Drug-transporter Interactions Research Articles

Beyond the ITC White Paper: Emerging Sciences in Drug Transporters and Opportunities for Drug Development

Over the recent years there has been a greater appreciation in the important roles drug transporters play in drug-drug interactions (DDI), safety and effectiveness of drugs. Notable consequence of this recognition includes the white paper published by the International Transporter Consortium (ITC) and the guidance documents drafted by regulatory agencies for investigating transporter-mediated DDIs during drug development. While DDIs as a result of transporter-mediated alterations in drug absorption, disposition, or excretion are typically undesirable, there are exceptions. When specific transporters selectively regulate the exposure of a drug at the site of action and/or toxicity, the use of these transporters as molecular targets has been proposed as a promising strategy for tissue-selective drug delivery to enhance efficacy or mitigate toxicity. Furthermore, membrane transporters play a pivotal role in the transport of nutrients and endogenous compounds into or out of cells to sustain cell survival. Genetic polymorphism of drug transporters as well as transporter-inhibiting drugs can alter the transporter functional activity and/or protein expression, causing transporter-specific diseases. Therefore, investigating drug-transporter interactions is a critical aspect in candidate drug selection, in order to enhance the pharmacological effects and/or prevent the unintended off-target toxicity. The goal of this review is to provide the drug discovery scientists with a cadre of concepts beyond the ITC White Paper that facilitate rational drug design for optimal safety and efficacy. To that end, this review focuses on the following aspects: 1) regulatory landscape on drug transporter-mediated DDIs, 2) transporter related organ toxicity, 3) utility of drug transporters for target organ delivery, and 4) to highlight the diseases known thus far that are associated with variants of transporter genes.

Dynamic morphology and cytoskeletal protein changes during spontaneous inside-out vesiculation of red blood cell membranes

Vesicle preparations from cell plasma membranes, red blood cells in particular, are extensively used in transport and enzymic studies and in the fields of drug delivery and drug-transport interactions. Here we investigated the role of spectrin–actin, the main components of the red cell cortical cytoskeleton, in a particular mechanism of vesicle generation found to be relevant to the egress process of Plasmodium falciparum merozoites from infected red blood cells. Plasma membranes from red blood cells lysed in ice-cold media of low ionic strength and free of divalent cations spontaneously and rapidly vesiculate upon incubation at 37 °C rendering high yields of inside-out vesicles. We tested the working hypothesis that the dynamic shape transformations resulted from changes in spectrin–actin configuration within a disintegrating cytoskeletal mesh. We showed that cytoskeletal-free membranes behave like a two-dimensional fluid lacking shape control, that spectrin–actin remain attached to vesiculating membranes for as long as spontaneous movement persists, that most of the spectrin–actin detachment occurs terminally at the time of vesicle sealing and that naked membrane patches increasingly appear during vesiculation. These results support the proposed role of spectrin–actin in spontaneous vesiculation. The implications of these results to membrane dynamics and to the mechanism of merozoite egress are discussed.

AMINO ACID TRANSPORTERS IN DRUG DISCOVERY

Administered drugs interact with membrane transporters of epithelia, Blood Brain Barrier and other districts influencing their delivery and efficacy. Drugs can also be used as inhibitors of transporters involved in human pathology. Drug-transporter interactions are responsible of off-target effects contributing to toxicity. High Throughput Screening technologies increased the potential applications in therapy or in predicting side effects. These strategies will be helpful in reducing animal experimentation. The identification of transporters important for drug absorption, delivery and side effect production and the best technologies for studying interactions are the main goals in this field. Amino acid transporters are not yet considered in human therapy in spite of their involvement in several pathologies. The function of the amino acid transporters EAAT1, ASCT2, GLYT2, GLYT1, B0AT1, LAT1 and LAT2 is so far well characterized. Some structural data on these transporters have also been obtained by bioinformatics. Interactions of these proteins with several drugs have been well defined at the molecular level. Large scale and, in some cases, high throughput screening of pharmacological compounds make these transporters of particular interest and potential application in human health.

Assessment of Drug Transporter Function Using Fluorescent Cell Imaging

ATP-binding cassette (ABC) proteins, including the breast cancer resistance protein (BCRP) and multidrug resistance proteins (MDRs), actively transport structurally diverse chemicals from a number of tissues and are being increasingly cited as mediators of clinically relevant drug-drug interactions. The potential outcomes of concomitantly administering two drugs that interact at the same transporter include altered disposition and toxicity and/or efficacy of one or both of the drugs. Research demonstrating the role of transporters in clinical pharmacokinetics has shed light on the need for in vitro screening methods that detect drug-transporter interactions during preclinical development. This unit describes cell-based procedures for detecting functional inhibitors of BCRP and MDR1 by measuring fluorescent substrate accumulation in suspended cells using an automated cell counter, which offers convenience, sensitivity, and speed in measuring intracellular fluorescence and identifying new inhibitors. An alternative method is provided for making similar measurements using a spectrophotometer with fluorescence detection capabilities.

Abstract 885: Human organic cation transporter 1 (hOCT1) as a mediator of bendamustine cytotoxic action.

Abstract Bendamustine hydrochloride is a cytotoxic agent that encompasses structural features of alkylating and nucleoside-derived drugs. This drug is currently used in the treatment of several haemotological malignancies; among them Chronic Lymphocytic Leukemia (CLL). CLL is the most common leukemia in Western countries and is characterized by the accumulation of monoclonal CD5+ B cells in the blood, bone marrow, lymph nodes, spleen and other organs. The pathways facilitating the uptake of bendamustine hydrochloride into target cells still remain poorly understood. Here we have addressed this issue by monitoring the interaction of the drug with selected plasma membrane transporter candidates, which might be relevant to bendamustine pharmacokinetics and pharmacodynamics. We have used a panel of HEK293 cells stably expressing selected drug transporter proteins to prove that bendamustine is able to interact with human Organic Cation Transporter 1 (hOCT1) with a Ki of 140+/-10 μM, as deduced from cis-inhibition studies using tritiated 1-methyl-4-phenylpyridinium (MPP+) as hOCT model substrate. Genetic variants known to modify substrate/transporter interactions, some of them with high allelic frequency in humans, were also tested and, in particular, hOCT1M420Del showed a significant decrease in its affinity for the drug (Ki value 234+/-24 μM, p<0.05 vs the wild type transporter). The whole panel of hOCT1 and hOCT1 allelic variants were also tested for bendamustine-induced cytotoxicity and data consistent with bendamustine being a hOCT1 substrate were obtained. This was based upon the finding that hOCT1 expression triggered increased sensitivity to the drug and variants known to decrease drug-transporter affinity interactions were also shown to induce resistance. hOCT1 is expressed in CLL cells and genotyping of a cohort of 125 CLL patients is now allowing the analysis of the relationships between particular genetic variants and ex vivo bendamustine-induced cytotoxicity, thus opening the possibility that hOCT1 genotyping might be a useful tool in optimizing bendamustine-based therapies. Citation Format: Cristina Arimany-Nardi, Arnau Montraveta, Eriong Lee-Vergés, Hermann Koepsell, Dolors Colomer, Marçal Pastor-Anglada. Human organic cation transporter 1 (hOCT1) as a mediator of bendamustine cytotoxic action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 885. doi:10.1158/1538-7445.AM2013-885

In Vitro and In Vivo Human Metabolism of Degarelix, a Gonadotropin-Releasing Hormone Receptor Blocker

Degarelix is a decapeptide that shows high affinity/selectivity to human gonadotropin-releasing hormone receptors and has been approved for the treatment of advanced prostate cancer in the United States, European Union, and Japan. To investigate the metabolism of degarelix in humans, in vitro metabolism was addressed in liver tissue and in vivo metabolism was studied in plasma and excreta samples collected in clinical studies. In addition, drug transporter interaction potential of degarelix with selected efflux transporters and uptake transporters was studied using in vitro membrane vesicle-based assays and whole cell-based assays. In vitro degradation was observed in fresh hepatocytes; less than 25% of the initial concentration of degarelix remained after incubation at 37°C for 2 hours. One metabolite was detected, representing a truncated nonapeptide of degarelix. The same metabolite was also detected at low concentrations in plasma. The in vivo investigations also showed that degarelix is excreted unchanged via the urine but is undergoing extensive sequential peptidic degradation during its elimination via the hepato-biliary pathway. No unique human metabolites of degarelix were detected in the circulation or in the excreta. Degarelix did not show any interaction with selected efflux transporters and uptake transporters up to concentrations representing 200 times the clinical concentration. Because degarelix does not seem to interact with the cytochrome P450 enzyme system as substrate, inhibitor, or inducer and does not show any interaction with hepatic and renal uptake and efflux transporters, the risk for pharmacokinetic drug-drug interactions with this compound is highly unlikely.

Computational Prospecting for Drug–Transporter Interactions

Membrane transporters are recognized as important determinants of drug disposition, action, and adverse events, yet our understanding of the molecular determinants of drug–transporter interactions is limited. Here, we discuss how computational analysis of transporter amino acid sequence, transporter structure, and ligand chemistry can be used to explore transporter function at the molecular level and to predict interactions with drug molecules. Clinical Pharmacology & Therapeutics (2013); 94 1, 30–32. doi:10.1038/clpt.2013.67

Structure–Activity Relationship for FDA Approved Drugs As Inhibitors of the Human Sodium Taurocholate Cotransporting Polypeptide (NTCP)

The hepatic bile acid uptake transporter sodium taurocholate cotransporting polypeptide (NTCP) is less well characterized than its ileal paralog, the apical sodium dependent bile acid transporter (ASBT), in terms of drug inhibition requirements. The objectives of this study were (a) to identify FDA approved drugs that inhibit human NTCP, (b) to develop pharmacophore and Bayesian computational models for NTCP inhibition, and (c) to compare NTCP and ASBT transport inhibition requirements. A series of NTCP inhibition studies were performed using FDA approved drugs, in concert with iterative computational model development. Screening studies identified 27 drugs as novel NTCP inhibitors, including irbesartan (Ki = 11.9 μM) and ezetimibe (Ki = 25.0 μM). The common feature pharmacophore indicated that two hydrophobes and one hydrogen bond acceptor were important for inhibition of NTCP. From 72 drugs screened in vitro, a total of 31 drugs inhibited NTCP, while 51 drugs (i.e., more than half) inhibited ASBT. Hence, while there was inhibitor overlap, ASBT unexpectedly was more permissive to drug inhibition than was NTCP, and this may be related to NTCP possessing fewer pharmacophore features. Findings reflected that a combination of computational and in vitro approaches enriched the understanding of these poorly characterized transporters and yielded additional chemical probes for possible drug-transporter interaction determinations.

Drug-transporter interaction testing in drug discovery and development

The human body consists of several physiological barriers that express a number of membrane transporters. For an orally absorbed drug the intestinal, hepatic, renal and blood-brain barriers are of the greatest importance. The ATP-binding cassette (ABC) transporters that mediate cellular efflux and the solute carrier transporters that mostly mediate cellular uptake are the two superfamilies responsible for membrane transport of vast majority of drugs and drug metabolites. The total number of human transporters in the two superfamilies exceeds 400, and about 40-50 transporters have been characterized for drug transport. The latest Food and Drug Administration guidance focuses on P-glycoprotein, breast cancer resistance protein, organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, organic cation transporter 2 (OCT2), and organic anion transporters 1 (OAT1) and OAT3. The European Medicines Agency’s shortlist additionally contains the bile salt export pump, OCT1, and the multidrug and toxin extrusion transporters, multidrug and toxin extrusion protein 1 (MATE1) and MATE2/MATE2K. A variety of transporter assays are available to test drugtransporter interactions, transporter-mediated drugdrug interactions, and transporter-mediated toxicity. The drug binding site of ABC transporters is accessible from the cytoplasm or the inner leaflet of the plasma membrane. Therefore, vesicular transport assays utilizing inside-out vesicles are commonly used assays, where the directionality of transport results in drugs being transported into the vesicle. Monolayer assays utilizing polarized cells expressing efflux transporters are the test systems suggested by regulatory agencies. However, in some monolayers, uptake transporters must be coexpressed with efflux transporters to assure detectable transport of low passive permeability drugs. For uptake transporters mediating cellular drug uptake, utilization of stable transfectants have been suggested. In vivo animal models complete the testing battery. Some issues, such as in vivo relevance, gender difference, age and ontogeny issues can only be addressed using in vivo models. Transporter specificity is provided by using knock-out or mutant models. Alternatively, chemical knock-outs can be employed. Compensatory changes are less likely when using chemical knockouts. On the other hand, specific inhibitors for some uptake transporters are not available, limiting the options to genetic knock-outs. © 2013 Baishideng. All rights reserved.

Computational Modeling to Accelerate the Identification of Substrates and Inhibitors for Transporters That Affect Drug Disposition

We have seen the increased use of computational approaches to predict drug interactions with human transporters that affect drug disposition and may lead to toxicity. These predominantly ligand-based methods use limited experimental data but provide new insights into structure activity relationships (SARs). The promiscuity of ligand interaction with transporters represents a challenge to computational methods. Development of models capable of identifying new transport substrates and unwanted drug-drug interactions requires novel applications of current computational methods. The clinical importance of efflux and uptake transporters in drug disposition is widely acknowledged [1–5] and membrane transporter anomalies are the basis for certain clinical disorders. Consequently, increasing attention is being paid to the potential for transport-based toxicity in vivo, and to unwanted drug-drug interactions [6–9] and consequences from polymorphic transport activity [10]. Furthermore, with hundreds of transporters yet to be characterized, the potential exists for many new drug targets to be discovered [8]. Computational models could therefore enable repurposing of already approved drugs [11] as well as predict the potential for, and ultimately preempt undesirable effects [12–14] that are based on drug-transporter interactions.

Insights into binding events of GABA- and Tiagabine- analogues in the Gamma-Aminobutyric Acid Transporter 1 by means of Molecular Modelling

The human transporters for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) hGAT-1, 2, and 3, and hBGT-1 belong to the neurotransmittersodium symporter (NSS) family of membrane transport proteins. hGAT-1 has been a target for the design of antiepileptic therapeutics [1], with tiagabine (Gabitril ® ) being the only GAT inhibitor on the market. The lack of specific inhibitors for the other hGAT subtypes, results from a still missing detailed understanding of the molecular basis of drug-transporter interactions of the respective subtypes. We aim at elucidating plausible binding modes for ligands of each subtype, respectively. In our first studies, we built up homology models for hGAT-1 in the occluded and outward-facing conformation based on the respective high resolution structures of the leucine transporter of Aquifex aeolicus (LeuT) (pdb-codes: 2A65 and 3F3A). Afterwards, the natural substrate GABA was docked into the occluded state model and tiagabine into the outward-facing model by making use of the Induced Fit Docking module of Schrodinger, LLC. Both models were further subject to extensive Molecular Dynamics (MD) simulation studies (GROMACS 4.5.3 was used). By the aid of MD simulations we could detect the existence of conserved water molecules into the GABA (occluded) and tiagabine (open-to-out) binding sites, respectively. Average structures from the equilibrated trajectories were extracted and subsequently served for further docking experiments. Docking of small ligands (GABA, Guvacine and R-/S Nipecotic Acid) into the occluded state model nicely indicated their preference to bind in an extended conformation (also demonstrated by long-term MD with GABA). MD and Docking of tiagabine and analogues into the open-to-out state model revealed a common binding mode. Additionally, our studies will be extended to the other hGAT subtypes so as to elucidate the secret of subtype selectivity of GABA Transporters.

Associations Between ABCC2 Polymorphisms and Cisplatin Disposition and Efficacy

ABCC2 (MRP2, cMOAT) expression has been implicated in cisplatin resistance in vitro. In mice, cisplatin disposition and toxicity were unaffected by Abcc2 knockout (Abcc2−/−). Moreover, in cancer patients (n = 237), cisplatin pharmacokinetics (P > 0.12) and efficacy (P > 0.41) were not associated with seven of the single-nucleotide polymorphisms (SNPs) in ABCC2. These SNPs were also not correlated with ABCC2 expression in the NCI60 panel (P > 0.26) or with cisplatin-induced cytotoxicity (P = 0.21). These findings highlight the importance of verifying drug-transporter interactions with in vitro tests in humans.

Drug-Permeability and Transporter Assays in Caco-2 and Mdck Cell Lines

The human colon adenocarcinoma Caco-2 and Madin-Darby canine kidney epithelial cell lines provide in vitro tools to assess a drug's permeability and transporter interactions during discovery and development. The cells, when cultured on semiporous filters, form confluent monolayers that model the intestinal epithelial barrier for permeability, transporter and drug-interaction assays. The applications of these assays in pharmaceutical research include qualitative prediction and ranking of absorption, determining mechanism(s) of permeability, formulation effects on drug permeability, and the potential for transporter-mediated drug-drug interactions. This review focuses on recent examples of Caco-2 and Madin-Darby canine kidney cells assays for drug permeability including transfected and knock-down cells, miniaturization and automation, and assay combinations to better understand and predict intestinal drug absorption.

Nonsteroidal Anti-Inflammatory Drugs May Reduce Enterohepatic Recirculation of Mycophenolic Acid in Patients With Childhood-Onset Systemic Lupus Erythematosus

The large interindividual differences observed in mycophenolic acid (MPA) pharmacokinetics (MPA-PK) are in part attributed to the large variability in enterohepatic recirculation (EHC) of the drug. The main metabolite of MPA, MPA glucuronide is actively secreted into the bile via the multidrug resistance-associated protein 2 (MRP2) transporter. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit the MRP2 transporter, which can alter EHC and drug exposure. Here, we evaluated the effects of this potential drug-transporter interaction on MPA-PK in a cohort of patients with childhood-onset systemic lupus erythematosus on mycophenolate mofetil therapy. Full MPA concentration-time profiles and demographics including comedications were available for 19 patients with childhood-onset systemic lupus erythematosus. Concentrations at predose (C(trough)), 9 hour (C₉), and nadir (C(nadir); defined as the lowest concentration between C(max) and C₉), and area under the curve (AUC₀₋₁₂ and AUC₆₋₁₂) were assessed using standard methods (WinNonlin5.1). AUC6-12/AUC₀₋₁₂ and C₉/C(nadir) ratios were used to evaluate the effects of NSAID treatment on MPA-PK. Eleven out of 19 patients were on NSAID treatment and did not show visual evidence of EHC in their PK profile. In contrast, patients not on NSAID therapy showed evidence of EHC-related MPA concentration increase in the later part of their PK profiles, typically after 6 hours. This phenomenon could be well characterized by the C₉/C(nadir) ratio, which was significantly lower in the NSAID-treated cohort (P < 0.01). These preliminary data suggest that the concomitant intake of NSAIDs may lower EHC of MPA possibly through the inhibition of MRP2 transport of MPA-G. Further mechanism-based studies are needed to further elucidate this potential transporter interaction.

Towards a structural understanding of drug and peptide transport within the proton-dependent oligopeptide transporter (POT) family.

One of the principal aims of modern drug design is the targeted delivery of drugs within the body, such as to the central nervous system, combined with their exclusion from the liver and kidneys, which break down foreign molecules and subsequently eliminate them. Many of the commonly prescribed drugs are transported into cells and across the plasma membrane via endogenous membrane transporters, whose principal roles are the uptake of essential nutrients for metabolism. In many cases, such drug transport is serendipitous as they are simply mistaken as 'natural' compounds. Many of these transporters could, however, be targeted more efficiently, improving drug absorption, distribution and retention. The molecular details of these drug-transporter interactions, however, are at best poorly understood, in large part through the absence of any high-resolution structural information. To address this issue, we recently determined the structure of a prokaryotic peptide transporter, PepTSo from Shewanella oneidensis, which shares a high degree of sequence similarity and functional characteristics with the human PepT1 and PepT2 proteins. PepT1 and PepT2 contribute significantly to the oral bioavailability and pharmacokinetic properties of a number of important drug families, including antibiotics, antivirals and anticancer agents. The crystal structure of PepTSo provides the first high-resolution model of a drug importer and provides the starting point for understanding drug and peptide transport within the human body.

Towards a structural understanding of drug and peptide transport within the proton-dependent oligopeptide transporter (POT) family

One of the principal aims of modern drug design is the targeted delivery of drugs within the body, such as to the central nervous system, combined with their exclusion from the liver and kidneys, which break down foreign molecules and subsequently eliminate them. Many of the commonly prescribed drugs are transported into cells and across the plasma membrane via endogenous membrane transporters, whose principal roles are the uptake of essential nutrients for metabolism. In many cases, such drug transport is serendipitous as they are simply mistaken as 'natural' compounds. Many of these transporters could, however, be targeted more efficiently, improving drug absorption, distribution and retention. The molecular details of these drug-transporter interactions, however, are at best poorly understood, in large part through the absence of any high-resolution structural information. To address this issue, we recently determined the structure of a prokaryotic peptide transporter, PepTSo from Shewanella oneidensis, which shares a high degree of sequence similarity and functional characteristics with the human PepT1 and PepT2 proteins. PepT1 and PepT2 contribute significantly to the oral bioavailability and pharmacokinetic properties of a number of important drug families, including antibiotics, antivirals and anticancer agents. The crystal structure of PepTSo provides the first high-resolution model of a drug importer and provides the starting point for understanding drug and peptide transport within the human body.

Environmental and Genetic Factors Affecting Transport of Imatinib by OATP1A2

The bioavailability of orally administered imatinib is >90%, although the drug is monocationic under the acidic conditions in the duodenum. In vitro, we found that imatinib is transported by the intestinal uptake carrier organic anion transporting polypeptide (OATP1A2) and that this process is sensitive to pH, rosuvastatin, and genetic variants. However, in a study in patients with cancer, imatinib absorption was not associated with OATP1A2 variants and was unaffected by rosuvastatin. These findings highlight the importance of verifying in a clinical setting the drug-transporter interactions observed in in vitro tests.

Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P‐glycoprotein (P‐gp) inhibition screen

The evaluation of interactions between drug candidates and transporters such as P-glycoprotein (P-gp) has gained considerable interest in drug discovery and development. Inhibition of P-gp can be assessed by performing bi-directional permeability studies with in vitro P-gp-expressing cellular model systems such as Caco-2 (human colon carcinoma) cells, using digoxin as a substrate probe. Existing methodologies include either assaying (3)H-digoxin with liquid scintillation counting (LSC) detection or assaying non-labeled digoxin with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis at a speed of several minutes per sample. However, it is not feasible to achieve a throughput high enough using these approaches to sustain an early liability screen that generates more than a thousand samples on a daily basis. To address this challenge, we developed an ultrafast (9 s per sample) bioanalytical method for digoxin analysis using RapidFire™, an on-line solid-phase extraction (SPE) system, with MS/MS detection. A stable isotope labeled analog, d3-digoxin, was used as internal standard to minimize potential ionization matrix effect during the RF-MS/MS analysis. The RF-MS/MS method was more than 16 times faster than the LC-MS/MS method but demonstrated similar sensitivity, selectivity, reproducibility, linearity and robustness. P-gp inhibition results of multiple validation compounds obtained with this RF-MS/MS method were in agreement with those generated by both the LC-MS/MS method and the (3)H-radiolabel assay. This method has been successfully deployed to assess P-gp inhibition potential as an important early liability screen for drug-transporter interaction.

Predicting the Response of CML Patients to Tyrosine Kinase Inhibitor Therapy

As of 2011, the choice of tyrosine kinase inhibitor (TKI) for the patient with newly diagnosed chronic-phase chronic myelogenous leukemia (CP-CML) is no longer limited to imatinib but can be expanded to include nilotinib and dasatinib. Since 2000, imatinib has demonstrated remarkable efficacy in the majority of chronic-phase patients. Nilotinib and dasatinib, both more potent TKIs, are likely to produce quicker and deeper molecular responses, but there are no established criteria for choosing the best inhibitor for each patient. We now need to establish clearly defined recommendations to address this new stage, in which individualized therapy in the front-line should become a reality. Likely to be paramount in this setting are assays that directly assess the efficacy of the protein-drug and drug-transporter interactions, taking into account factors intrinsic to the patient, factors related to disease stage, and the amount of drug freely available in the plasma.

Use of Transporter Knockdown Caco-2 Cells to Investigate the In Vitro Efflux of Statin Drugs

The objective of the present study was to determine the efflux transporters responsible for acid and lactone statin drug efflux using transporter knockdown Caco-2 cells. The bidirectional transport was determined in Caco-2 cell monolayers in which the expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), or multidrug resistance associated protein 2 (MRP2) was knocked down by transduction with lentivirus containing human transporter-targeted small hairpin RNAs (shRNAs). Cells transduced with lentivirus containing nontargeted shRNA served as the vector control. Atorvastatin, lovastatin, and rosuvastatin displayed extremely low apical-to-basolateral (A-to-B) transport, which made the P(app,A-B) values too unreliable to calculate the efflux ratio. Thus, transport comparisons were performed using the B-to-A permeability (P(app,B-A)) values. Presented in the order of vector control, P-gp, BCRP, and MRP2 knockdown Caco-2 cells, the P(app,B-A) values (×10(-6), cm/s) were 28.1 ± 1.3, 8.6 ± 2.9, 20.3 ± 1.8, and 21.5 ± 1.6 for atorvastatin; 96.1 ± 7.1, 25.3 ± 3.5, 57.3 ± 9.8, and 48.2 ± 2.3 for fluvastatin; and 14.1 ± 1.9, 4.6 ± 1.7, 5.8 ± 0.7, and 6.6 ± 1.8 for rosuvastatin, respectively. Lovastatin and simvastatin showed no efflux in the vector control or knockdown cell monolayers in either lactone or acid forms. Results indicate that atorvastatin, fluvastatin, and rosuvastatin were transported by P-gp, BCRP, and MRP2. On the other hand, neither the lactone nor the resulting acid of lovastatin and simvastatin was transported by P-gp, BCRP, or MRP2. The current study demonstrated that the transporter knockdown Caco-2 cells are useful tools for studying drug-transporter interactions and should help eliminate some of the ambiguity associated with the identification of drug-transporter interactions based on chemical inhibitors alone.