Modulation of monepantel secretion into milk by soy isoflavones.

Monepantel (MNP), a novel anthelmintic drug from amino-acetonitrile derivatives, is a substrate for breast cancer resistance protein (BCRP). BCRP-mediated milk secretion of drugs can be altered by isoflavones. In this study, we aimed to show how soy isoflavones and BCRP inhibitors genistein (GEN) and daidzein (DAI) can modulate the secretion of MNP into milk. Moreover, we observed that the expression of BCRP in the lactating mammary gland of sheep was significantly higher than in non-lactating sheep using Western blot analysis. These properties of MNP and MNPSO2 (monepantel sulfone, the major active metabolite of MNP), identified as a BCRP substrate in determining the interaction with BCRP, were examined by vesicular transport (VT) inhibition assays. In pharmacokinetic studies, we demonstrated the transport of MNP into milk in three experimental groups: G1 fed standard forage; G2 fed soy-enriched forage; G3 fed standard forage paired with orally administered exogenous GEN and DAI. The concentrations of MNP and MNPSO2 were analyzed by high-performance liquid chromatography. Compared to the control group (3.27 ± 1.13 vs. 5.46 ± 2.23), the AUC (0-840 h) milk/plasma ratio decreased by 40% in the soy-enriched diet group. The concentrations of GEN and DAI were determined using liquid chromatography coupled with tandem mass spectrometry in soy. A VT inhibition assay was conducted to determine the IC50 values for MNP and MNPSO2 as BCRP inhibitors. This study showed that milk excretion of a BCRP substrate, such as monepantel, can be diminished by the presence of isoflavones in the diet.

Cladribine as a Potential Object of Nucleoside Transporter-Based Drug Interactions.

Cladribine is a nucleoside analog that is phosphorylated in its target cells (B and T-lymphocytes) to its active triphosphate form (2-chlorodeoxyadenosine triphosphate). Cladribine tablets 10 mg (Mavenclad®), administered for up to 10 days per year in 2 consecutive years (3.5-mg/kg cumulative dose over 2 years), are used to treat patients with relapsing multiple sclerosis. Cladribine has been shown to be a substrate of various nucleoside transporters (NTs). Intestinal absorption and distribution of cladribine throughout the body appear to be essentially mediated by equilibrative NTs (ENTs) and concentrative NTs (CNTs), specifically by ENT1, ENT2, ENT4, CNT2 (low affinity), and CNT3. Other efficient transporters of cladribine are the ABC efflux transporters, specifically breast cancer resistance protein, which likely modulates the oral absorption and renal excretion of cladribine. A key transporter for the intracellular uptake of cladribine into B and T-lymphocytes is ENT1 with ancillary contributions of ENT2 and CNT2. Transporter-based drug interactions affecting absorption and target cellular uptake of a prodrug such as cladribine are likely to reduce systemic bioavailability and target cell exposure, thereby possibly hampering clinical efficacy. In order to manage optimized therapy, i.e., to ensure uncompromised target cell uptake to preserve the full therapeutic potential of cladribine, it is important that clinicians are aware of the existence of NT-inhibiting medicinal products, various lifestyle drugs, and food components. This article reviews the existing knowledge on inhibitors of NT, which may alter cladribine absorption, distribution, and uptake into target cells, thereby summarizing the existing knowledge on optimized methods of administration and concomitant drugs that should be avoided during cladribine treatment.

Role of Hepatocyte Transporters in Drug-Induced Liver Injury (DILI)-In Vitro Testing.

Bile acids and bile salts (BA/BS) are substrates of both influx and efflux transporters on hepatocytes. Canalicular efflux transporters, such as BSEP and MRP2, are crucial for the removal of BA/BS to the bile. Basolateral influx transporters, such as NTCP, OATP1B1/1B3, and OSTα/ß, cooperate with canalicular transporters in the transcellular vectorial flux of BA/BS from the sinusoids to the bile. The blockage of canalicular transporters not only impairs the bile flow but also causes the intracellular accumulation of BA/BS in hepatocytes that contributes to, or even triggers, liver injury. In the case of BA/BS overload, the efflux of these toxic substances back to the blood via MRP3, MRP4, and OST α/ß is considered a relief function. FXR, a key regulator of defense against BA/BS toxicity suppresses de novo bile acid synthesis and bile acid uptake, and promotes bile acid removal via increased efflux. In drug development, the early testing of the inhibition of these transporters, BSEP in particular, is important to flag compounds that could potentially inflict drug-induced liver injury (DILI). In vitro test systems for efflux transporters employ membrane vesicles, whereas those for influx transporters employ whole cells. Additional in vitro pharmaceutical testing panels usually include cellular toxicity tests using hepatocytes, as well as assessments of the mitochondrial toxicity and accumulation of reactive oxygen species (ROS). Primary hepatocytes are the cells of choice for toxicity testing, with HepaRG cells emerging as an alternative. Inhibition of the FXR function is also included in some testing panels. The molecular weight and hydrophobicity of the drug, as well as the steady-state total plasma levels, may positively correlate with the DILI potential. Depending on the phase of drug development, the physicochemical properties, dosing, and cut-off values of BSEP IC50 ≤ 25-50 µM or total Css,plasma/BSEP IC50 ≥ 0.1 may be an indication for further testing to minimize the risk of DILI liability.

Review of Transporter Substrate, Inhibitor, and Inducer Characteristics of Cladribine.

Cladribine is a nucleoside analog that is phosphorylated in its target cells (B- and T-lymphocytes) to its active adenosine triphosphate form (2-chlorodeoxyadenosine triphosphate). Cladribine tablets 10 mg (Mavenclad®) administered for up to 10 days per year in 2 consecutive years (3.5-mg/kg cumulative dose over 2 years) are used to treat patients with relapsing multiple sclerosis. The ATP-binding cassette, solute carrier, and nucleoside transporter substrate, inhibitor, and inducer characteristics of cladribine are reviewed in this article. Available evidence suggests that the distribution of cladribine across biological membranes is facilitated by a number of uptake and efflux transporters. Among the key ATP-binding cassette efflux transporters, only breast cancer resistance protein has been shown to be an efficient transporter of cladribine, while P-glycoprotein does not transport cladribine well. Intestinal absorption, distribution throughout the body, and intracellular uptake of cladribine appear to be exclusively mediated by equilibrative and concentrative nucleoside transporters, specifically by ENT1, ENT2, ENT4, CNT2 (low affinity), and CNT3. Renal excretion of cladribine appears to be most likely driven by breast cancer resistance protein, ENT1, and P-glycoprotein. The latter may play a role despite its poor cladribine transport efficiency in view of the renal abundance of P-glycoprotein. There is no evidence that solute carrier uptake transporters such as organic anion transporting polypeptides, organic anion transporters, and organic cation transporters are involved in the transport of cladribine. Available in vitro studies examining the inhibitor characteristics of cladribine for a total of 13 major ATP-binding cassette, solute carrier, and CNT transporters indicate that in vivo inhibition of any of these transporters by cladribine is unlikely.

Modulation of Urate Transport by Drugs.

BACKGROUND: Serum urate (SU) levels in primates are extraordinarily high among mammals. Urate is a Janus-faced molecule that acts physiologically as a protective antioxidant but provokes inflammation and gout when it precipitates at high concentrations. Transporters play crucial roles in urate disposition, and drugs that interact with urate transporters either by intention or by accident may modulate SU levels. We examined whether in vitro transporter interaction studies may clarify and predict such effects. METHODS: Transporter interaction profiles of clinically proven urate-lowering (uricosuric) and hyperuricemic drugs were compiled from the literature, and the predictive value of in vitro-derived cut-offs like Cmax/IC50 on the in vivo outcome (clinically relevant decrease or increase of SU) was assessed. RESULTS: Interaction with the major reabsorptive urate transporter URAT1 appears to be dominant over interactions with secretory transporters in determining the net effect of a drug on SU levels. In vitro inhibition interpreted using the recommended cut-offs is useful at predicting the clinical outcome. CONCLUSIONS: In vitro safety assessments regarding urate transport should be done early in drug development to identify candidates at risk of causing major imbalances. Attention should be paid both to the inhibition of secretory transporters and inhibition or trans-stimulation of reabsorptive transporters, especially URAT1.

Inhibition of ABCG2/BCRP-mediated transport-correlation analysis of various expression systems and probe substrates.

BCRP / ABCG2 is a key determinant of pharmacokinetics of substrate drugs. Several BCRP substrates and inhibitors are of low passive permeability, and the vesicular transport assay works well in this permeability space. Membranes were prepared from BCRP-HEK293, MCF-7/MX, and baculovirus-infected Sf9 cells with (BCRP-Sf9-HAM), and without (BCRP-Sf9) cholesterol loading. Km values for three substrates - estrone-3-sulfate, sulfasalazine, topotecan - correlated well between the four expression systems. In contrast, a 10-20-fold range in Vmax values was observed, with BCRP-HEK293 membranes possessing the largest dynamic range. IC50 values of the different test systems were similar to each other, with 94.4% of pairwise comparisons being within 3-fold. Substrate dependent inhibition showed somewhat greater variation, as 81.4% of IC50 values in the BCRP-HEK293 membranes were within 3-fold in pairwise comparisons. Overall, BCRP-HEK293 membranes demonstrated the highest activity. The IC50 values showed good concordance but substrate dependent inhibition was observed for some drugs.

Prediction of Drug-Induced Hyperbilirubinemia by In Vitro Testing.

Bilirubin, the end product of heme catabolism, is produced continuously in the body and may reach toxic levels if accumulates in the serum and tissues; therefore, a highly efficient mechanism evolved for its disposition. Normally, unconjugated bilirubin enters hepatocytes through the uptake transporters organic anion transporting polypeptide (OATP) 1B1 and 1B3, undergoes glucuronidation by the Phase II enzyme UDP glucuronosyltransferase 1A1 (UGT1A1), and conjugated forms are excreted into the bile by the canalicular export pump multidrug resistance protein 2 (MRP2). Any remaining conjugated bilirubin is transported back to the blood by MRP3 and passed on for uptake and excretion by downstream hepatocytes or the kidney. The bile salt export pump BSEP as the main motor of bile flow is indirectly involved in bilirubin disposition. Genetic mutations and xenobiotics that interfere with this machinery may impede bilirubin disposition and cause hyperbilirubinemia. Several pharmaceutical compounds are known to cause hyperbilirubinemia via inhibition of OATP1Bs, UGT1A1, or BSEP. Herein we briefly review the in vitro prediction methods that serve to identify drugs with a potential to induce hyperbilirubinemia. In vitro assays can be deployed early in drug development and may help to minimize late-stage attrition. Based on current evidence, drugs that behave as mono- or multispecific inhibitors of OATP1B1, UGT1A1, and BSEP in vitro are at risk of causing clinically significant hyperbilirubinemia. By integrating inhibition data from in vitro assays, drug serum concentrations, and clinical reports of hyperbilirubinemia, predictor cut-off values have been established and are provisionally suggested in this review. Further validation of in vitro readouts to clinical outcomes is expected to enhance the predictive power of these assays.

Age-Related Functional and Expressional Changes in Efflux Pathways at the Blood-Brain Barrier.

During the last decade, several articles have reported a relationship between advanced age and changes in the integrity of the blood-brain barrier (BBB). These changes were manifested not only in the morphology and structure of the cerebral microvessels but also in the expression and function of the transporter proteins in the luminal and basolateral surfaces of the capillary endothelial cells. Age-associated downregulation of the efflux pumps ATP-binding cassette transporters (ABC transporters) resulted in increased permeability and greater brain exposure to different xenobiotics and their possible toxicity. In age-related neurodegenerative pathologies like Alzheimer's disease (AD), the amyloid-ß (Aß) clearance decreased due to P-glycoprotein (P-gp) dysfunction, leading to higher brain exposure. In stroke, however, an enhanced P-gp function was reported in the cerebral capillaries, making it even more difficult to perform effective neuroprotective therapy in the infarcted brain area. This mini-review article focuses on the efflux functions of the transporters and receptors of the BBB in age-related brain pathologies and also in healthy aging.

ABCG2/BCRP: variants, transporter interaction profile of substrates and inhibitors.

INTRODUCTION: ABCG2 has a broad substrate specificity and is one of the most important efflux proteins modulating pharmacokinetics of drugs, nutrients and toxicokinetics of toxicants. ABCG2 is an important player in transporter-mediated drug-drug interactions (tDDI). Areas covered: The aims of the review are i) to cover transporter interaction profile of substrates and inhibitors that can be utilized to test interaction of drug candidates with ABCG2, ii) to highlight main characteristics of in vitro testing and iii) to describe the structural basis of the broad substrate specificity of the protein. Preclinical data utilizing Abcg2/Bcrp1 knockouts and clinical studies showing effect of ABCG2 c.421C>A polymorphism on pharmacokinetics of drugs have provided evidence for a broad array of drug substrates and support drug - ABCG2 interaction testing. A consensus on using rosuvastatin and sulfasalazine as intestinal substrates for clinical studies is in the formation. Other substrates relevant to the therapeutic area can be considered. Monolayer efflux assays and vesicular transport assays have been extensively utilized in vitro. Expert opinion: Clinical substrates display complex pharmacokinetics due to broad interaction profiles with multiple transporters and metabolic enzymes. Substrate-dependent inhibition has been observed for several inhibitors. Harmonization of in vitro and in vivo testing makes sense. However, rosuvastatin and sulfasalazine are not efficiently transported in either MDCKII or LLC-PK1-based monolayers. Caco-2 monolayer assays and vesicular transport assays are potential alternatives.

Determination of Reference Values of MDR-ABC Transporter Activities in CD3+ Lymphocytes of Healthy Volunteers Using a Flow Cytometry Based Method.

BACKGROUND: MDR transporters are important biomarkers of drug resistance in cancer and in autoimmune conditions. We determined the MDR1, MRP1 and BCRP activity in CD3+ lymphocytes using a flow cytometry based method from 120 healthy volunteers in order to describe normal reference values of the activity of these transporters. The effects of gender and age were also determined. METHODS: The Solvo MDQ Kit™ was used for measurements. In this assay, fluorescent reporter substrates (Calcein-AM for MDR1 and MRP1 and mitoxantrone for BCRP, respectively) are trapped in the cytoplasm and pumped out by MDR proteins depending on the presence or absence of specific inhibitors (verapamil for MDR1 and MRP1, indomethacin for MRP1 and KO134 for BCRP, respectively), allowing for quantitative, standardized assessment. Cell surface staining was applied to select CD3+ cells. RESULTS: MAF values of MRP1 and BCRP are independent from age. MAFC and MAF of MDR1 show negative correlation with the age of the studied subjects (P = 0.003, r = -0.27 and P = 0.0001, r = -0.34, respectively). No difference was detected in any of the four MAF values between men and women. Gender does not affect the presence or lack of correlation between MAF values and age. CONCLUSIONS: The determination of the functional activity of MDR-ABC transporters is achievable using a flow cytometry based standardized method. Having established the normal range of MAF values on CD3+ lymphocytes of a healthy population, our results allow for the development of novel flow cytometry based diagnostic tools. © 2018 International Clinical Cytometry Society.

Inhibitor selectivity of CNTs and ENTs.

The concentrative nucleoside transporters (CNT; solute carrier family 28 (SLC28)) and the equilibrative nucleoside transporters (ENT; solute carrier family 29 (SLC29)) are important therapeutic targets but may also mediate toxicity or adverse events. To explore the relative role of the base and the monosaccharide moiety in inhibitor selectivity we selected compounds that either harbor an arabinose moiety or a cytosine moiety, as these groups had several commercially available drug members. The screening data showed that more compounds harboring a cytosine moiety displayed potent interactions with the CNTs than compounds harboring the arabinose moiety. In contrast, ENTs showed a preference for compounds with an arabinose moiety. The correlation between CNT1 and CNT3 was good as five of six compounds displayed IC50 values within the threefold threshold and one displayed a borderline 4-fold difference. For CNT1 and CNT2 as well as for CNT2 and CNT3 only two of six IC50 values correlated and one displayed a borderline 4-fold difference. Interestingly, of the six compounds that potently interacted with both ENT1 and ENT2 only nelarabine displayed selectivity. Our data show differences between inhibitor selectivities of CNTs and ENTs as well as differences within the CNT family members.

Correlation Analysis of Potential Breast Cancer Resistance Protein Probes in Different Monolayer Systems.

Breast cancer resistance protein (BCRP) is a point of interest in drug-drug interaction safety testing. Therefore, a consensus probe that can be applied as victim in multiple experimental settings is of great benefit. Identification of candidates has been driven by the amount and quality of available clinical data, and as a result, drugs such as sulfasalazine and rosuvastatin have been suggested. In this article, the in vitro performance of 5 possible alternatives was evaluated: atorvastatin, chlorothiazide, dantrolene, topotecan, and teriflunomide, and benchmarked against sulfasalazine and rosuvastatin in reference in vitro assays for BCRP drug-drug interaction testing. Based on the results, teriflunomide is proposed as an alternate in vitro BCRP probe.

Human OATP1B1 (SLCO1B1) transports sulfated bile acids and bile salts with particular efficiency.

Human OATP1B1 is highly expressed at the basolateral membrane of the hepatocyte. It plays an important role in the sodium-independent transport of bile acids and bile salts and contributes to the systemic clearance of many drugs. In this study, the interaction of at least one representative of all major chemical classes of bile acids and bile salts, which include the bile acid chenodeoxycholate (CDC), monovalent (amidated) bile salts glycochenodeoxycholate (GCDC), taurochenodeoxycholate (TCDC) and taurocholate (TC), a sulfated bile acid 3-sulfo-chenodeoxycholate (3S-CDC) and a divalent (amidated and sulfated) bile salt 3-sulfo-glycolithocholate (3S-GLC) were tested with OATP1B1 overexpressed in HEK293 cells. All bile acid derivatives except for CDC showed an efficient transport by OATP1B1. 3S-GLC gave the lowest KM (0.708 ±â€¯0.125 µM) and 3S-CDC showed the highest Vmax value (158 ±â€¯87.3 pmol/mg protein/min). The ranking of Clint values (3S-GLC > 3S-CDC > TCDC > GCDC > TC) also showed a preference for sulfated derivatives. In summary, human OATP1B1 transports sulfate esters of bile acids and bile salts more efficiently than monovalent bile salts.

Baicalin is a substrate of OATP2B1 and OATP1B3.

The use and significance of baicalin, the main bioactive component found in Radix Scutellaria, have been on the rise due to its interesting pharmacological properties. Baicalin, a low passive permeability compound, is directly absorbed from the upper intestine and its hepatic elimination is dominant. However, interaction but no transport studies have implicated organic anion­transporting polypeptides in its cellular uptake. By using mammalian cells stably expressing the uptake transporters of interest, we are showing that baicalin is a potent substrate of Organic anion­transporting polypeptide 2B1 (OATP2B1) and less potent substrate of OATP1B3. OATP2B1 and OATP1B3 transport baicalin and may play a role in the hepatic uptake of baicalin formed in the intestine.

Kinetic characterization of bile salt transport by human NTCP (SLC10A1).

The transport of bile acids facilitated by NTCP is an important factor in establishing bile flow. In this study, we examine the kinetics associated with human NTCP-dependent transport of two quantitatively important bile acids comprising the human bile acid pool, chenodeoxycholic acid and glycine-chenodeoxycholate, and secondary bile salt, 3-sulfo-glycolithocholate of potential toxicological significance. The study employed human NTCP overexpressing Chinese Hamster Ovary cells and results compared with taurocholate, a prototypical bile salt commonly used in transporter studies. GCDC and 3S-GLC but not CDCA were transported by NTCP. The efficient uptake of GCDC, TCA and 3S-GLC by NTCP enabled the determination of kinetics. GCDC displayed a lower KM (0.569±0.318µM) than TCA (6.44±3.83µM) and 3S-GLC (3.78±1.17µM). The apparent CLint value for GCDC was 20-fold greater (153±53µl/mg protein/min) than the apparent CLint for TCA (6.92±4.72µl/mg protein/min) and apparent CLint for 3S-GLC (8.05±1.33µl/mg protein/min). These kinetic results provide important complementary data on the substrate selectivity and specificity of NTCP to transport bile acids. NTCP transports GCDC with greater efficiency than TCA and has the same efficacy for 3S-GLC and TCA.

Abcb1a (P-glycoprotein) limits brain exposure of the anticancer drug candidate seliciclib in vivo in adult mice.

Seliciclib displayed limited brain exposure in vivo in adult rats with mature blood-brain barrier (BBB). Selicilib was shown to be a specific substrate of human ABCB1 in vitro. To demonstrate that ABCB1/Abcb1 can limit brain exposure in vivo in mice we are showing that seliciclib is a substrate of mouse Abcb1a, the murine ABCB1 ortholog expressed in the BBB as LLC-PK-Abcb1a cells displayed an efflux ratio (ER) of 15.31±3.54 versus an ER of 1.44±0.10 in LLC-PK1-mock cells. Additionally, in the presence of LY335979, an ABCB1/Abcb1a specific inhibitor, the observed ER for seliciclib in the LLC-PK1-mMdr1a cells decreased to 1.05±0.25. To demonstrate in vivo relevance of seliciclib transport by Abcb1a mouse brain microdialysis experiments were carried out that showed that the AUCbrain/AUCblood ratio of 0.143 in anesthetized mice increased about two-fold to 0.279 in the presence of PSC833 another ABCB1/Abcb1a specific inhibitor. PSC833 also increased the brain exposure (AUCbrain) of seliciclib close to 2-fold (136 vs 242) in awake mice. In sum, Abcb1a significantly decreases seliciclib permeability in vitro and is partly responsible for limited brain exposure of seliciclib in vivo in mice.

Species specificity profiling of rat and human organic cation/carnitine transporter Slc22a5/SLC22A5 (Octn2/OCTN2).

The purpose of this study was to characterize the uptake of carnitine, the physiological substrate, and the uptake of 3-(2,2,2-trimethylhydrazinium)propionate, a consensus substrate by rat Octn2 and human OCTN2 transporters as well as to characterize drug-mediated inhibition of l-carnitine uptake by the rat and human orthologs overexpressed in CHO-K1 cells. l-carnitine and 3-(2,2,2-trimethylhydrazinium)propionate were found to be a lower affinity substrate for rat Octn2 (KM = 32.66 ± 5.11 µM and 23.62 ± 4.99 µM respectively) than for human OCTN2 (KM = 3.08 ± 0.74 µM and 7.98 ± 0.63 µM). The intrinsic clearance (CLint) value for carnitine was higher for the human than for the rat transporter (22.82 ± 5.57 ml/min*mg vs 4.008 ± 0.675 ml/min*mg). For 3-(2,2,2-trimethylhydrazinium)propionate, in contrast, the CLint value for rat Octn2 was higher than for human OCTN2 (323.9 ± 72.8 ml/min*mg vs 65.11 ± 5.33 ml/min*mg). Furthermore, many pharmacologically important drugs were shown to affect l-carnitine transport by Octn2/OCTN2. The correlation between the IC50 datasets for the rat and human transporter resulted in an r value of 0.47 (p > 0.05). However, the greatest difference was less than seven-fold and 13 of 15 compounds yielded a difference less than 3-fold. Thus, the transporters from these two species showed an overlapping but somewhat different substrate and inhibitor specificity.

Membrane Transporters in Physiological Barriers of Pharmacological Importance.

Membrane transporters expressed in barrier forming cell types provide a dual filtration system as unwanted xenobiotics are effluxed by ABC transporters, and compounds essential for the organism, such as nutrients or physiological substrates, are taken up by influx transporters. The majority of efflux transporters apically-localized in barrier forming cell types are ABC transporters that may limit absorption or distribution, and promote excretion. Pharmaceutical scientists are increasingly aware of the limitations these efflux transporters represent. Influx transporters are also critically important, as apically-located influx transporters may counteract the effect of co-localized efflux transporters, promoting absorption or reabsorption, as well as facilitating distribution of low passive permeability substrates into tissues that are otherwise heavily guarded by efflux transporters. In excretory organs, basolaterally-localized influx transporters cooperate with apically-localized efflux trransporters to efficiently drive transcellular movement of xenobiotics and their metabolites. Pharmacological inhibition of absorption or reabsorption of unwanted nutrients and endobiotics has become a great opportunity for pharmaceutical development. For drug developers, these transporters also offer the opportunity to target specific organs and cell types. Targeting drugs to cells and tissues harboring the pharmacological target not only makes drugs more efficient, but can also make them less toxic, as it allows for administration of lower doses and less distribution of drugs into non-target organs.

Investigating ABCB1-Mediated Drug-Drug Interactions: Considerations for In vitro and In vivo Assay Design.

BACKGROUND: ABCB1 is a key ABC efflux transporter modulating the pharmacokinetics of a large percentage of drugs. ABCB1 is also a site of transporter mediated drug-drug interactions (tDDI). It is the transporter most frequently tested for tDDIs both in vitro and in the clinic. OBJECTIVE: Understanding the limitations of various in vitro and in vivo models, therefore, is crucial. In this review we cover regulatory aspects of ABCB1 mediated drug transport as well as inhibition and the available models and methods. We also discuss protein structure and mechanistic aspects of transport as ABCB1 displays complex kinetics that involves multiple binding sites, potentiation of transport and probe-dependent IC50 values. RESULTS: Permeability of drugs both passive and mediated by transporters is also a covariate that modulates apparent kinetic values. Levels of expression as well as lipid composition of the expression system used in in vitro studies have also been acknowledged as determinates of transporter activity. ABCB1-mediated clinical tDDIs are often complex as multiple transporters as well as metabolic enzymes may play a role. This complexity often masks the role of ABCB1 in tDDIs. CONCLUSION: It is expected that utilization of in vitro data will further increase with the refinement of simulations. It is also anticipated that transporter humanized preclinical models have a significant impact and utility.

Multiple ABC Transporters Efflux Baicalin.

Baicalein, the aglycone formed by hydrolysis of baicalin in the intestine, is well absorbed by passive diffusion but subjected to extensive intestinal glucuronidation. Efflux of baicalin, the low passive permeability glucuronide of baicalein from enterocytes, likely depends on a carrier-mediated transport. The present study was designed to explore potential drug-herb interaction by investigating the inhibitory effect of baicalin on the transport of reporter substrates by transporters and to identify the transporters responsible for the efflux of baicalin from enterocytes and hepatocytes. The interaction of baicalin with specific ABC transporters was studied using membranes from cells overexpressing human BCRP, MDR1, MRP2, MRP3 and MRP4. Baicalin was tested for its potential to inhibit vesicular transport by these transporters. The transport of baicalin by the selected transporters was also investigated. Transport by BCRP, MRP3 and MRP4 was inhibited by baicalin with an IC50 of 3.41 ± 1.83 µM, 14.01 ± 2.51 µM and 14.39 ± 5.69 µM respectively. Inhibition of MDR1 (IC50 = 94.84 ± 31.10 µM) and MRP2 (IC50 = 210.13 ± 110.49 µM) was less potent. MRP2 and BCRP are the apical transporters of baicalin that may mediate luminal efflux in enterocytes and biliary efflux in hepatocytes. The basolateral efflux of baicalin is likely mediated by MRP3 and MRP4 both in enterocytes and hepatocytes. Via inhibition of transport by ABC transporters, baicalin could interfere with the absorption and disposition of drugs.