart blood vessels, with a recent study in rats indicating that the P-gp/BCRP inhibitor cilostazol increased donepezil accumulation in heart tissue, suggesting that, at the concentrations used, this was due to a BCRP-mediated DDI.
Expression of uptake transporters relevant to drug development has also been reported, including OCT1-3, OCTNs and OATPs. OCTN2 is expressed throughout the myocardium and in the blood vessels, mediating the transport of carnitine and some organic cations, with loss of function mutations leading to primary systemic carnitine deficiency and subsequent cardiomyopathy and skeletal muscle weakness. Thus, inhibition of OCTN2 by administered drugs could lead to carnitine deficiency. The role of OATP2B1 (SLCO2B1) has been illustrated using samples of human heart tissue, with its likely functional role in the uptake of the antidiabetic drug glyburide suggested using whole-body PET imaging of baboons. A further approach using gene therapy has demonstrated the functionality of OCT1: by enhancing expression of uptake transporters (OCT1) using adenoviral constructs, delivery of paclitaxel into heart muscle cells was increased, suggesting that this type of approach could improve drug delivery to the heart.
1.3.4 Skin
The skin can be used to administer both local and systemic drugs, with drug penetration depending not only on the physicochemical properties of the drug but also the method of delivery and excipients used. As skin may be exposed to various environmental xenobiotics, as well as exhibiting adverse reactions to some topically administered drugs, there has been ongoing research into the presence of both metabolising enzymes and membrane transporters.
Human skin keratinocytes have been shown to contain a range of influx and efflux transporters capable of drug transport, although expression is more limited than in other organs. Recent studies with total human skin using reverse transcription polymerase chain reaction (RT-PCR) techniques have shown expression of several ABC transporters, including members of the ABCA, ABCB (e.g. MDR3, ABCB4), ABCC (e.g. MRP1, MRP4 and MRP5), ABCD, ABCE and ABCG sub-families, with P-gp and BCRP being barely detectable. Of these, one paper concluded that MRP1 was of particular importance for drug uptake in skin, with expression being 15-fold greater in skin compared with hepatocytes. Using a functional analysis in skin, the authors found that the MRP inhibitors verapamil and MK571 decreased the skin absorption of rhodamine 123, vinblastine and LTC4.
A subsequent RT-PCR analysis for the SLC gene super family indicated expression of various transporters, including OATP2B1, OATP4A1 (SLCO4A1), MCT1 and MCT2 (SLC16A7), with only slight expression of OCT and OAT family members such as OCT2, OCT3, OCTN2 and OAT3. Expression was found to be very variable between samples for both SLC and ABC transporters, which may be one reason for the observed inter-individual differences in skin therapy and the potential for drug-induced skin diseases in some patients.
1.4 Organotypic In vitro Technologies
There has also been a growing interest within the pharmaceutical industry to adopt new approaches that could potentially help address some of the challenges associated with the development of NMEs, with drug failure in clinical trials often being attributed to the poor predictive power of existing preclinical in vivo and in vitro models. With respect to the latter, current approaches still employ conventional two-dimensional (2D) cell cultures, a system that was developed almost a century ago. However, despite their demonstrated value in biomedical research, these systems cannot reproduce the tissue-specific, differentiated functions and interdependencies of the many cell types that are found in vivo, nor can they accurately predict in vivo tissue functions and drug activities. In light of these limitations, interest has focused on more complex 2D models, incorporating multiple cell types or involving cell patterning, and in three-dimensional (3D) models, which better represent the spatial and chemical complexity of living tissues. Some emerging examples of novel approaches currently being investigated are presented in the following sections. It should be noted however that, for all of these systems, studies into their transporter expression and function are still required.
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Excerpted from Drug Transporters Volume 2 by Glynis Nicholls, Kuresh Youdim. Copyright © 2016 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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