Membrane transporters in drug development.
Journal: 2010/April - Nature Reviews Drug Discovery
ISSN: 1474-1784
Abstract:
Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labelling.
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Nat Rev Drug Discov 9(3): 215-236

Membrane transporters in drug development

+16 authors

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.

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selected human transport proteins for drugs and endogenous substances

Transporters in plasma membrane domains of intestinal epithelia, hepatocytes, kidney proximal tubules and brain capillary endothelial cells are presented. Those coloured in red indicate that the selected transporters are described in detail in this manuscript. Those coloured in blue indicate that the transport proteins are of importance but are not described in this manuscript. a | Intestinal epithelia contain in their apical (luminal) membrane several uptake transporters including one or more members of the organic anion transporting polypeptide (OATP) family; peptide transporter 1 (PEPT1; SLC15A1); ileal apical sodium/bile acid co-transporter (ASBT; SLC10A2); and monocarboxylic acid transporter 1 (MCT1; SLC16A1). The apical ATP-dependent efflux pumps include multidrug resistance protein 2 (MRP2; ABCC2); breast cancer resistance protein (BCRP; ABCG2); and P-glycoprotein (P-gp; MDR1, ABCB1). The basolateral membrane of intestinal epithelia contains organic cation transporter 1 (OCT1; SLC22A1); heteromeric organic solute transporter (OSTα–OSTβ); and MRP3 (ABCC3). b | Human hepatocyte uptake transporters in the basolateral (sinusoidal) membrane include the sodium/taurocholate co-transporting peptide (NTCP; SLC10A1); three members of the OATP family (OATP1B1 (SLCO1B1), OATP1B3 (SLCO1B3) and OATP2B1 (SLCO2B1)); organic anion transporter 2 (OAT2; SLC22A7) and OAT7 (SLC22A9); and OCT1. Efflux pumps in the hepatocyte basolateral membrane include MRP3, MRP4 (ABCC4) and MRP6 (ABCC6). Apical (canalicular) efflux pumps of the hepatocyte comprise P-gp; bile-salt export pump (BSEP or SPGP; ABCB11); BCRP (ABCG2); and MRP2. In addition, multidrug and toxin extrusion protein 1 (MATE1; SLC47A1) is located in the apical hepatocyte membrane. c | Kidney proximal tubules contain in the apical (luminal) membrane OAT4 (SLC22A11); urate transporter 1 (URAT1; SCL22A12); PEPT1 and PEPT2 (SLC15A2); MRP2 and MRP4; MATE1 and MATE2-K (SLC47A2); P-gp; organic cation/ergothioneine transporter (OCTN1; SLC22A4); and organic cation/carnitine transporter (OCTN2; SLC22A5). Basolateral uptake transporters in proximal tubule epithelia include OATP4C1 (SLCO4C1); OCT2; and OAT1, OAT2 and OAT3 (SLC22A8). d | Apical (luminal) transport proteins of brain capillary endothelial cells contributing to the function of the blood–brain barrier include the uptake transporters OATP1A2 and OATP2B1; and the efflux pumps P-gp, BCRP, MRP4 and MRP5 (ABCC5). Note that localization of transporters to particular membranes and tissues is sometimes controversial; therefore, the International Transporter Consortium erred on the conservative side in only showing the localization of transporters for which good evidence exists.

Table 1

SLC transporters of emerging clinical importance in the absorption and disposition of drugs

Transporter/
alias (Gene)
Selected substratesSelected
inhibitors
Organs/cellsComments
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-prolineIntestinal
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
Can potentially be used for in vivo (clinical) studies.

Table 2

ABC transporters of emerging clinical importance in the absorption and disposition of drugs

Transporter/alias
(Gene)
Selected substratesSelected
inhibitors
Organs/cellsComments
MDR1/P-gp, ABCB1 (ABCB1)Digoxin, loperamide, berberine, irinotecan, doxorubicin, vinblastine, paclitaxel, fexofenadineCyclosporine, quinidine, tariquidar, verapamilIntestinal enterocytes, kidney proximal tubule, hepatocytes (canalicular), brain endothelia
  • Has a role in absorption, disposition and excretion

  • Has a role in clinical drug–drug interactions

BCRP/MXR (ABCG2)Mitoxantrone, methotrexate, topotecan, imatinib, irinotecan, statins, sulphate conjugates, porphyrinsOestrone-17β-oestradiol, fumitre - morgin CIntestinal enterocytes, hepatocytes (canalicular), kidney proximal tubule, brain endothelia, placenta, stem cells, mammary glands (lactating)
  • Has a role in absorption, disposition and excretion

  • Has clinically relevant genetic polymorphisms

  • Has a role in clinical drug–drug interactions

BSEP/SPGP, cBAT, ABCB11 (ABCB11)Taurocholic acid, pravastatin, bile acidsCyclosporin A, rifampicin, glibenclamideHepatocytes (canalicular)
  • Has a role in excretion

  • Has clinically relevant genetic polymorphisms

  • Has a role in clinical drug–drug interactions

MRP2/ABCC2, cMOAT (ABCC2)Glutathione and glucuronide conjugates, methotrexate, etoposide, mitoxantrone, valsartan, olmesartan, glucuronidated SN-38Cyclosporine, delaviridine, efavirenz, emtricitabineHepatocytes (canalicular), kidney (proximal tubule, luminal), enterocytes (luminal)
  • Has a role in absorption, disposition and excretion

  • Has clinically relevant Genetic polymorphisms

  • Has a role in clinical drug–drug interactions

MRP3/ABCC3 (ABCC3)Oestradiol-17β-glucuronide, methotrexate, fexofenadine, glucuronate conjugatesDelaviridine, efavirenz, emtricitabineHepatocytes (sinusoidal), intestinal enterocytes (basolateral)
  • • Has a role in disposition

MRP4/ABCC4 (ABCC4)Adefovir, tenofovir, cyclic AMP, dehydroepiandrostero ne sulphate, methotrexate, topotecan, furosemide, cyclic GMP, bile acids plus glutathioneCelecoxib, diclofenacKidney proximal tubule (luminal), choroid plexus, hepatocytes (sinusoidal), platelets
  • • Has a role in disposition and excretion

MDR3/ABCB4 (ABCB4)Phosphatidylcholine, paclitaxel, digoxin, vinblastineVerapamil, cyclosporineHepatocytes (canalicular)
  • Has a role in disposition

  • Has a role in clinical drug–drug interactions

ABC, ATP-binding cassette.

Can potentially be used for in vivo (clinical) studies.

Table 3

Selected transporter-mediated clinical drug–drug interactions

Implicated transporter*Interacting drugAffected drugClinical pharmacokinetic impact on affected drug
Organic anion transporting polypeptidesCyclosporinePravastatinAUC ↑890% and Cmax ↑678%102,204
CyclosporineRosuvastatinAUC ↑610%205
CyclosporinePitavastatinAUC ↑ 360% and Cmax ↑560%206
Rifampicin (single dose)GlyburideAUC ↑125%207
Rifampicin (single dose)BosentanCtrough ↑ 500%208
Lopinavir/ritonavirBosentanDay 4: Ctrough ↑ 4,700%208; day 10: Ctrough ↑ 400%208
Lopinavir/ritonavirRosuvastatinAUC ↑107% and Cmax ↑365%209
Organic anion transportersProbenecidCidofovirCLr ↓32%210,211
ProbenecidFurosemideCLr ↓66%210
ProbenecidAcyclovirCLr ↓32% and AUC ↑40%210,212
Organic cation transportersCimetidineMetforminAUC ↑50% and CLr ↓ 27%213,214
CimetidinePindololCLr ↓~34%215
CimetidineVareniclineAUC ↑29%216
CimetidinePilsicainideAUC ↑33%, CLr ↓28%217
CimetidinePilsicainideCLr ↓41%218
CimetidineDofetilideCLr ↓33%219
P-glycoproteinQuinidineDigoxinCLr ↓34–48%220,221
RitonavirDigoxinAUC ↑86%222
DronedaroneDigoxinAUC ↑157% and Cmax ↑75%223
RanolazineDigoxinAUC ↑60% and Cmax ↑46%224
Breast cancer resistance proteinGF120918TopotecanAUC ↑143%225
Implicated transporter refers to the likely transporter; however, because the studies are carried out in vivo it is not possible to assign specific transporters to the drug–drug interaction.
Percent change refers to the difference between the area under the curve (AUC), or Cmax, in the presence and the absence of the inhibitor (interacting drug) normalized to the AUC in the absence of the inhibitor. For clearance values (CLr), the values are normalized for the absence of the inhibitor. Ctrough is the minimum drug concentration observed after administration of a dose of the drug and the concentration prior to the administration of a subsequent dose.

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 substrates5,1721.

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 mutagenesis2325.

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.

Box 2 | Decision trees for P-lycoprotein or BCRP substrate interactions

Although the current US Food and Drug Administration draft Drug Interaction Guidance provides a decision tree specifically for P-glycoprotein (P-gp; also known as MDR1, ABCB1)1415183, we broadened the decision tree here and in BOX 3 to include both P-gp and breast cancer resistance protein (BCRP; also known as ABCG2). This is because similar in vitro methodologies and criteria are used to suggest clinical drug–drug interaction (DDI) studies for these two transporters. Generally, most P-gp substrates are organic cations or neutral molecules, relatively hydrophobic, and have a range of molecular masses (200 daltons to greater than 1,000 daltons). BCRP substrates tend to overlap with P-gp but also include acids or drug conjugates that are not good substrates for P-gp. A new molecular entity (NME) is considered to be a potential P-gp or BCRP substrate if the efflux ratio — basal to apical (B-A) to apical to basal (A–B) — is ≥ 2 in an epithelial cell system that expresses one or both transporters (see footnote and (a) in the figure). A net flux ratio cut-off higher than 2 or a relative ratio to positive controls may be used to avoid false positives if a ratio of 2 is deemed non-discriminative as supported by prior experience with the cell system used. Additional corroboration that an NME may be a P-gp or BCRP substrate can be achieved with the use of inhibitors; information especially valuable if non-transfected cells are not included as controls for endogenous transport activity. Reduction of the flux ratio by the P-gp (or BCRP) inhibitors should be greater than 50% (see (b) in the figure). If the flux ratio is not reduced by P-gp (or BCRP) inhibitors, then other efflux transporters may be responsible for the observed net flux (see (d) in the figure).

Box 3 | Decision trees for P-glycoprotein or BCRP inhibitor interactions

A new molecular entity (NME) is considered to be a potential P-glycoprotein (P-gp; also known as MDR1, ABCB1) and/or breast cancer resistance protein (BCRP; also known as ABCG2) inhibitor if the net flux ratio of a P-gp (or BCRP) probe substrate is decreased in the presence of the NME using a bi-directional transport assay (see footnote 1 below). Generally, the potency of inhibition is reported only as an IC50 value, which is dependent on the test system used. There are several practical challenges in evaluating whether NMEs that inhibit efflux transporters in vitro are also inhibitors in vivo. Orally administered drugs that are classified as in vitro efflux inhibitors provide the dual challenge of considering both intestinal efflux inhibition, in which intestinal drug concentrations will be the highest, and tissue efflux inhibition, in which circulating concentrations will be more relevant. Zhang et al.15 proposed that drugs exhibiting an [I]1/IC50 ≥ 0.1 or [I]2/IC50 ≥10 should be evaluated to determine whether inhibition occurs in vivo. In that publication, [I]1 is the mean NME steady-state total Cmax at the highest clinical dose, and [I]2 is the theoretical maximal gastrointestinal NME concentration after oral administration calculated as the highest clinical dose (mg) in a volume of 250 ml15. The [I]1/IC50 ≥ 0.1 calculation is familiar to those working on metabolic inhibition, as this approach has been used for many years.

In this paper, to be consistent with decision trees for other transporters (for example, organic cation transporter 2 (OCT2) and organic anion transporting polypeptide (OATP)) and the notion that unbound drug is the pharmacologically active species, mean steady-state Cmax values for unbound drug following administration of the highest proposed clinical dose is used for [I]1 in the decision tree (see footnote 2 for further discussion). Using this approach, a drug (molecular mass of 500 daltons) with an in vitro IC50 value of 10 µM will exceed an [I]1/IC50 value > 0.1 at an unbound Cmax > 1 µM (assumes fraction unbound (fu) = 0.1 and Cmax = 10 µM). This suggests that there is limited drug interaction potential with P-gp or BCRP. By contrast, for the same drug, an [I]2/IC50 value > 10 will be exceeded at a dose of approximately 12 mg or greater. Thus, a drug with an inhibition potency of approximately 10 µM or less in vitro is likely to exceed [I]2/IC50 value > 10 as many drugs require oral doses greater than 12 mg.

If an NME meets either [I]1/IC50 ≥ 0.1 (for example, IC50 is ≤ 10-fold the unbound Cmax value) or [I]2/IC50 ≥ 10, an in vivo drug interaction study with digoxin is recommended. Digoxin is a unique drug (see footnote 3), and may not be the best reference for decisions regarding interaction studies with other P-gp substrate drugs. Fenner et al.188 analysed drug interactions with digoxin and suggested that interactions of digoxin with P-gp inhibitors were limited (for example, most inhibitors caused < 2-fold changes in plasma concentrations of digoxin). However, owing to its narrow therapeutic index, interactions that alter the digoxin AUC > 1.25-fold are clinically important for digoxin. Therefore, the interpretation of the significance of P-gp for drug–drug interactions should not be over-extrapolated based on the importance for digoxin safety, as most other drugs have a much larger therapeutic index. In addition, if the NME inhibits a cytochrome P450 enzyme as well as P-gp, selection of a drug that is a dual substrate of cytochrome P450 and P-gp may be deemed appropriate. If the NME is a pure P-gp inhibitor, other than for clarifying a digoxin dose adjustment requirement, no drug–drug interaction studies may be indicated depending on the therapeutic area of the NME and its drug labeling considerations.

For an NME that is a BCRP inhibitor, possible candidate probe substrates include sulphasalazine189, rosuvastatin190, pitavastatin41, ciprofloxacin47 and dipyridamole191. However, many of these remain to be tested as selective BCRP probe substrates in clinical studies.

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Note 1: A unidirectional assay based on the probe substrate can also be considered188. Inside-out vesicles expressing P-gp or BCRP, or transfected cell monolayers grown on solid support with appropriate controls (for example, inhibitors and positive controls, as described in Section 2), can also be used for inhibition assays. Note 2: If fu cannot be accurately determined owing to high protein binding, then assume fu = 0.01, to err on the conservative side. Alternatively, unbound portal vein concentrations may be used and might be more applicable to liver uptake192193. Note 3: Digoxin has an absolute bioavailability of approximately 70% from immediate release tablets. By contrast, the absolute bioavailability from a liquid-filled formulation (for example, Lanoxicaps) is approximately 90%. Therefore, consideration should also be given to the dosage form of digoxin used in an interaction study. In addition, digoxin may be a substrate for other transporters1592188194. Hence, caution is advised when extrapolating results from digoxin to other P-gp substrates.

The conclusions from in vitro efflux studies are dependent on the cell line used. For example, for cells heterologously expressing P-gp or BCRP (for example, MDCK or LLC-PK1), in which expression of the transporters can be high, the efflux ratio would be expected to be much higher than 2 for strong substrates. A classification of relevant substrates versus non-substrates will depend on the assay system. Validation studies of in vitro assay systems using known weak and strong substrates and, where possible, correlations with in vivo model systems, should be established. Meaningful efflux ratios are dependent on the transporter expression; thus, results should be related to a reference compound. In cell lines such as Caco-2, which express multiple uptake and efflux transporters, reduction of efflux in the presence of an inhibitor may support efflux by P-gp and/or BCRP, depending on the selectivity of the inhibitor used (for example, cyclosporine inhibits multiple transporters).

If in vitro experiments suggest that the NME is a P-gp and/or BCRP substrate (see (c) in the figure), preclinical and clinical information should be assessed to determine whether a clinical in vivo DDI study is warranted. In particular, the relative contribution of the transporter-mediated pathway to the overall clearance of the drug is the primary determinant of whether an inhibitor will have a major effect on the disposition of the NME. For example, the pharmacokinetics of an NME that has high solubility, high permeability and/or is highly metabolized are less likely to be affected by a co-administered drug that is a P-gp inhibitor15173174. Therefore, an in vivo interaction study may not be needed. By contrast, an NME that has poor solubility, limited permeability and is metabolically stable (eliminated primarily as the parent compound) is more likely to demonstrate a pharmacokinetic change in the presence of an inhibitor. The possible clinical consequence of such an interaction needs to be assessed based on careful evaluation of available exposure–response data186187. The recommended clinical study with a known P-gp inhibitor should consider the therapeutic use of the NME; however, cyclosporine is a reasonable choice, as it is known to inhibit P-gp in vivo. As noted previously, because cyclosporine inhibits multiple transporters, care must be taken in the design and interpretation of DDI studies in which it is used as an inhibitor.

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This figure shows a decision tree for P-gp and a similar tree could be used for BCRP. Although flux systems have traditionally been used to determine whether an NME is a substrate of P-gp or BCRP, inside-out vesicles expressing P-gp or BCRP, or transfected cell monolayers grown on solid support with appropriate controls (for example, inhibitors and positive controls) as described in Section 2, also can be used.

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 BCRP3840. 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 topotecan4955. 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 OCTs7,5760 and OATs57,6163.

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) cells6569. 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 activity8,7274. 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 electroneutral8587. OATP1B1 was cloned by several groups8891 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 rifampicin8691. 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%, respectively88,104106. 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, respectively88,104106. 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 substrates5,1721.

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 mutagenesis2325.

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.

Box 2 | Decision trees for P-lycoprotein or BCRP substrate interactions

Although the current US Food and Drug Administration draft Drug Interaction Guidance provides a decision tree specifically for P-glycoprotein (P-gp; also known as MDR1, ABCB1)1415183, we broadened the decision tree here and in BOX 3 to include both P-gp and breast cancer resistance protein (BCRP; also known as ABCG2). This is because similar in vitro methodologies and criteria are used to suggest clinical drug–drug interaction (DDI) studies for these two transporters. Generally, most P-gp substrates are organic cations or neutral molecules, relatively hydrophobic, and have a range of molecular masses (200 daltons to greater than 1,000 daltons). BCRP substrates tend to overlap with P-gp but also include acids or drug conjugates that are not good substrates for P-gp. A new molecular entity (NME) is considered to be a potential P-gp or BCRP substrate if the efflux ratio — basal to apical (B-A) to apical to basal (A–B) — is ≥ 2 in an epithelial cell system that expresses one or both transporters (see footnote and (a) in the figure). A net flux ratio cut-off higher than 2 or a relative ratio to positive controls may be used to avoid false positives if a ratio of 2 is deemed non-discriminative as supported by prior experience with the cell system used. Additional corroboration that an NME may be a P-gp or BCRP substrate can be achieved with the use of inhibitors; information especially valuable if non-transfected cells are not included as controls for endogenous transport activity. Reduction of the flux ratio by the P-gp (or BCRP) inhibitors should be greater than 50% (see (b) in the figure). If the flux ratio is not reduced by P-gp (or BCRP) inhibitors, then other efflux transporters may be responsible for the observed net flux (see (d) in the figure).

Box 3 | Decision trees for P-glycoprotein or BCRP inhibitor interactions

A new molecular entity (NME) is considered to be a potential P-glycoprotein (P-gp; also known as MDR1, ABCB1) and/or breast cancer resistance protein (BCRP; also known as ABCG2) inhibitor if the net flux ratio of a P-gp (or BCRP) probe substrate is decreased in the presence of the NME using a bi-directional transport assay (see footnote 1 below). Generally, the potency of inhibition is reported only as an IC50 value, which is dependent on the test system used. There are several practical challenges in evaluating whether NMEs that inhibit efflux transporters in vitro are also inhibitors in vivo. Orally administered drugs that are classified as in vitro efflux inhibitors provide the dual challenge of considering both intestinal efflux inhibition, in which intestinal drug concentrations will be the highest, and tissue efflux inhibition, in which circulating concentrations will be more relevant. Zhang et al.15 proposed that drugs exhibiting an [I]1/IC50 ≥ 0.1 or [I]2/IC50 ≥10 should be evaluated to determine whether inhibition occurs in vivo. In that publication, [I]1 is the mean NME steady-state total Cmax at the highest clinical dose, and [I]2 is the theoretical maximal gastrointestinal NME concentration after oral administration calculated as the highest clinical dose (mg) in a volume of 250 ml15. The [I]1/IC50 ≥ 0.1 calculation is familiar to those working on metabolic inhibition, as this approach has been used for many years.

In this paper, to be consistent with decision trees for other transporters (for example, organic cation transporter 2 (OCT2) and organic anion transporting polypeptide (OATP)) and the notion that unbound drug is the pharmacologically active species, mean steady-state Cmax values for unbound drug following administration of the highest proposed clinical dose is used for [I]1 in the decision tree (see footnote 2 for further discussion). Using this approach, a drug (molecular mass of 500 daltons) with an in vitro IC50 value of 10 µM will exceed an [I]1/IC50 value > 0.1 at an unbound Cmax > 1 µM (assumes fraction unbound (fu) = 0.1 and Cmax = 10 µM). This suggests that there is limited drug interaction potential with P-gp or BCRP. By contrast, for the same drug, an [I]2/IC50 value > 10 will be exceeded at a dose of approximately 12 mg or greater. Thus, a drug with an inhibition potency of approximately 10 µM or less in vitro is likely to exceed [I]2/IC50 value > 10 as many drugs require oral doses greater than 12 mg.

If an NME meets either [I]1/IC50 ≥ 0.1 (for example, IC50 is ≤ 10-fold the unbound Cmax value) or [I]2/IC50 ≥ 10, an in vivo drug interaction study with digoxin is recommended. Digoxin is a unique drug (see footnote 3), and may not be the best reference for decisions regarding interaction studies with other P-gp substrate drugs. Fenner et al.188 analysed drug interactions with digoxin and suggested that interactions of digoxin with P-gp inhibitors were limited (for example, most inhibitors caused < 2-fold changes in plasma concentrations of digoxin). However, owing to its narrow therapeutic index, interactions that alter the digoxin AUC > 1.25-fold are clinically important for digoxin. Therefore, the interpretation of the significance of P-gp for drug–drug interactions should not be over-extrapolated based on the importance for digoxin safety, as most other drugs have a much larger therapeutic index. In addition, if the NME inhibits a cytochrome P450 enzyme as well as P-gp, selection of a drug that is a dual substrate of cytochrome P450 and P-gp may be deemed appropriate. If the NME is a pure P-gp inhibitor, other than for clarifying a digoxin dose adjustment requirement, no drug–drug interaction studies may be indicated depending on the therapeutic area of the NME and its drug labeling considerations.

For an NME that is a BCRP inhibitor, possible candidate probe substrates include sulphasalazine189, rosuvastatin190, pitavastatin41, ciprofloxacin47 and dipyridamole191. However, many of these remain to be tested as selective BCRP probe substrates in clinical studies.

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Note 1: A unidirectional assay based on the probe substrate can also be considered188. Inside-out vesicles expressing P-gp or BCRP, or transfected cell monolayers grown on solid support with appropriate controls (for example, inhibitors and positive controls, as described in Section 2), can also be used for inhibition assays. Note 2: If fu cannot be accurately determined owing to high protein binding, then assume fu = 0.01, to err on the conservative side. Alternatively, unbound portal vein concentrations may be used and might be more applicable to liver uptake192193. Note 3: Digoxin has an absolute bioavailability of approximately 70% from immediate release tablets. By contrast, the absolute bioavailability from a liquid-filled formulation (for example, Lanoxicaps) is approximately 90%. Therefore, consideration should also be given to the dosage form of digoxin used in an interaction study. In addition, digoxin may be a substrate for other transporters1592188194. Hence, caution is advised when extrapolating results from digoxin to other P-gp substrates.

The conclusions from in vitro efflux studies are dependent on the cell line used. For example, for cells heterologously expressing P-gp or BCRP (for example, MDCK or LLC-PK1), in which expression of the transporters can be high, the efflux ratio would be expected to be much higher than 2 for strong substrates. A classification of relevant substrates versus non-substrates will depend on the assay system. Validation studies of in vitro assay systems using known weak and strong substrates and, where possible, correlations with in vivo model systems, should be established. Meaningful efflux ratios are dependent on the transporter expression; thus, results should be related to a reference compound. In cell lines such as Caco-2, which express multiple uptake and efflux transporters, reduction of efflux in the presence of an inhibitor may support efflux by P-gp and/or BCRP, depending on the selectivity of the inhibitor used (for example, cyclosporine inhibits multiple transporters).

If in vitro experiments suggest that the NME is a P-gp and/or BCRP substrate (see (c) in the figure), preclinical and clinical information should be assessed to determine whether a clinical in vivo DDI study is warranted. In particular, the relative contribution of the transporter-mediated pathway to the overall clearance of the drug is the primary determinant of whether an inhibitor will have a major effect on the disposition of the NME. For example, the pharmacokinetics of an NME that has high solubility, high permeability and/or is highly metabolized are less likely to be affected by a co-administered drug that is a P-gp inhibitor15173174. Therefore, an in vivo interaction study may not be needed. By contrast, an NME that has poor solubility, limited permeability and is metabolically stable (eliminated primarily as the parent compound) is more likely to demonstrate a pharmacokinetic change in the presence of an inhibitor. The possible clinical consequence of such an interaction needs to be assessed based on careful evaluation of available exposure–response data186187. The recommended clinical study with a known P-gp inhibitor should consider the therapeutic use of the NME; however, cyclosporine is a reasonable choice, as it is known to inhibit P-gp in vivo. As noted previously, because cyclosporine inhibits multiple transporters, care must be taken in the design and interpretation of DDI studies in which it is used as an inhibitor.

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This figure shows a decision tree for P-gp and a similar tree could be used for BCRP. Although flux systems have traditionally been used to determine whether an NME is a substrate of P-gp or BCRP, inside-out vesicles expressing P-gp or BCRP, or transfected cell monolayers grown on solid support with appropriate controls (for example, inhibitors and positive controls) as described in Section 2, also can be used.

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 substrates5,1721.

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 mutagenesis2325.

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.

Box 2 | Decision trees for P-lycoprotein or BCRP substrate interactions

Although the current US Food and Drug Administration draft Drug Interaction Guidance provides a decision tree specifically for P-glycoprotein (P-gp; also known as MDR1, ABCB1)1415183, we broadened the decision tree here and in BOX 3 to include both P-gp and breast cancer resistance protein (BCRP; also known as ABCG2). This is because similar in vitro methodologies and criteria are used to suggest clinical drug–drug interaction (DDI) studies for these two transporters. Generally, most P-gp substrates are organic cations or neutral molecules, relatively hydrophobic, and have a range of molecular masses (200 daltons to greater than 1,000 daltons). BCRP substrates tend to overlap with P-gp but also include acids or drug conjugates that are not good substrates for P-gp. A new molecular entity (NME) is considered to be a potential P-gp or BCRP substrate if the efflux ratio — basal to apical (B-A) to apical to basal (A–B) — is ≥ 2 in an epithelial cell system that expresses one or both transporters (see footnote and (a) in the figure). A net flux ratio cut-off higher than 2 or a relative ratio to positive controls may be used to avoid false positives if a ratio of 2 is deemed non-discriminative as supported by prior experience with the cell system used. Additional corroboration that an NME may be a P-gp or BCRP substrate can be achieved with the use of inhibitors; information especially valuable if non-transfected cells are not included as controls for endogenous transport activity. Reduction of the flux ratio by the P-gp (or BCRP) inhibitors should be greater than 50% (see (b) in the figure). If the flux ratio is not reduced by P-gp (or BCRP) inhibitors, then other efflux transporters may be responsible for the observed net flux (see (d) in the figure).

Box 3 | Decision trees for P-glycoprotein or BCRP inhibitor interactions

A new molecular entity (NME) is considered to be a potential P-glycoprotein (P-gp; also known as MDR1, ABCB1) and/or breast cancer resistance protein (BCRP; also known as ABCG2) inhibitor if the net flux ratio of a P-gp (or BCRP) probe substrate is decreased in the presence of the NME using a bi-directional transport assay (see footnote 1 below). Generally, the potency of inhibition is reported only as an IC50 value, which is dependent on the test system used. There are several practical challenges in evaluating whether NMEs that inhibit efflux transporters in vitro are also inhibitors in vivo. Orally administered drugs that are classified as in vitro efflux inhibitors provide the dual challenge of considering both intestinal efflux inhibition, in which intestinal drug concentrations will be the highest, and tissue efflux inhibition, in which circulating concentrations will be more relevant. Zhang et al.15 proposed that drugs exhibiting an [I]1/IC50 ≥ 0.1 or [I]2/IC50 ≥10 should be evaluated to determine whether inhibition occurs in vivo. In that publication, [I]1 is the mean NME steady-state total Cmax at the highest clinical dose, and [I]2 is the theoretical maximal gastrointestinal NME concentration after oral administration calculated as the highest clinical dose (mg) in a volume of 250 ml15. The [I]1/IC50 ≥ 0.1 calculation is familiar to those working on metabolic inhibition, as this approach has been used for many years.

In this paper, to be consistent with decision trees for other transporters (for example, organic cation transporter 2 (OCT2) and organic anion transporting polypeptide (OATP)) and the notion that unbound drug is the pharmacologically active species, mean steady-state Cmax values for unbound drug following administration of the highest proposed clinical dose is used for [I]1 in the decision tree (see footnote 2 for further discussion). Using this approach, a drug (molecular mass of 500 daltons) with an in vitro IC50 value of 10 µM will exceed an [I]1/IC50 value > 0.1 at an unbound Cmax > 1 µM (assumes fraction unbound (fu) = 0.1 and Cmax = 10 µM). This suggests that there is limited drug interaction potential with P-gp or BCRP. By contrast, for the same drug, an [I]2/IC50 value > 10 will be exceeded at a dose of approximately 12 mg or greater. Thus, a drug with an inhibition potency of approximately 10 µM or less in vitro is likely to exceed [I]2/IC50 value > 10 as many drugs require oral doses greater than 12 mg.

If an NME meets either [I]1/IC50 ≥ 0.1 (for example, IC50 is ≤ 10-fold the unbound Cmax value) or [I]2/IC50 ≥ 10, an in vivo drug interaction study with digoxin is recommended. Digoxin is a unique drug (see footnote 3), and may not be the best reference for decisions regarding interaction studies with other P-gp substrate drugs. Fenner et al.188 analysed drug interactions with digoxin and suggested that interactions of digoxin with P-gp inhibitors were limited (for example, most inhibitors caused < 2-fold changes in plasma concentrations of digoxin). However, owing to its narrow therapeutic index, interactions that alter the digoxin AUC > 1.25-fold are clinically important for digoxin. Therefore, the interpretation of the significance of P-gp for drug–drug interactions should not be over-extrapolated based on the importance for digoxin safety, as most other drugs have a much larger therapeutic index. In addition, if the NME inhibits a cytochrome P450 enzyme as well as P-gp, selection of a drug that is a dual substrate of cytochrome P450 and P-gp may be deemed appropriate. If the NME is a pure P-gp inhibitor, other than for clarifying a digoxin dose adjustment requirement, no drug–drug interaction studies may be indicated depending on the therapeutic area of the NME and its drug labeling considerations.

For an NME that is a BCRP inhibitor, possible candidate probe substrates include sulphasalazine189, rosuvastatin190, pitavastatin41, ciprofloxacin47 and dipyridamole191. However, many of these remain to be tested as selective BCRP probe substrates in clinical studies.

An external file that holds a picture, illustration, etc.
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Note 1: A unidirectional assay based on the probe substrate can also be considered188. Inside-out vesicles expressing P-gp or BCRP, or transfected cell monolayers grown on solid support with appropriate controls (for example, inhibitors and positive controls, as described in Section 2), can also be used for inhibition assays. Note 2: If fu cannot be accurately determined owing to high protein binding, then assume fu = 0.01, to err on the conservative side. Alternatively, unbound portal vein concentrations may be used and might be more applicable to liver uptake192193. Note 3: Digoxin has an absolute bioavailability of approximately 70% from immediate release tablets. By contrast, the absolute bioavailability from a liquid-filled formulation (for example, Lanoxicaps) is approximately 90%. Therefore, consideration should also be given to the dosage form of digoxin used in an interaction study. In addition, digoxin may be a substrate for other transporters1592188194. Hence, caution is advised when extrapolating results from digoxin to other P-gp substrates.

The conclusions from in vitro efflux studies are dependent on the cell line used. For example, for cells heterologously expressing P-gp or BCRP (for example, MDCK or LLC-PK1), in which expression of the transporters can be high, the efflux ratio would be expected to be much higher than 2 for strong substrates. A classification of relevant substrates versus non-substrates will depend on the assay system. Validation studies of in vitro assay systems using known weak and strong substrates and, where possible, correlations with in vivo model systems, should be established. Meaningful efflux ratios are dependent on the transporter expression; thus, results should be related to a reference compound. In cell lines such as Caco-2, which express multiple uptake and efflux transporters, reduction of efflux in the presence of an inhibitor may support efflux by P-gp and/or BCRP, depending on the selectivity of the inhibitor used (for example, cyclosporine inhibits multiple transporters).

If in vitro experiments suggest that the NME is a P-gp and/or BCRP substrate (see (c) in the figure), preclinical and clinical information should be assessed to determine whether a clinical in vivo DDI study is warranted. In particular, the relative contribution of the transporter-mediated pathway to the overall clearance of the drug is the primary determinant of whether an inhibitor will have a major effect on the disposition of the NME. For example, the pharmacokinetics of an NME that has high solubility, high permeability and/or is highly metabolized are less likely to be affected by a co-administered drug that is a P-gp inhibitor15173174. Therefore, an in vivo interaction study may not be needed. By contrast, an NME that has poor solubility, limited permeability and is metabolically stable (eliminated primarily as the parent compound) is more likely to demonstrate a pharmacokinetic change in the presence of an inhibitor. The possible clinical consequence of such an interaction needs to be assessed based on careful evaluation of available exposure–response data186187. The recommended clinical study with a known P-gp inhibitor should consider the therapeutic use of the NME; however, cyclosporine is a reasonable choice, as it is known to inhibit P-gp in vivo. As noted previously, because cyclosporine inhibits multiple transporters, care must be taken in the design and interpretation of DDI studies in which it is used as an inhibitor.

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This figure shows a decision tree for P-gp and a similar tree could be used for BCRP. Although flux systems have traditionally been used to determine whether an NME is a substrate of P-gp or BCRP, inside-out vesicles expressing P-gp or BCRP, or transfected cell monolayers grown on solid support with appropriate controls (for example, inhibitors and positive controls) as described in Section 2, also can be used.

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 BCRP3840. 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 topotecan4955. 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 BCRP3840. 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 topotecan4955. 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 OCTs7,5760 and OATs57,6163.

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) cells6569. 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 activity8,7274. 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 OCTs7,5760 and OATs57,6163.

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) cells6569. 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 activity8,7274. 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 electroneutral8587. OATP1B1 was cloned by several groups8891 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 rifampicin8691. 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 involvi