Vesicular Transport Assay Research Articles

Study on the Effect of Pharmaceutical Excipient PEG400 on the Pharmacokinetics of Baicalin in Cells Based on MRP2, MRP3, and BCRP Efflux Transporters.

Pharmaceutical excipient PEG400 is a common component of traditional Chinese medicine compound preparations. Studies have demonstrated that pharmaceutical excipients can directly or indirectly influence the disposition process of active drugs in vivo, thereby affecting the bioavailability of drugs. In order to reveal the pharmacokinetic effect of PEG400 on baicalin in hepatocytes and its mechanism, the present study first started with the effect of PEG400 on the metabolic disposition of baicalin at the hepatocyte level, and then the effect of PEG400 on the protein expression of baicalin-related transporters (BCRP, MRP2, and MRP3) was investigated by using western blot; the effect of MDCKII-BCRP, MDCKII-BCRP, MRP2, and MRP3 was investigated by using MDCKII-BCRP, MDCKII-MRP2, and MDCKII-MRP3 cell monolayer models, and membrane vesicles overexpressing specific transporter proteins (BCRP, MRP2, and MRP3), combined with the exocytosis of transporter-specific inhibitors, were used to study the effects of PEG400 on the transporters in order to explore the possible mechanisms of its action. The results demonstrated that PEG400 significantly influenced the concentration of baicalin in hepatocytes, and the AUC0-t of baicalin increased from 75.96 ± 2.57 μg·h/mL to 106.94 ± 2.22 μg·h/mL, 111.97 ± 3.98 μg·h/mL, and 130.42 ± 5.26 μg·h/mL (p ˂ 0.05). Furthermore, the efflux rate of baicalin was significantly reduced in the vesicular transport assay and the MDCKII cell model transport assay, which indicated that PEG400 had a significant inhibitory effect on the corresponding transporters. In conclusion, PEG400 can improve the bioavailability of baicalin to some extent by affecting the efflux transporters and thus the metabolic disposition of baicalin in the liver.

Inhibition of efflux transporters by poly ADP-ribose polymerase inhibitors.

Poly ADP-ribose polymerase (PARP) inhibitors have been approved for the treatment of various cancers. They share a similar mechanism of action but have differences in pharmacokinetic characteristics and potential for drug-drug interactions (DDI). This study evaluated the potential ATP-binding cassette transporter-mediated interactions between PARP inhibitors (niraparib, olaparib and rucaparib) and statins (atorvastatin and rosuvastatin). We studied the inhibitory activity of PARP inhibitors on breast cancer resistance protein (BCRP), multidrug resistance-associated protein 3 (MRP3) and P-glycoprotein (P-gp) using vesicular transport assays and determined the concentrations required for 50% inhibition (IC50 ). Then, we predicted the increase of statin exposure followed by the administration of PARP inhibitors using a mechanistic static model. Rucaparib was the strongest inhibitor of BCRP-mediated rosuvastatin transport (IC50 13.7 μM), followed by niraparib (42.6 μM) and olaparib (216 μM). PARP inhibitors did not affect MRP3. While niraparib appeared to inhibit P-gp, the inhibition showed large variability. The inhibition of intestinal BCRP by rucaparib, niraparib and olaparib was predicted to elevate rosuvastatin exposure by 52%, 37% and 24%, respectively. The interactions between PARP inhibitors and rosuvastatin are probably of minor clinical significance alone, but combined with other predisposing factors, they may increase the risk of rosuvastatin-associated adverse effects.

The inhibitor preincubation effect is universal to SLC transporter assays and is only partially eliminated in the presence of extracellular protein.

Variation in the methodology of in vitro transporter inhibition assays causes wide divergence in reported IC50/Ki data. Notably, although potentiation of transporter inhibition by preincubation (PTIP) has been described, current guidelines do not specifically recommend inhibitor preincubation, only encourage sponsors to follow emerging literature. To clarify how generally preincubation should be considered in transporter inhibition studies, and whether PTIP can be solely explained by protein binding of the respective inhibitors, we performed in vitro inhibition assays on SLC and ABC transporters scarcely or not covered in prior research, and examined the effect of extracellular protein in preincubation and washout experiments. In SLC assays without extracellular protein, a 30-minute preincubation caused significant >2-fold change of IC50 in 21/33 transporter-inhibitor combinations involving 19 evolutionarily disparate transporters. The preincubation effect correlated with inhibitor properties like protein binding and aqueous solubility. In vesicular transport assays of MDR1, BCRP, MRP2, and BSEP, sizable PTIP was observed for only 2/23 combinations, and preincubation was practically inconsequential in BCRP or MDR1 monolayer assays. In SLC assays, PTIP partly persisted in the presence of 5% albumin, indicating that the absence of extracellular protein does not fully explain PTIP. The presence of protein, however, complicated the interpretation of results. Overall, while preincubating without protein may overpredict inhibitory potency, adding protein compromises clarity, and omitting preincubation altogether may miss clinically relevant inhibitors. Therefore, we propose that protein-free preincubation should be considered in all SLC inhibition assays. ABC inhibition seems less commonly affected by preincubation, but conclusions require further investigation. Significance Statement Drugs may inhibit transporter proteins in the body, which may precipitate drug interactions. In vitro transporter inhibition assays help predict such drug interactions. Some inhibitors act more potently when preincubated with the transporter prior to the assay. Here we argue that this effect is not a mere in vitro artefact due to the lack of plasma proteins and should be considered in all uptake inhibition assays to model the worst-case scenario. Preincubation in efflux transporter inhibition assays is likely dispensable.

Novel inhibitors of breast cancer resistance protein (BCRP, ABCG2) among marketed drugs

Drug-drug interactions (DDIs) are a major concern for the safe use of medications. Breast cancer resistance protein (BCRP) is a clinically relevant ATP-binding cassette (ABC) transporter for drug disposition. Inhibition of BCRP increases the plasma concentrations of BCRP substrate drugs, which potentially could lead to adverse drug reactions. The aim of the present study was to identify BCRP inhibitors amongst a library of 232 commonly used drugs and anticancer drugs approved by the United States Food and Drug Administration (FDA). BCRP inhibition studies were carried out using the vesicular transport assay. We found 75 drugs that reduced the relative transport activity of BCRP to less than 25% of the vehicle control and were categorized as strong inhibitors. The concentration required for 50% inhibition (IC50) was determined for 13 strong inhibitors that were previously poorly characterized for BCRP inhibition. The IC50 ranged from 1.1 to 11 µM, with vemurafenib, dabigatran etexilate and everolimus being the strongest inhibitors. According to the drug interaction guidance documents from the FDA and the European Medicines Agency (EMA), in vivo DDI studies are warranted if the theoretical intestinal luminal concentration of a drug exceeds its IC50 by tenfold. Here, the IC50 values for eight of the drugs were 100-fold lower than their theoretical intestinal luminal concentration. Moreover, a mechanistic static model suggested that vemurafenib, bexarotene, dabigatran etexilate, rifapentine, aprepitant, and ivacaftor could almost fully inhibit intestinal BCRP, increasing the exposure of concomitantly administered rosuvastatin over 90%. Therefore, clinical studies are warranted to investigate whether these drugs cause BCRP-mediated DDIs in humans.

Deciphering the chemical constituents of Shengjiang Xiexin decoction by ultrahigh-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry and the impact of 20 characteristic components on multidrug resistance-associated protein 2 in the vesicular transport assay.

Shengjiang Xiexin decoction, a traditional Chinese medical formula, has been utilized to alleviate the delayed-onset diarrhea induced by irino tecan. However, the chemical constituents of this formula and the activities of its constituents remain unclear. In this study, ultrahigh-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry was employed to comprehensively analyze the chemical constituents of Shengjiang Xiexin decoction. A total of 270 components, including flavonoids, coumarins, triterpenoids, alkaloids, diarylheptanoids and others, were identified or characterized. Multidrug resistance-associated protein 2 is an efflux transporter responsible for regulating drug absorption. A total of 20 characteristic components from the formula were selected to evaluate their effects on the function of multidrug resistance-associated protein 2 using the vesicular transport assay. Glycyrrhizic acid and glycyrrhetinic acid were identified as potential multidrug resistance-associated protein 2 inhibitors, while 9 flavonoid aglycones increased the uptake of the substrate [3 H]-estradiol 17-β-glucuronide in the vesicles. This was the first systematic investigation of the chemical constituents from Shengjiang Xiexin decoction and the effect of its characteristic components on the transporter. The results offered a basis for further exploring the detoxification mechanisms of this formula and its interactions with other drugs.

Multidrug resistance-associated protein 2 (MRP2) is an efflux transporter of EGCG and its metabolites in the human small intestine

Green tea polyphenols have various beneficial effects on human health, such as antiobesity and anti-carcinogenesis. (-)-Epigallocatechin-gallate (EGCG) is one of the major potent green tea catechins; however, detailed mechanisms of EGCG transport and metabolism in the human small intestine remain unknown due to lack of a suitable model. We investigated metabolite profiles of EGCG in the fresh human duodenal biopsy, cryopreserved human duodenal mucosal enterocytes and Caco-2 cells, and found that EGCG was readily metabolized into methylated and sulphate conjugates, which are major metabolites in these models. Next, we examined possible efflux transporters of EGCG and its metabolites using specific inhibitors of MRP2, P-gp and BCRP in Caco-2 cell monolayers. MRP2 was thereby identified as an efflux transporter, and further analysis using MRP2-knockout Caco-2 cells and vesicular transport assays confirmed that MRP2 is a selective efflux transporter of EGCG and its metabolites. Assuming that functional inhibition of MRP2 would result in efficient uptake of EGCG, we screened for MRP2 functional blockade and identified quercetin, which led to increased intracellular accumulation and basal transport of EGCG in Caco-2 cells. This result suggested that co-administration of quercetin and EGCG would enable efficient transport of EGCG in the human intestine. Therefore, we performed co-oral administration of quercetin and EGCG in human subjects to examine whether this occurred in humans. These studies demonstrated that MRP2 is a selective transporter of EGCG and conjugates and Caco-2 is a model to examine transport mechanisms and metabolites of polyphenols in the human small intestine.

Tetrahydrocannabinol and Its Major Metabolites Are Not (or Are Poor) Substrates or Inhibitors of Human P-Glycoprotein [ATP-Binding Cassette (ABC) B1] and Breast Cancer Resistance Protein (ABCG2).

(-)-Δ9-Tetrahydrocannabinol (THC) is the primary psychoactive constituent of cannabis. In humans, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) are psychoactive and nonpsychoactive circulating metabolites of THC, respectively. Whether these cannabinoids are substrates or inhibitors of human P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) is unknown. Previous animal studies suggest that THC and its metabolites could be substrates of these transporters. Therefore, we performed Transwell, cellular accumulation, and vesicular transport assays, at pharmacologically relevant concentrations of these cannabinoids, using Madin-Darby canine kidney (MDCK) II cells or plasma membrane vesicles overexpressing human P-gp or BCRP. Neither THC nor 11-OH-THC was found to be a substrate or inhibitor of P-gp or BCRP. The efflux ratio of THC-COOH in MDCKII-BCRP cells was 1.6, which was significantly decreased to 1.0 by the BCRP inhibitor Ko143. Likewise, cellular accumulation of THC-COOH was significantly increased 1.6-fold in the presence versus absence of Ko143. THC-COOH also significantly inhibited BCRP-mediated transport of Lucifer yellow, a BCRP substrate; however, THC-COOH was neither a substrate nor an inhibitor of P-gp. Collectively, these results indicate that THC and 11-OH-THC are not substrates or inhibitors (at pharmacologically relevant concentrations) of either P-gp or BCRP. THC-COOH is a weak substrate and inhibitor of BCRP, but not of P-gp. Accordingly, we predict that P-gp/BCRP will not modulate the disposition of these cannabinoids in humans. In addition, use of these cannabinoids will not result in P-gp- or BCRP-based drug interactions. SIGNIFICANCE STATEMENT: This study systematically investigated whether Δ9-tetrahydrocannabinol (THC) and its major metabolites, 11-hydroxy-THC and 11-nor-9-carboxy-THC, are substrates and/or inhibitors of human P-gp and BCRP at pharmacologically relevant concentrations. The results obtained are highly valuable for mechanistic understanding and prediction of the roles of P-gp and BCRP in determining the human pharmacokinetics, tissue distribution, and drug interactions of cannabinoids.

Tetrahydrocannabinol and Its Major Metabolites are Not (or are poor) Substrates and Inhibitors of Human P‐glycoprotein (P‐gp/MDR1/ABCB1) and Breast Cancer Resistance Protein (BCRP/ABCG2)

Cannabis consumption in the United States is rising. (-)-Δ9-tetrahydrocannabinol (THC) is the primary psychoactive constituent of cannabis. In humans, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) are psychoactive and non-psychoactive circulating metabolites of THC, respectively. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are ATP-binding cassette (ABC) efflux transporters important in drug disposition. Previous animal studies suggest that THC and its metabolites could be substrates of P-gp and BCRP orthologs in mice and macaques; however, whether these cannabinoids are substrates or inhibitors of human P-gp and BCRP is unknown. Therefore, we performed transwell transport, cellular accumulation, and vesicular transport assays using Madin-Darby canine kidney II (MDCKII) cells or plasma membrane vesicles overexpressing human P-gp or BCRP to determine whether THC, 11-OH-THC or THC-COOH, at pharmacologically relevant concentrations, are substrates or inhibitors of these human ABC transporters. Neither THC nor 11-OH-THC was found to be a substrate or inhibitor of P-gp or BCRP. The efflux ratio of THC-COOH in MDCKII-BCRP cells was 1.6, which was significantly decreased to 1.0 by the BCRP inhibitor Ko143. Likewise, cellular accumulation of THC-COOH was significantly increased 1.6-fold in the presence vs. absence of Ko143. THC-COOH also significantly inhibited BCRP-mediated transport of lucifer yellow, a BCRP substrate; however, THC-COOH was neither a substrate nor an inhibitor of P-gp. Collectively, these results indicate that THC and 11-OH-THC are not substrates or inhibitors (at pharmacologically relevant concentrations) of either P-gp or BCRP. THC-COOH is a weak substrate and inhibitor of BCRP, but not of P-gp. Based on these data we predict that P-gp or BCRP will not modulate the brain/fetal distribution or intestinal absorption of these cannabinoids in humans. In addition, significant in vivo drug interactions between these cannabinoids and P-gp or BCRP inhibitors/inducers are unlikely.

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.

Functional Expression of Multidrug Resistance Protein 4 MRP4/ABCC4

To study the function and structure of membrane proteins, high quantities of pure and stable protein are needed. One of the first hurdles in accomplishing this is expression of the membrane protein at high levels and in a functional state. Membrane proteins are naturally expressed at low levels, so finding a suitable host for overexpression is imperative. Multidrug resistance protein 4 (MRP4) or ATP-binding cassette subfamily C member 4 (ABCC4) is a multi-transmembrane protein that is able to transport a range of organic anionic compounds (both endogenous and xenobiotic) out of the cell. This versatile transporter has been linked with extracellular signaling pathways and cellular protection, along with conferring drug resistance in cancers. Here we report the use of MRP4 as a case study to be expressed in three different expression systems: mammalian, insect, and yeast cells, to gain the highest yield possible. Interestingly, using the baculovirus expression system with Sf9 insect cells produced the highest protein yields. Vesicular transport assays were used to confirm that MRP4 expressed in Sf9 was functional using a fluorescent cAMP analogue (fluo-cAMP) instead of the traditional radiolabeled substrates. MRP4 transported fluo-cAMP in an ATP-dependent manner. The specificity of functional expression of MRP4 was validated by the use of nonhydrolyzable ATP analogues and MRP4 inhibitor MK571. Functionally expressed MRP4 in Sf9 cells can now be used in downstream processes such as solubilization and purification in order to better understand its function and structure.

A Change in Bile Flow: Looking Beyond Transporter Inhibition in the Development of Drug-induced Cholestasis.

Drug-induced Liver Injury (DILI) has received increasing attention over the past decades, as it represents the leading cause of drug failure and attrition. One of the most prevalent and severe forms of DILI involves the toxic accumulation of bile acids in the liver, known as Drug-induced Cholestasis (DIC). Traditionally, DIC is studied by exploring the inhibition of hepatic transporters such as Bile Salt Export Pump (BSEP) and multidrug resistance-associated proteins, predominantly through vesicular transport assays. Although this approach has identified numerous drugs that alter bile flow, many DIC drugs do not demonstrate prototypical transporter inhibition, but rather are associated with alternative mechanisms. We undertook a focused literature search on DIC and biliary transporters and analyzed peer-reviewed publications over the past two decades or so. We have summarized the current perception regarding DIC, biliary transporters, and transcriptional regulation of bile acid homeostasis. A growing body of literature aimed to identify alternative mechanisms in the development of DIC has been evaluated. This review also highlights current in vitro approaches used for prediction of DIC. Efforts have continued to focus on BSEP, as it is the primary route for hepatic biliary clearance. In addition to inhibition, drug-induced BSEP repression or the combination of these two has emerged as important alternative mechanisms leading to DIC. Furthermore, there has been an evolution in the approaches to studying DIC including 3D cell cultures and computational modeling.

Involvement of H+-gradient dependent transporter in PGE2 release from A549 cells

The purpose of this study was to identify the transporter involved in the release of prostaglandin E2 (PGE2). In the present study, transport assays were conducted using membrane vesicles prepared from human lung adenocarcinoma A549 cells, thus enabling identification of the novel exporter present in A549 cells. PGE2 transport into A549 vesicles was higher in the presence of a proton (H+)-gradient, thus suggesting the involvement of PGE2H+ symporter in PGE2 transport. Results from our experiments showed enhanced PGE2 release in A549 cells in the presence of H+-gradient ([H+]extracellular < [H+]intracellular). Moreover, in vesicular transport assays, H+-gradient-dependent transport of PGE2 did not show saturation up to 500 μM PGE2, and 10 mM aromatic monocarboxylic acids (acetylsalicylic acid, salicylic acid, and p-nitrobenzoic acid) significantly inhibited PGE2 transport by 62–70%. These results suggest, the involvement of monocarboxylate transporters in the H+-gradient-dependent PGE2 export.

Endogenous, cholesterol-activated ATP-dependent transport in membrane vesicles from Spodoptera frugiperda cells

Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda -derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,′7′-dichlorofluorescein (CDCF), estradiol-17β-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a Km of 8.06 ± 1.11 μM. The observed CDCF transport was inhibited by known inhibitors of human ABCC transporters, but not by ABCB1 and ABCG2 inhibitors verapamil and Ko143, respectively. Two candidate genes for ABCC-type transporters in the S. frugiperda genome (SfABCC2 and SfABCC3) were identified based on sequence analysis as a hypothesis to explain the observed endogenous ABCC-type transport in Sf9 vesicles. Although further studies are needed to verify the role of SfABCC2 and SfABCC3 in Sf9 vesicles, the findings of this study highlight the need to carefully characterize background transport in Sf9 derived membrane vesicles to avoid false positive substrate findings for human ABC transporters studied with this overexpression system.

Major glucuronide metabolites of testosterone are primarily transported by MRP2 and MRP3 in human liver, intestine and kidney

Testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG) and dihydrotestosterone glucuronide (DHTG) are the major metabolites of testosterone (T), which are excreted in urine and bile. Glucuronides can be deconjugated to active androgen in gut lumen after biliary excretion, which in turn can affect physiological levels of androgens. The goal of this study was to quantitatively characterize the mechanisms by which TG, AG, EtioG and DHTG are eliminated from liver, intestine, and kidney utilizing relative expression factor (REF) approach. Using vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP, we first identified that TG, AG, EtioG, and DHTG were primarily substrates of MRP2 and MRP3, although lower levels of transport were also observed with MDR1 and BCRP vesicles. The transport kinetic analyses revealed higher intrinsic clearances of TG by MRP2 and MRP3 as compared to that of DHTG, AG, and EtioG. MRP3 exhibited higher affinity for the transport of the studied glucuronides than MRP2. We next quantified the protein abundances of these efflux transporters in vesicles and compared the same with pooled total membrane fractions isolated from human tissues by quantitative LC–MS/MS proteomics. The fractional contribution of individual transporters (ft) was estimated by proteomics-based physiological scaling factors, i.e., transporter abundance in whole tissue versus vesicles, and corrected for inside-out vesicles (determined by 5′-nucleotidase assay). The glucuronides of inactive androgens, AG and EtioG were preferentially transported by MRP3, whereas the glucuronides of active androgens, TG and DHTG were mainly transported by MRP2 in liver. Efflux by bile canalicular transport may indicate the potential role of enterohepatic recirculation in regulating the circulating active androgens after deconjugation in the gut. In intestine, MRP3 possibly contributes most to the efflux of these glucuronides. In kidney, all studied glucuronides seemed to be preferentially effluxed by MRP2 and MDR1 (for EtioG). These REF based analysis need to be confirmed with in vivo findings. Overall, characterization of the efflux mechanisms of T glucuronide metabolites is important for predicting the androgen disposition and interindividual variability, including drug-androgen interaction in humans. The mechanistic data can be extrapolated to other androgen relevant organs (e.g. prostate, testis and placenta) by integrating these data with quantitative tissue proteomics data.

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

ABSTRACTIntroduction: 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.

Assay calibration to refine prediction of P-GP mediated DDI based on in vitro vesicular transport assay data

Assay calibration to refine prediction of P-GP mediated DDI based on in vitro vesicular transport assay data

Efflux transport of estrogen glucuronides by human MRP2, MRP3, MRP4 and BCRP

Estrone, estradiol and estriol are endogenous human estrogens that are rapidly conjugated with glucuronic acid in both intestinal and hepatic epithelial cells. The resulting glucuronides, estrone-3-glucuronide (E1-G), estradiol-3- and 17-glucuronides (E2-3G and E2-17G), as well as estriol-3- and 16-glucuronides (E3-3G and E3-16G) are found in human plasma and urine. Unlike E2-17G, the efflux transport of other estrogen glucuronides by human transporters has not yet been investigated comprehensively. We have studied the transport of E1-G, E2-3G, E3-3G, E3-16G and estrone-3-sulfate (E1-S), another important estrogen conjugate, using the vesicular transport assay with recombinant human MRP2, MRP3, MRP4, MDR1 and BCRP that were expressed in insect cells. The transport screening assays revealed that whereas E1-S was a good and specific substrate for BCRP, the less transporter-specific conjugates, E1-G and E2-3G, were still transported by BCRP at 10-fold higher rates than E1-S. BCRP also transported E3-16G at higher rates than the studied MRPs, while it transported E3-3G at lower rates than MRP3. MRP2 exhibited lower or equal transport rates of E1-G, E2-3G, E3-3G and E3-16G in comparison to MRP3 and BCRP in the screening assays, mainly due to its high Km values, between 180 and 790 μM. MRP3 transported all the tested glucuronides at rather similar rates, at Km values below 20 μM, but lower Vmax values than other transporters. In the case of E3-3G, MRP3 was the most active transporter in the screening assay. MRP4 transported only E3-16G at considerable rates, while none of the tested estrogen conjugates was transported by MDR1 at higher rates than control vesicles. These new results, in combination with previously reported in vivo human data, stimulate our understanding on the substrate specificity and role of efflux transporters in disposition of estrogen glucuronides in humans.

Selectivity in the Efflux of Glucuronides by Human Transporters: MRP4 Is Highly Active toward 4-Methylumbelliferone and 1-Naphthol Glucuronides, while MRP3 Exhibits Stereoselective Propranolol Glucuronide Transport.

Xenobiotic and endobiotic glucuronides, which are generated in hepatic and intestinal epithelial cells, are excreted via efflux transporters. Multidrug resistance proteins 2-4 (MRP2-MRP4) and the breast cancer resistance protein (BCRP) are efflux transporters that are expressed in these polarized cells, on either the basolateral or apical membranes. Their localization, along with expression levels, affects the glucuronide excretion pathways. We have studied the transport of three planar cyclic glucuronides and glucuronides of the two propranolol enantiomers, by the vesicular transport assay, using vesicles from baculovirus-infected insect cells expressing human MRP2, MRP3, MRP4, or BCRP. The transport of estradiol-17β-glucuronide by recombinant MRP2-4 and BCRP, as demonstrated by kinetic values, were within the ranges previously reported. Our results revealed high transport rates and apparent affinity of MRP4 toward the glucuronides of 4-methylumbelliferone, 1-naphthol, and 1-hydroxypyrene (Km values of 168, 13, and 3 μM, respectively) in comparison to MRP3 (Km values of 278, 98, and 8 μM, respectively). MRP3 exhibited lower rates, but stereoselective transport of propranolol glucuronides, with higher affinity toward the R-enantiomer than the S-enantiomer (Km values 154 vs 434 μM). The glucuronide of propranolol R-enantiomer was not significantly transported by either MRP2, MRP4, or BCRP. Of the tested small glucuronides in this study, BCRP transported only 1-hydroxypyrene glucuronide, at very high rates and high apparent affinity (Vmax and Km values of 4400 pmol/mg/min and 11 μM). The transport activity of MRP2 with all of the studied small glucuronides was relatively very low, even though it transported the reference compound, estradiol-17β-glucuronide, at a high rate (Vmax = 3500 pmol/mg/min). Our results provide new information, at the molecular level, of efflux transport of the tested glucuronides, which could explain their disposition in vivo, as well as provide new tools for in vitro studies of MRP3, MRP4, and BCRP.

A structure-activity relationship study of ABCC2 inhibitors

Multidrug resistance associated protein 2 (MRP2/ABCC2) is a membrane transport protein that can potentially affect the disposition of many substrate drugs and their metabolites. Recently, we studied the interaction of a library of 432 compounds with ABCC2, and the structure-activity relationship (SAR) of a subset of 64 compounds divided into four scaffolds (Wissel, G. et al., 2015. Bioorg Med Chem., 23(13), pp.3513–25). We have now expanded this test set by investigating 114 new compounds, of which 71 are representative of the previous four scaffolds and 43 compounds belong to a new scaffold. Interaction with ABCC2 was assessed by measuring the compounds effect on 5(6)-carboxy-2′,7′-dichlorofluorescein transport in the vesicular transport assay. In line with our previous study, we observed that anionic charge is not essential for inhibition of ABCC2 transport, even though it often increases the inhibitory activity within the analogue series. Additionally, we found that halogen substitutions often increase the inhibitory activity. The results confirm the importance of structural features such as aromaticity and lipophilicity for ABCC2 inhibitory activity.

Inhibition of Breast Cancer Resistance Protein and Multidrug Resistance Associated Protein 2 by Natural Compounds and Their Derivatives.

The food and dietary supplements we consume contain a wide variety of plant secondary metabolites and other compounds, which, like drugs, can be absorbed, metabolized, distributed, and excreted from the body. In the intestine, these compounds can interact with transport proteins such as the multidrug resistance associated protein 2 (MRP2, ABCC2) and the breast cancer resistance protein (BCRP, ABCG2) that regulate the absorption of drugs and other compounds. Inhibition of these transporters by dietary components could lead to increased exposure and adverse effects of concomitantly administered drugs. Therefore, we screened a library of 124 natural compounds and their derivatives using the vesicular transport assay to evaluate their inhibitory potential on MRP2 and BCRP. Of the library compounds, 36% were identified as BCRP inhibitors, whereas the number was only 3.2% for MRP2. BCRP inhibitors are described by higher molecular weight, number of rings, aromaticity, and LogD7.4 than noninhibitors. IC50 values were measured for six dual inhibitors, among which three novel inhibitors, gossypin, nordihydroguaiaretic acid, and octyl gallate, were identified. Our results confirm that flavonoids are avid inhibitors of BCRP, and flavones and flavonols appear to be important subclasses of flavonoids for this inhibition. The strong inhibition of BCRP transport by some compounds suggests that their presence at high levels in the diet could cause food-drug interactions, but this seems to be a minor cause of concern for MRP2.