Membrane transporters in drug development
Section 1: Overview of transporters
A limited number of transporters, including several uptake transporters from the SLC superfamily6 and some ATP-dependent efflux pumps from the ABC superfamily2, have been given priority in the context of this article. Their selection was based on practical considerations and on clinical evidence that these transport proteins influence, to different degrees, drug disposition and/or side effects. FIGURE 1 and TABLES 1,,22 describe the selected transporters as well as other can be explained, in part or in full, by modulation of important transporters. Note that clinical data documenting the importance of these transport proteins with respect to drug disposition and/or toxicity continues to emerge. TABLES 1,,22 include clinically relevant information with respect to DDIs and genetic polymorphisms. Examples of clinically relevant DDIs that can be explained, in part or in full, by modulation of transporter activity are compiled in TABLE 3. Below, we present an overview of P-gp, breast cancer resistance protein (BCRP; also known as ABCG2); organic cation transporters (OCTs) and organic anion transporters (OATs); and organic anion transporting polypeptides (OATPs). For ease of reading we refer to MDR1/P-gp as P-gp throughout this report. Although evidence is available demonstrating that various members of the multidrug resistance protein (MRP) and the multidrug and toxin extrusion transporter (MATE) families are involved in drug disposition, as mentioned in various sections, because of the need to limit the scope of this manuscript, they are not discussed in detail here. Because of the extensive body of literature obtained over many years on the interaction of drugs with P-gp, as well as the vast experience in studying this transporter in the industry, P-gp will be described in more detail than other transporters. Many issues discussed for P-gp are relevant for other transporters. In this article capitalized letters are used for human genes and proteins (for example, SLCO and OATP), whereas only the initial letter is capitalized for rodent genes and proteins (for example, Abcg2 and Bcrp). The standard human gene nomenclature is listed in TABLES 1,,2,2, but, in general, the transport proteins or gene products responsible for transport function are referred to by the names commonly used in the field.
Table 1
Transporter/ alias (Gene) | Selected substrates | Selected inhibitors | Organs/cells | Comments |
---|---|---|---|---|
OATP1B1/O ATP-C, OATP2, LST- 1 (SLCO1B1) | Bromosulphophthalein, oestrone-3-sulphate, oestradiol-17β-glucuronide, statins, repaglinide, valsartan, olmesartan, bilirubin glucuronide, bilirubin, bile acids | Saquinavir, ritonavir, lopinavir, rifampicin, cyclosporine* | Hepatocytes (sinusoidal) | • Has a role in disposition and excretion • Has clinically relevant polymorphisms • Has a role in clinical drug–drug interactions |
OATP1B3/O ATP-8 (SLCO1B3) | Bromosulphophthalein, cholecysto kinin 8, statins, digoxin, fexofenadine, telmisartan glucuronide, telmisartan, valsartan, olmesartan, oestradiol-17- β-glucuronide, bile acids | Rifampicin, cyclosporine, ritonavir, lopinavir* | Hepatocytes (sinusoidal) | • Has a role in disposition and excretion |
OAT1 (SLC22A6) | Para-aminohippurate, adefovir, cidofovir, zidovudine, lamivudine, zalcitabine, acyclovir, tenofovir, ciprofloxacin, methotrexate* | Probenecid, novobiocin | Kidney proximal tubule, placenta | • Has a role in disposition and excretion Has a role in clinical drug–drug interactions |
OAT3 (SLC22A8) | Oestrone-3-sulphate, non- steroidal anti-inflammatory drugs, cefaclor, ceftizoxime, furosemide, bumetanide* | Probenecid, novobiocin | Kidney proximal tubule, choroid plexus, blood–brain barrier | • Has a role in disposition and excretion • Has a role in clinical drug–drug interactions |
OCT2 (SLC22A2) | N-Methylpyridinium, tetraethylammonium, metformin, pindolol, procainamide, ranitidine amantadine, amiloride, oxaliplatin, varenicline* | Cimetidine, pilsicainide, cetirizine, testosterone, quinidine | Kidney proximal tubule, neurons | • Has a role in disposition and excretion • Has clinically relevant genetic polymorphisms • Has a role in clinical drug–drug interactions |
OATP1A2/O ATP-A (SLCO1A2) | Oestrone-3-sulphate, dehydroepiandrosterone sulphate, fexofenadine, bile salts, methotrexate, bromosulphophthalein, ouabain, digoxin, levofloxacin, statins* | Naringin, ritonavir, lopinavir, saquinavir, rifampicin* | Brain capillaries endothelia, cholangiocyte s, distal nephron | • Has role in disposition and excretion |
OATP2B1/O ATP-B (SLCO2B1) | Oestrone-3-sulphate, bromosulphophthalein, taurocholate, statins, fexofenadine, glyburide, taurocholate | Rifampicin, cyclosporine* | Hepatocytes (sinusoidal), endothelia | • Has a role in disposition and excretion • Has a role in clinical drug–drug interactions |
OCT1 (SLC22A1) | Tetraethylammonium, N- methylpyridinium, metformin, oxaliplatin | Quinine, quinidine, disopyramide | Hepatocytes (sinusoidal), intestinal enterocytes | • Has a role in disposition and excretion • Has clinically relevant genetic polymorphisms • Has a role in clinical drug–drug interactions |
PEPT1 (SLC15A1) | Glycylsarcosine, cephalexin, cefadroxil, bestatin, valacyclovir, enalapril, aminolevulinic acid, captopril, dipeptides, tripeptides | Glycyl-proline | Intestinal enterocytes, kidney proximal tubule | • Has a role in absorption, disposition and excretion • Has a role in clinical drug–drug interactions |
PEPT2 (SLC15A2) | Glycylsarcosine, cephalexin, cefadroxil, bestatin, valacyclovir, enalapril, aminolevulinic acid, captopril, dipeptides, tripeptides | Zofenopril, fosinopril | Kidney proximal tubule, choroid plexus, lung | • Has a role in excretion MATE1 |
(SLC47A1) | Metformin, N- methylpyridinium, tetraethylammonium | Quinidine, cimetidine, procainamide | Kidney proximal tubule, liver (canalicular membrane), skeletal muscle | • Has a role in disposition and excretion • Has a role in clinical drug–drug interactions |
MATE2-K (SLC47A2) | Metformin, N- methylpyridinium, tetraethylammonium | Cimetidine, quinidine, pramipexole | Kidney proximal tubule | • Has a role in disposition and excretion |
Table 2
Transporter/alias (Gene) | Selected substrates | Selected inhibitors | Organs/cells | Comments |
---|---|---|---|---|
MDR1/P-gp, ABCB1 (ABCB1) | Digoxin, loperamide, berberine, irinotecan, doxorubicin, vinblastine, paclitaxel, fexofenadine | Cyclosporine, quinidine, tariquidar, verapamil | Intestinal enterocytes, kidney proximal tubule, hepatocytes (canalicular), brain endothelia |
|
BCRP/MXR (ABCG2) | Mitoxantrone, methotrexate, topotecan, imatinib, irinotecan, statins, sulphate conjugates, porphyrins | Oestrone-17β-oestradiol, fumitre - morgin C | Intestinal enterocytes, hepatocytes (canalicular), kidney proximal tubule, brain endothelia, placenta, stem cells, mammary glands (lactating) |
|
BSEP/SPGP, cBAT, ABCB11 (ABCB11) | Taurocholic acid, pravastatin, bile acids | Cyclosporin A, rifampicin, glibenclamide | Hepatocytes (canalicular) |
|
MRP2/ABCC2, cMOAT (ABCC2) | Glutathione and glucuronide conjugates, methotrexate, etoposide, mitoxantrone, valsartan, olmesartan, glucuronidated SN-38 | Cyclosporine, delaviridine, efavirenz, emtricitabine | Hepatocytes (canalicular), kidney (proximal tubule, luminal), enterocytes (luminal) |
|
MRP3/ABCC3 (ABCC3) | Oestradiol-17β-glucuronide, methotrexate, fexofenadine, glucuronate conjugates | Delaviridine, efavirenz, emtricitabine | Hepatocytes (sinusoidal), intestinal enterocytes (basolateral) |
|
MRP4/ABCC4 (ABCC4) | Adefovir, tenofovir, cyclic AMP, dehydroepiandrostero ne sulphate, methotrexate, topotecan, furosemide, cyclic GMP, bile acids plus glutathione | Celecoxib, diclofenac | Kidney proximal tubule (luminal), choroid plexus, hepatocytes (sinusoidal), platelets |
|
MDR3/ABCB4 (ABCB4) | Phosphatidylcholine, paclitaxel, digoxin, vinblastine | Verapamil, cyclosporine | Hepatocytes (canalicular) |
|
ABC, ATP-binding cassette.
Table 3
Implicated transporter* | Interacting drug | Affected drug | Clinical pharmacokinetic impact on affected drug‡ |
---|---|---|---|
Organic anion transporting polypeptides | Cyclosporine | Pravastatin | AUC ↑890% and Cmax ↑678%102,204 |
Cyclosporine | Rosuvastatin | AUC ↑610%205 | |
Cyclosporine | Pitavastatin | AUC ↑ 360% and Cmax ↑560%206 | |
Rifampicin (single dose) | Glyburide | AUC ↑125%207 | |
Rifampicin (single dose) | Bosentan | Ctrough ↑ 500%208 | |
Lopinavir/ritonavir | Bosentan | Day 4: Ctrough ↑ 4,700%208; day 10: Ctrough ↑ 400%208 | |
Lopinavir/ritonavir | Rosuvastatin | AUC ↑107% and Cmax ↑365%209 | |
Organic anion transporters | Probenecid | Cidofovir | CLr ↓32%210,211 |
Probenecid | Furosemide | CLr ↓66%210 | |
Probenecid | Acyclovir | CLr ↓32% and AUC ↑40%210,212 | |
Organic cation transporters | Cimetidine | Metformin | AUC ↑50% and CLr ↓ 27%213,214 |
Cimetidine | Pindolol | CLr ↓~34%215 | |
Cimetidine | Varenicline | AUC ↑29%216 | |
Cimetidine | Pilsicainide | AUC ↑33%, CLr ↓28%217 | |
Cimetidine | Pilsicainide | CLr ↓41%218 | |
Cimetidine | Dofetilide | CLr ↓33%219 | |
P-glycoprotein | Quinidine | Digoxin | CLr ↓34–48%220,221 |
Ritonavir | Digoxin | AUC ↑86%222 | |
Dronedarone | Digoxin | AUC ↑157% and Cmax ↑75%223 | |
Ranolazine | Digoxin | AUC ↑60% and Cmax ↑46%224 | |
Breast cancer resistance protein | GF120918 | Topotecan | AUC ↑143%225 |
P-glycoprotein
General description
P-gp mediates the ATP-dependent export of drugs from cells. It is expressed in the luminal membrane of the small intestine and blood–brain barrier, and in the apical membranes of excretory cells such as hepatocytes and kidney proximal tubule epithelia. P-gp has an important role in limiting entry of various drugs into the central nervous system. In addition, it also plays a part in the intestinal absorption and in the biliary and urinary excretion of drugs. The level of expression and functionality of P-gp can be modulated by inhibition and induction, which can affect the pharmacokinetics, efficacy, safety or tissue levels of P-gp substrates5,17–21.
Substrate and inhibitor selectivity
Initially discovered as a result of its interaction with multiple anticancer drugs, P-gp is responsible for the efflux across biological membranes of a broad range of therapeutic drugs. Recently, a high-resolution structure of the mouse P-gp has been described, which revealed distinct binding sites for drugs22. A select number of substrates and inhibitors of human P-gp are shown in TABLE 2. P-gp substrates are generally hydrophobic molecules, of which many are cationic. Multiple binding sites for substrates and inhibitors on P-gp have been identified using site-directed mutagenesis23–25.
Methodology for evaluating function
Cell lines that express P-gp and inside-out membrane vesicles prepared from these cell lines can be used to determine whether a drug is a P-gp substrate or inhibitor. As P-gp is localized in the apical plasma membrane in polarized cell monolayers, a high efflux ratio of basal-to-apical to apical-to-basal (the so called B-A/A-B ratio) indicates a potentially significant role for P-gp in transporting drugs across cell monolayers. A high efflux ratio in vitro has been shown to correlate well with studies that have demonstrated a role for P-gp in drug penetration to the central nervous system in mice26,27. By contrast, a high efflux ratio does not always translate into poor oral absorption. The involvement of P-gp in absorption of a drug is more pronounced if the drug has a poor apparent permeability coefficient (Papp), or in cases in which there is interplay between metabolism and efflux.
Mice deficient in Mdr1a or Mdr1a/b are widely used as powerful tools for assessing the role of P-gp in vivo9. Knockout mice can be used as a reference for complete inhibition of P-gp, and represent the ‘worst case’ scenario. Sasongko et al.28 demonstrated that the AUCbrain/ AUCblood ratio of C-verapamil in humans increased by less than twofold (88 ± 20%) in the presence of a high intravenous dose of the P-gp inhibitor cyclosporine. By contrast, an almost eightfold (770%) increase was observed in the Mdr1a/b knockout mice compared with control mice. These data can be interpreted in multiple ways, but illustrate the challenge in predicting human transporter-mediated DDIs based on preclinical animal data.
Clinical significance
The clinical significance of a P-gp inhibitor can be investigated in humans by assessing the P-gp-mediated clearance or exposure of a probe substrate in the presence of the inhibitor. Digoxin is transported by P-gp in vitro and should be considered in DDI studies with new molecular entities (NMEs) that are inhibitors of P-gp (TABLE 3, BOX 2). Current clinical data indicate that there are no consistent examples in which inhibition of P-gp in the blood–brain barrier resulted in adverse effects2931. It is therefore difficult to extrapolate the data obtained for P-gp inhibitors in knockout mice to humans to indicate the potential for a clinically significant DDI at the human blood–brain barrier31.
Findings from many studies on the effect of ABCB1 polymorphisms20 on P-gp substrates have not been consistently reproduced; therefore, routine application of ABCB1 polymorphism analysis to clinical studies is not warranted at this time. Studies with larger numbers of samples may be needed to clarify the role of ABCB1 polymorphisms in pharmacokinetics and pharmacodynamics32.
BCRP
General description
BCRP is a ‘half ABC transporter’ consisting of 655 amino acids and six transmembrane domains33. BCRP was identified originally as a determinant of multidrug resistance in cancer cell lines in vitro34,35. BCRP is expressed in the gastrointestinal tract, liver, kidney, brain endothelium, mammary tissue, testis and placenta. It has a role in limiting oral bioavailability and transport across the blood–brain barrier, blood– testis barrier and the maternal–fetal barrier of some selected substrates36,37. The physiological functions of BCRP include the extrusion of porphyrins from haematopoietic cells and hepatocytes, as well as the secretion of vitamin B2 (riboflavin) and possibly other vitamins (such as biotin and vitamin K) into breast milk36.
Substrate and inhibitor selectivity
BCRP actively extrudes a broad range of endogenous and exogenous substrates across biological membranes37. TABLE 2 lists selected substrates and inhibitors of human BCRP. High-speed screening and quantitative structure–activity relationship (QSAR) analysis methods suggest that one amine bonded to one carbon of a heterocyclic ring is an important component for drug interactions with BCRP. In addition, fused heterocyclic ring(s) and two substituents on a carbocyclic ring of the fused heterocyclic ring(s) are also important chemical moieties for the interaction with BCRP38–40. Many protein kinase inhibitors such as imatinib (Gleevec; Novartis) carry such structural components. Substrates of BCRP include pitavastatin (Livalo; Kowa Pharmaceuticals America)41 and phytoestrogens, such as genistein, daidzein and coumestrol42.
Methodology for evaluating function
Polarized and non-polarized cell lines expressing BCRP are used to assay BCRP-mediated transport and inhibition43. In addition, membrane vesicles containing BCRP that constitutively express the transporter can also be used43. Prazosin and cimetidine can be used as a positive control in cell lines, whereas several relatively polar, hydrophilic substrates (for example, methotrexate, oestrone 3-sulphate and sulphasalazine) can be used as controls in vesicular transport assays. Since the initial publications of the Bcrpknockout mouse36,37, drug disposition studies in Abcg2 knockout mice have delineated an important role for intestinal Bcrp as a rate-determining barrier for the oral bioavailability of several drugs. These include topotecan (hycamtin; GlaxoSmithKline)44 and sulphasalazine (Salazopyrin; Pharmacia), which had 10-fold to 110-fold increase in relative AUC45. BCRP is also a moderate determinant of the bioavailability of nitrofurantoin46, some fluoroquinolones47 and imatinib48.
Clinical significance
Recent clinical studies have demonstrated that subjects with reduced BCRP expression levels, correlating with the Q141K variant, are at increased risk for gefitinib (Iressa; AstraZeneca)-induced diarrhoea and altered pharmacokinetics of 9-aminocamptothecin, diflomotecan, irinotecan (Camptosar; Pfizer), rosuvastatin (Crestor; AstraZeneca), sulphasalazine and topotecan49–55. Inter-individual differences in BCRP function probably contribute to variable bioavailability, exposure (AUC and Cmax), and pharmacological response of drugs that are BCRP substrates. The most significant clinical effects are likely to be for drugs that have a low bioavailability and have a narrow therapeutic index.
OCTs and OATs
General description
A distinct family of proteins within the SLC superfamily is encoded by 22 genes of the human SLC22A family, and includes the electrogenic OCTs (isoforms 1–3) and the oATs (significant isoforms in humans include oAT1–4 and 7, and URAT1)6,7,56,57. The genes encoding OCT1–3 and OATs encode proteins that are 542–556 amino acids long with 12 predicted transmembrane-spanning domains57. The tissue distribution and localization of OCTs and OATs are summarized in TABLE 1 and FIG. 1. There are several published reviews of the molecular characteristics, expression and function of OCTs7,57–60 and OATs57,61–63.
Substrate and inhibitor selectivity
TABLe 1 summarizes various compounds that interact with human OCTs and OATs. OCTs transport relatively hydrophilic, low molecular mass organic cations. Properties of inhibitors of OCT1 and OCT2 have been identified and include a net positive charge and high lipophilicity59,64. OAT1, OAT3 and OAT4 support exchange of intracellular 2-oxoglutarate for extracellular substrate62. OAT1 and OAT3 mediate the basolateral entry step in renal secretion of many organic anions, and have distinct selectivities for different structural classes of type I organic anions. That is, monovalent (or selected divalent) anions that are less than 500 daltons58; although OAT3 can also transport some positively charged drugs such as cimetidine.
Methodology for evaluating function
In vitro assays that are commonly used to characterize OCT function include Xenopus laevis oocytes, membrane vesicles and cell lines derived from various tissues including proximal tubule (Caki-1), placenta (BeWo) and colon carcinoma (Caco-2) cells65–69. These are in addition to various human embryonic kidney (HEK293) cell lines expressing recombinant OCTs68,73,83. Functional assessment of OATs generally uses heterologous expression in cultured cells or X. laevis oocytes, and such systems have been used to develop structure–activity relationships for the interaction of OATs70,71. Studies performed in Oct knockout mice have demonstrated the pharmacokinetic, pharmacological and physiological relevance of OCTs in organic cation disposition and activity8,72–74. Studies using Oat1 and Oat3 knockout mice have also allowed an initial assessment of the influence of each transporter on the renal handling of selected compounds, and have led to the identification of endogenous OAT substrates75,76.
Clinical significance
Recent studies suggest that genetic variation in OCT1 may be a significant determinant of inter-individual variability in the disposition and response to cationic drug substrates, particularly metformin73,77. However, such studies have been controversial78. Recent studies suggest that the activity of OCT1 is positively associated with the degree to which patients with chronic myeloid leukaemia respond to imatinib79,80. In Chinese and Korean populations, a common OCT2 variant (A270S) has been associated with a significant reduction in renal clearance of metformin81,82, but the effect of this variant may differ depending on the ethnic group83. By contrast, genetic variants in the OATs have not been associated with changes in drug disposition. DDIs with the OATs and the OCTs can occur, and may result in reduced renal clearance (TABLE 3). Creatinine, which is often used to assess filtration clearance, undergoes renal tubular secretion, which could be inhibited by inhibitors of OCT2. Therefore, other methods should be used to assess filtration clearance of NMEs in DDI studies involving OCT2 inhibitors. DDIs involving the OATs and the OCTs are discussed in more detail in Section 3.
OATPs
General description
The OATPs (SLCOs) represent a superfamily of important membrane transport proteins that mediate the sodium-independent transport of a diverse range of amphiphilic organic compounds. These include bile acids, steroid conjugates, thyroid hormones, anionic peptides, numerous drugs and other xenobiotic substances84 (TABLE 1). The general predicted OATP structure consists of proteins with 12 transmembrane domains85. The mechanism of transport appears to consist of anion exchange by coupling the cellular uptake of substrate with the efflux of endogenous intracellular substances such as bicarbonate in a process that seems to be electroneutral85–87. OATP1B1 was cloned by several groups88–91 and its localization along with other OATPs is shown in FIG. 1. Interestingly, OATP family members are poorly conserved evolutionarily and orthologues for human oATPs may not exist in rodents.
Substrate and inhibitor selectivity
OATP1B1 transports a broad range of compounds such as bile acids; sulphate and glucuronate conjugates; thyroid hormones; peptides; and drugs such as methotrexate and HMG-CoA reductase inhibitors (TABLE 1). Similar to OATP1B1, OATP1B3 also transports bile acids; monoglucuronosyl bilirubin; bromosulphophthalein; steroid conjugates; peptide deltorphin II92; the hepatotoxic cyclic peptide the thyroid hormones T3 and T4; leukotriene C4; and amanitin; and the cardiac glycosides digoxin and ouadrugs such as methotrexate and rifampicin86–91. However, bain92. It is also involved in the uptake of the angiotensin OATP1B3 also exhibits unique transport properties in II receptor antagonist telmisartan and its glucuronide that it is able to mediate the cellular uptake of the opioid conjugate91, and the selective uptake of the intestinal peptide cholecystokinin 8 (refs 9394). TABLE 1 lists additional details on the substrate specificity of OATP1B3, and the specificities of OATP1A2 and OATP2B1 are also noted.
Methodology for evaluating function
In vitro assessment of OATP transporter function has relied on a number of transient and stable heterologous expression systems84. These include X. laevis oocytes, recombinant virus, or stable cell lines expressing an individual or multiple transporters95. In addition, stable expression of OATP transporters in polarized cells such as MDCK-II has been reported, typically in combination with efflux transporters such as MRP2 (refs 9697). Isolated hepatocytes with OATP inhibitors can also be used to study OATP transport98,99. More recently, in vivo drug disposition profiles from Oatp1b2 knockout mouse models have been reported100,101 and may reflect, in part, the activity of both human OATP1B1 and OATP1B3. Additional studies are needed to more fully validate the utility of rodent models to predict human OATP-mediated drug disposition.
Clinical significance
The clinical relevance and DDIs related to OATPs (TABLe 3) have been noted only for certain OATPs; for example, OATP1B1 and OATP1B3 expressed primarily in the liver. DDIs involving the OATPs have focused primarily on OATP1B1. Inhibition of OATP1B1-mediated hepatic uptake appears to contribute to the significant increase in statin (for example, rosuvastatin) concentrations in blood after cyclosporine administration102,103 (TABLE 3). Because cyclosporine is an inhibitor of multiple transporters, the specific transporter involved in the clinical DDI cannot be ascertained; although OATP1B1 is a likely candidate. In addition, a series of functional polymorphisms of OATP1B1 have been characterized88. The frequencies of the SLCO1B1 388G allele in Caucasians, African Americans, and Asians is approximately 40%, 75% and 60%, respectively88,104–106. Another common single nucleotide polymorphism is 521T>C in codon 174, which has frequencies of approximately 15%, 2% and 15% in Caucasians, African Americans, and Asians, respectively88,104–106. The c.388G allele often occurs in a haplotype with the c.521C allele. These haplotypes — c.388G-c521T (*1B), c.388A-c.521C (*5) and c.388G-c.521C (*15) — have different activities and also occur at different frequencies in various ethnic groups. In addition, the promoter variants (−11187G>A and −10499A>C) may be in linkage disequilibrium with common coding region single nucleotide polymorphisms104.
Pharmacokinetic studies indicate that individuals with the SLCO1B1*5 or *15 haplotypes have increased exposure to statin drugs such as pravastatin106, pitavastatin107, simvastatin acid108, atorvastatin109 and rosuvastatin110. They also have increased exposure to other drugs such as repaglinide111, atrasentan112, irinotecan113 and ezetimibe (Zetia; Merck/Schering–Plough)114. The *5 haplotype is rare (with a frequency of 2% in Caucasians and is absent in individuals of other ancestries), whereas the *15 haplotype is more common (16% in Caucasians, 2% in sub-Saharan Africans and 9–12% in Asians)104. However, because of the large number of patients on statins, even less common variants may have an effect in many individuals. Compelling clinical evidence supporting an important role for SLCO1B1 polymorphisms has come from a genome-wide association study of simvastatin-induced myopathy115.
P-glycoprotein
General description
P-gp mediates the ATP-dependent export of drugs from cells. It is expressed in the luminal membrane of the small intestine and blood–brain barrier, and in the apical membranes of excretory cells such as hepatocytes and kidney proximal tubule epithelia. P-gp has an important role in limiting entry of various drugs into the central nervous system. In addition, it also plays a part in the intestinal absorption and in the biliary and urinary excretion of drugs. The level of expression and functionality of P-gp can be modulated by inhibition and induction, which can affect the pharmacokinetics, efficacy, safety or tissue levels of P-gp substrates5,17–21.
Substrate and inhibitor selectivity
Initially discovered as a result of its interaction with multiple anticancer drugs, P-gp is responsible for the efflux across biological membranes of a broad range of therapeutic drugs. Recently, a high-resolution structure of the mouse P-gp has been described, which revealed distinct binding sites for drugs22. A select number of substrates and inhibitors of human P-gp are shown in TABLE 2. P-gp substrates are generally hydrophobic molecules, of which many are cationic. Multiple binding sites for substrates and inhibitors on P-gp have been identified using site-directed mutagenesis23–25.
Methodology for evaluating function
Cell lines that express P-gp and inside-out membrane vesicles prepared from these cell lines can be used to determine whether a drug is a P-gp substrate or inhibitor. As P-gp is localized in the apical plasma membrane in polarized cell monolayers, a high efflux ratio of basal-to-apical to apical-to-basal (the so called B-A/A-B ratio) indicates a potentially significant role for P-gp in transporting drugs across cell monolayers. A high efflux ratio in vitro has been shown to correlate well with studies that have demonstrated a role for P-gp in drug penetration to the central nervous system in mice26,27. By contrast, a high efflux ratio does not always translate into poor oral absorption. The involvement of P-gp in absorption of a drug is more pronounced if the drug has a poor apparent permeability coefficient (Papp), or in cases in which there is interplay between metabolism and efflux.
Mice deficient in Mdr1a or Mdr1a/b are widely used as powerful tools for assessing the role of P-gp in vivo9. Knockout mice can be used as a reference for complete inhibition of P-gp, and represent the ‘worst case’ scenario. Sasongko et al.28 demonstrated that the AUCbrain/ AUCblood ratio of C-verapamil in humans increased by less than twofold (88 ± 20%) in the presence of a high intravenous dose of the P-gp inhibitor cyclosporine. By contrast, an almost eightfold (770%) increase was observed in the Mdr1a/b knockout mice compared with control mice. These data can be interpreted in multiple ways, but illustrate the challenge in predicting human transporter-mediated DDIs based on preclinical animal data.
Clinical significance
The clinical significance of a P-gp inhibitor can be investigated in humans by assessing the P-gp-mediated clearance or exposure of a probe substrate in the presence of the inhibitor. Digoxin is transported by P-gp in vitro and should be considered in DDI studies with new molecular entities (NMEs) that are inhibitors of P-gp (TABLE 3, BOX 2). Current clinical data indicate that there are no consistent examples in which inhibition of P-gp in the blood–brain barrier resulted in adverse effects2931. It is therefore difficult to extrapolate the data obtained for P-gp inhibitors in knockout mice to humans to indicate the potential for a clinically significant DDI at the human blood–brain barrier31.
Findings from many studies on the effect of ABCB1 polymorphisms20 on P-gp substrates have not been consistently reproduced; therefore, routine application of ABCB1 polymorphism analysis to clinical studies is not warranted at this time. Studies with larger numbers of samples may be needed to clarify the role of ABCB1 polymorphisms in pharmacokinetics and pharmacodynamics32.
General description
P-gp mediates the ATP-dependent export of drugs from cells. It is expressed in the luminal membrane of the small intestine and blood–brain barrier, and in the apical membranes of excretory cells such as hepatocytes and kidney proximal tubule epithelia. P-gp has an important role in limiting entry of various drugs into the central nervous system. In addition, it also plays a part in the intestinal absorption and in the biliary and urinary excretion of drugs. The level of expression and functionality of P-gp can be modulated by inhibition and induction, which can affect the pharmacokinetics, efficacy, safety or tissue levels of P-gp substrates5,17–21.
Substrate and inhibitor selectivity
Initially discovered as a result of its interaction with multiple anticancer drugs, P-gp is responsible for the efflux across biological membranes of a broad range of therapeutic drugs. Recently, a high-resolution structure of the mouse P-gp has been described, which revealed distinct binding sites for drugs22. A select number of substrates and inhibitors of human P-gp are shown in TABLE 2. P-gp substrates are generally hydrophobic molecules, of which many are cationic. Multiple binding sites for substrates and inhibitors on P-gp have been identified using site-directed mutagenesis23–25.
Methodology for evaluating function
Cell lines that express P-gp and inside-out membrane vesicles prepared from these cell lines can be used to determine whether a drug is a P-gp substrate or inhibitor. As P-gp is localized in the apical plasma membrane in polarized cell monolayers, a high efflux ratio of basal-to-apical to apical-to-basal (the so called B-A/A-B ratio) indicates a potentially significant role for P-gp in transporting drugs across cell monolayers. A high efflux ratio in vitro has been shown to correlate well with studies that have demonstrated a role for P-gp in drug penetration to the central nervous system in mice26,27. By contrast, a high efflux ratio does not always translate into poor oral absorption. The involvement of P-gp in absorption of a drug is more pronounced if the drug has a poor apparent permeability coefficient (Papp), or in cases in which there is interplay between metabolism and efflux.
Mice deficient in Mdr1a or Mdr1a/b are widely used as powerful tools for assessing the role of P-gp in vivo9. Knockout mice can be used as a reference for complete inhibition of P-gp, and represent the ‘worst case’ scenario. Sasongko et al.28 demonstrated that the AUCbrain/ AUCblood ratio of C-verapamil in humans increased by less than twofold (88 ± 20%) in the presence of a high intravenous dose of the P-gp inhibitor cyclosporine. By contrast, an almost eightfold (770%) increase was observed in the Mdr1a/b knockout mice compared with control mice. These data can be interpreted in multiple ways, but illustrate the challenge in predicting human transporter-mediated DDIs based on preclinical animal data.
Clinical significance
The clinical significance of a P-gp inhibitor can be investigated in humans by assessing the P-gp-mediated clearance or exposure of a probe substrate in the presence of the inhibitor. Digoxin is transported by P-gp in vitro and should be considered in DDI studies with new molecular entities (NMEs) that are inhibitors of P-gp (TABLE 3, BOX 2). Current clinical data indicate that there are no consistent examples in which inhibition of P-gp in the blood–brain barrier resulted in adverse effects2931. It is therefore difficult to extrapolate the data obtained for P-gp inhibitors in knockout mice to humans to indicate the potential for a clinically significant DDI at the human blood–brain barrier31.
Findings from many studies on the effect of ABCB1 polymorphisms20 on P-gp substrates have not been consistently reproduced; therefore, routine application of ABCB1 polymorphism analysis to clinical studies is not warranted at this time. Studies with larger numbers of samples may be needed to clarify the role of ABCB1 polymorphisms in pharmacokinetics and pharmacodynamics32.
BCRP
General description
BCRP is a ‘half ABC transporter’ consisting of 655 amino acids and six transmembrane domains33. BCRP was identified originally as a determinant of multidrug resistance in cancer cell lines in vitro34,35. BCRP is expressed in the gastrointestinal tract, liver, kidney, brain endothelium, mammary tissue, testis and placenta. It has a role in limiting oral bioavailability and transport across the blood–brain barrier, blood– testis barrier and the maternal–fetal barrier of some selected substrates36,37. The physiological functions of BCRP include the extrusion of porphyrins from haematopoietic cells and hepatocytes, as well as the secretion of vitamin B2 (riboflavin) and possibly other vitamins (such as biotin and vitamin K) into breast milk36.
Substrate and inhibitor selectivity
BCRP actively extrudes a broad range of endogenous and exogenous substrates across biological membranes37. TABLE 2 lists selected substrates and inhibitors of human BCRP. High-speed screening and quantitative structure–activity relationship (QSAR) analysis methods suggest that one amine bonded to one carbon of a heterocyclic ring is an important component for drug interactions with BCRP. In addition, fused heterocyclic ring(s) and two substituents on a carbocyclic ring of the fused heterocyclic ring(s) are also important chemical moieties for the interaction with BCRP38–40. Many protein kinase inhibitors such as imatinib (Gleevec; Novartis) carry such structural components. Substrates of BCRP include pitavastatin (Livalo; Kowa Pharmaceuticals America)41 and phytoestrogens, such as genistein, daidzein and coumestrol42.
Methodology for evaluating function
Polarized and non-polarized cell lines expressing BCRP are used to assay BCRP-mediated transport and inhibition43. In addition, membrane vesicles containing BCRP that constitutively express the transporter can also be used43. Prazosin and cimetidine can be used as a positive control in cell lines, whereas several relatively polar, hydrophilic substrates (for example, methotrexate, oestrone 3-sulphate and sulphasalazine) can be used as controls in vesicular transport assays. Since the initial publications of the Bcrpknockout mouse36,37, drug disposition studies in Abcg2 knockout mice have delineated an important role for intestinal Bcrp as a rate-determining barrier for the oral bioavailability of several drugs. These include topotecan (hycamtin; GlaxoSmithKline)44 and sulphasalazine (Salazopyrin; Pharmacia), which had 10-fold to 110-fold increase in relative AUC45. BCRP is also a moderate determinant of the bioavailability of nitrofurantoin46, some fluoroquinolones47 and imatinib48.
Clinical significance
Recent clinical studies have demonstrated that subjects with reduced BCRP expression levels, correlating with the Q141K variant, are at increased risk for gefitinib (Iressa; AstraZeneca)-induced diarrhoea and altered pharmacokinetics of 9-aminocamptothecin, diflomotecan, irinotecan (Camptosar; Pfizer), rosuvastatin (Crestor; AstraZeneca), sulphasalazine and topotecan49–55. Inter-individual differences in BCRP function probably contribute to variable bioavailability, exposure (AUC and Cmax), and pharmacological response of drugs that are BCRP substrates. The most significant clinical effects are likely to be for drugs that have a low bioavailability and have a narrow therapeutic index.
General description
BCRP is a ‘half ABC transporter’ consisting of 655 amino acids and six transmembrane domains33. BCRP was identified originally as a determinant of multidrug resistance in cancer cell lines in vitro34,35. BCRP is expressed in the gastrointestinal tract, liver, kidney, brain endothelium, mammary tissue, testis and placenta. It has a role in limiting oral bioavailability and transport across the blood–brain barrier, blood– testis barrier and the maternal–fetal barrier of some selected substrates36,37. The physiological functions of BCRP include the extrusion of porphyrins from haematopoietic cells and hepatocytes, as well as the secretion of vitamin B2 (riboflavin) and possibly other vitamins (such as biotin and vitamin K) into breast milk36.
Substrate and inhibitor selectivity
BCRP actively extrudes a broad range of endogenous and exogenous substrates across biological membranes37. TABLE 2 lists selected substrates and inhibitors of human BCRP. High-speed screening and quantitative structure–activity relationship (QSAR) analysis methods suggest that one amine bonded to one carbon of a heterocyclic ring is an important component for drug interactions with BCRP. In addition, fused heterocyclic ring(s) and two substituents on a carbocyclic ring of the fused heterocyclic ring(s) are also important chemical moieties for the interaction with BCRP38–40. Many protein kinase inhibitors such as imatinib (Gleevec; Novartis) carry such structural components. Substrates of BCRP include pitavastatin (Livalo; Kowa Pharmaceuticals America)41 and phytoestrogens, such as genistein, daidzein and coumestrol42.
Methodology for evaluating function
Polarized and non-polarized cell lines expressing BCRP are used to assay BCRP-mediated transport and inhibition43. In addition, membrane vesicles containing BCRP that constitutively express the transporter can also be used43. Prazosin and cimetidine can be used as a positive control in cell lines, whereas several relatively polar, hydrophilic substrates (for example, methotrexate, oestrone 3-sulphate and sulphasalazine) can be used as controls in vesicular transport assays. Since the initial publications of the Bcrpknockout mouse36,37, drug disposition studies in Abcg2 knockout mice have delineated an important role for intestinal Bcrp as a rate-determining barrier for the oral bioavailability of several drugs. These include topotecan (hycamtin; GlaxoSmithKline)44 and sulphasalazine (Salazopyrin; Pharmacia), which had 10-fold to 110-fold increase in relative AUC45. BCRP is also a moderate determinant of the bioavailability of nitrofurantoin46, some fluoroquinolones47 and imatinib48.
Clinical significance
Recent clinical studies have demonstrated that subjects with reduced BCRP expression levels, correlating with the Q141K variant, are at increased risk for gefitinib (Iressa; AstraZeneca)-induced diarrhoea and altered pharmacokinetics of 9-aminocamptothecin, diflomotecan, irinotecan (Camptosar; Pfizer), rosuvastatin (Crestor; AstraZeneca), sulphasalazine and topotecan49–55. Inter-individual differences in BCRP function probably contribute to variable bioavailability, exposure (AUC and Cmax), and pharmacological response of drugs that are BCRP substrates. The most significant clinical effects are likely to be for drugs that have a low bioavailability and have a narrow therapeutic index.
OCTs and OATs
General description
A distinct family of proteins within the SLC superfamily is encoded by 22 genes of the human SLC22A family, and includes the electrogenic OCTs (isoforms 1–3) and the oATs (significant isoforms in humans include oAT1–4 and 7, and URAT1)6,7,56,57. The genes encoding OCT1–3 and OATs encode proteins that are 542–556 amino acids long with 12 predicted transmembrane-spanning domains57. The tissue distribution and localization of OCTs and OATs are summarized in TABLE 1 and FIG. 1. There are several published reviews of the molecular characteristics, expression and function of OCTs7,57–60 and OATs57,61–63.
Substrate and inhibitor selectivity
TABLe 1 summarizes various compounds that interact with human OCTs and OATs. OCTs transport relatively hydrophilic, low molecular mass organic cations. Properties of inhibitors of OCT1 and OCT2 have been identified and include a net positive charge and high lipophilicity59,64. OAT1, OAT3 and OAT4 support exchange of intracellular 2-oxoglutarate for extracellular substrate62. OAT1 and OAT3 mediate the basolateral entry step in renal secretion of many organic anions, and have distinct selectivities for different structural classes of type I organic anions. That is, monovalent (or selected divalent) anions that are less than 500 daltons58; although OAT3 can also transport some positively charged drugs such as cimetidine.
Methodology for evaluating function
In vitro assays that are commonly used to characterize OCT function include Xenopus laevis oocytes, membrane vesicles and cell lines derived from various tissues including proximal tubule (Caki-1), placenta (BeWo) and colon carcinoma (Caco-2) cells65–69. These are in addition to various human embryonic kidney (HEK293) cell lines expressing recombinant OCTs68,73,83. Functional assessment of OATs generally uses heterologous expression in cultured cells or X. laevis oocytes, and such systems have been used to develop structure–activity relationships for the interaction of OATs70,71. Studies performed in Oct knockout mice have demonstrated the pharmacokinetic, pharmacological and physiological relevance of OCTs in organic cation disposition and activity8,72–74. Studies using Oat1 and Oat3 knockout mice have also allowed an initial assessment of the influence of each transporter on the renal handling of selected compounds, and have led to the identification of endogenous OAT substrates75,76.
Clinical significance
Recent studies suggest that genetic variation in OCT1 may be a significant determinant of inter-individual variability in the disposition and response to cationic drug substrates, particularly metformin73,77. However, such studies have been controversial78. Recent studies suggest that the activity of OCT1 is positively associated with the degree to which patients with chronic myeloid leukaemia respond to imatinib79,80. In Chinese and Korean populations, a common OCT2 variant (A270S) has been associated with a significant reduction in renal clearance of metformin81,82, but the effect of this variant may differ depending on the ethnic group83. By contrast, genetic variants in the OATs have not been associated with changes in drug disposition. DDIs with the OATs and the OCTs can occur, and may result in reduced renal clearance (TABLE 3). Creatinine, which is often used to assess filtration clearance, undergoes renal tubular secretion, which could be inhibited by inhibitors of OCT2. Therefore, other methods should be used to assess filtration clearance of NMEs in DDI studies involving OCT2 inhibitors. DDIs involving the OATs and the OCTs are discussed in more detail in Section 3.
General description
A distinct family of proteins within the SLC superfamily is encoded by 22 genes of the human SLC22A family, and includes the electrogenic OCTs (isoforms 1–3) and the oATs (significant isoforms in humans include oAT1–4 and 7, and URAT1)6,7,56,57. The genes encoding OCT1–3 and OATs encode proteins that are 542–556 amino acids long with 12 predicted transmembrane-spanning domains57. The tissue distribution and localization of OCTs and OATs are summarized in TABLE 1 and FIG. 1. There are several published reviews of the molecular characteristics, expression and function of OCTs7,57–60 and OATs57,61–63.
Substrate and inhibitor selectivity
TABLe 1 summarizes various compounds that interact with human OCTs and OATs. OCTs transport relatively hydrophilic, low molecular mass organic cations. Properties of inhibitors of OCT1 and OCT2 have been identified and include a net positive charge and high lipophilicity59,64. OAT1, OAT3 and OAT4 support exchange of intracellular 2-oxoglutarate for extracellular substrate62. OAT1 and OAT3 mediate the basolateral entry step in renal secretion of many organic anions, and have distinct selectivities for different structural classes of type I organic anions. That is, monovalent (or selected divalent) anions that are less than 500 daltons58; although OAT3 can also transport some positively charged drugs such as cimetidine.
Methodology for evaluating function
In vitro assays that are commonly used to characterize OCT function include Xenopus laevis oocytes, membrane vesicles and cell lines derived from various tissues including proximal tubule (Caki-1), placenta (BeWo) and colon carcinoma (Caco-2) cells65–69. These are in addition to various human embryonic kidney (HEK293) cell lines expressing recombinant OCTs68,73,83. Functional assessment of OATs generally uses heterologous expression in cultured cells or X. laevis oocytes, and such systems have been used to develop structure–activity relationships for the interaction of OATs70,71. Studies performed in Oct knockout mice have demonstrated the pharmacokinetic, pharmacological and physiological relevance of OCTs in organic cation disposition and activity8,72–74. Studies using Oat1 and Oat3 knockout mice have also allowed an initial assessment of the influence of each transporter on the renal handling of selected compounds, and have led to the identification of endogenous OAT substrates75,76.
Clinical significance
Recent studies suggest that genetic variation in OCT1 may be a significant determinant of inter-individual variability in the disposition and response to cationic drug substrates, particularly metformin73,77. However, such studies have been controversial78. Recent studies suggest that the activity of OCT1 is positively associated with the degree to which patients with chronic myeloid leukaemia respond to imatinib79,80. In Chinese and Korean populations, a common OCT2 variant (A270S) has been associated with a significant reduction in renal clearance of metformin81,82, but the effect of this variant may differ depending on the ethnic group83. By contrast, genetic variants in the OATs have not been associated with changes in drug disposition. DDIs with the OATs and the OCTs can occur, and may result in reduced renal clearance (TABLE 3). Creatinine, which is often used to assess filtration clearance, undergoes renal tubular secretion, which could be inhibited by inhibitors of OCT2. Therefore, other methods should be used to assess filtration clearance of NMEs in DDI studies involving OCT2 inhibitors. DDIs involving the OATs and the OCTs are discussed in more detail in Section 3.
OATPs
General description
The OATPs (SLCOs) represent a superfamily of important membrane transport proteins that mediate the sodium-independent transport of a diverse range of amphiphilic organic compounds. These include bile acids, steroid conjugates, thyroid hormones, anionic peptides, numerous drugs and other xenobiotic substances84 (TABLE 1). The general predicted OATP structure consists of proteins with 12 transmembrane domains85. The mechanism of transport appears to consist of anion exchange by coupling the cellular uptake of substrate with the efflux of endogenous intracellular substances such as bicarbonate in a process that seems to be electroneutral85–87. OATP1B1 was cloned by several groups88–