Alterations in zonal distribution and plasma membrane localization of hepatocyte bile acid transporters in patients with NAFLD.

BACKGROUND: NAFLD is highly prevalent with limited treatment options. Bile acids (BAs) increase in the systemic circulation and liver during NAFLD progression. Changes in plasma membrane localization and zonal distribution of BA transporters can influence transport function and BA homeostasis. However, a thorough characterization of how NAFLD influences these factors is currently lacking. This study aimed to evaluate the impact of NAFLD and the accompanying histologic features on the functional capacity of key hepatocyte BA transporters across zonal regions in human liver biopsies. METHODS: A novel machine learning image classification approach was used to quantify relative zonal abundance and plasma membrane localization of BA transporters (bile salt export pump [BSEP], sodium-taurocholate cotransporting polypeptide, organic anion transporting polypeptide [OATP] 1B1 and OATP1B3) in non-diseased (n = 10), NAFL (n = 9), and NASH (n = 11) liver biopsies. Based on these data, membrane-localized zonal abundance (MZA) measures were developed to estimate transporter functional capacity. RESULTS: NAFLD diagnosis and histologic scoring were associated with changes in transporter membrane localization and zonation. Increased periportal BSEPMZA (mean proportional difference compared to non-diseased liver of 0.090) and decreased pericentral BSEPMZA (-0.065) were observed with NASH and also in biopsies with higher histologic scores. Compared to Non-diseased Liver, periportal OATP1B3MZA was increased in NAFL (0.041) and NASH (0.047). Grade 2 steatosis (mean proportional difference of 0.043 when compared to grade 0) and grade 1 lobular inflammation (0.043) were associated with increased periportal OATP1B3MZA. CONCLUSIONS: These findings provide novel mechanistic insight into specific transporter alterations that impact BA homeostasis in NAFLD. Changes in BSEPMZA likely contribute to altered BA disposition and pericentral microcholestasis previously reported in some patients with NAFLD. BSEPMZA assessment could inform future development and optimization of NASH-related pharmacotherapies.

Membrane transporters in drug development and as determinants of precision medicine.

The effect of membrane transporters on drug disposition, efficacy and safety is now well recognized. Since the initial publication from the International Transporter Consortium, significant progress has been made in understanding the roles and functions of transporters, as well as in the development of tools and models to assess and predict transporter-mediated activity, toxicity and drug-drug interactions (DDIs). Notable advances include an increased understanding of the effects of intrinsic and extrinsic factors on transporter activity, the application of physiologically based pharmacokinetic modelling in predicting transporter-mediated drug disposition, the identification of endogenous biomarkers to assess transporter-mediated DDIs and the determination of the cryogenic electron microscopy structures of SLC and ABC transporters. This article provides an overview of these key developments, highlighting unanswered questions, regulatory considerations and future directions.

In vitro identification of decreased function phenotype ABCG2 variants.

Reduced activity of efflux transporter ABCG2, caused e.g., by inhibition or decreased function genetic variants, can increase drug absorption and plasma levels. ABCG2 has one clinically significant single nucleotide variant Q141K (c.421C>A), which leads to decreased protein levels and transport activity. In addition to Q141K, ABCG2 has over 500 rare (<1% minor allele frequency) nonsynonymous variants, but their functionality remains unknown. We studied the transport activity and abundance of 30 rare ABCG2 variants. The variants were transiently expressed in HEK293 cells. Transport activity and protein abundance were measured from inside-out crude membrane vesicles. Results were normalised to the reference ABCG2, while Q141K was used to categorise variants into decreased and normal function phenotypes based on their apparent transport activity. Fourteen variants (G80E, D128V, T434M, Q437R, C438R, C438W, C438Y, L479S, P480L, S486N, T512N, S519P, G553D and K647E) had similar or lower apparent transport activity than Q141K and thus were categorised as having a decreased function phenotype. Protein abundance could not explain all of the observed changes in transport activity: Only six variants (D128V, Q437R, C438R, S519P, G553D, and K647E) had similar or lower abundance compared to Q141K. The decreased function variants may increase systemic drug exposure and therefore cause interindividual variability in pharmacokinetics. In the future, in vitro phenotype classification may help to design personalised drug treatments.

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.

Considerations for Physiologically Based Modeling in Liver Disease: From Nonalcoholic Fatty Liver (NAFL) to Nonalcoholic Steatohepatitis (NASH).

Nonalcoholic fatty liver disease (NAFLD), representing a clinical spectrum ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), is rapidly evolving into a global pandemic. Patients with NAFLD are burdened with high rates of metabolic syndrome-related comorbidities resulting in polypharmacy. Therefore, it is crucial to gain a better understanding of NAFLD-mediated changes in drug disposition and efficacy/toxicity. Despite extensive clinical pharmacokinetic data in cirrhosis, current knowledge concerning pharmacokinetic alterations in NAFLD, particularly at different stages of disease progression, is relatively limited. In vitro-to-in vivo extrapolation coupled with physiologically based pharmacokinetic and pharmacodynamic (IVIVE-PBPK/PD) modeling offers a promising approach for optimizing pharmacologic predictions while refining and reducing clinical studies in this population. Use of IVIVE-PBPK to predict intra-organ drug concentrations at pharmacologically relevant sites of action is particularly advantageous when it can be linked to pharmacodynamic effects. Quantitative systems pharmacology/toxicology (QSP/QST) modeling can be used to translate pharmacokinetic and pharmacodynamic data from PBPK/PD models into clinically relevant predictions of drug response and toxicity. In this review, a detailed summary of NAFLD-mediated alterations in human physiology relevant to drug absorption, distribution, metabolism, and excretion (ADME) is provided. The application of literature-derived physiologic parameters and ADME-associated protein abundance data to inform virtual NAFLD population development and facilitate PBPK/PD, QSP, and QST predictions is discussed along with current limitations of these methodologies and knowledge gaps. The proposed methodologic framework offers great potential for meaningful prediction of pharmacological outcomes in patients with NAFLD and can inform both drug development and clinical practice for this population.

Effect of Carbidopa Dose on Levodopa Pharmacokinetics With and Without Catechol-O-Methyltransferase Inhibition in Healthy Subjects.

BACKGROUND AND OBJECTIVES: The treatment of Parkinson's disease (PD) is still symptomatic since disease-modifying treatments for PD are not available. Oral levodopa is the gold standard for the treatment of PD motor symptoms. However, incomplete and fluctuating plasma exposure of levodopa leads to suboptimal treatment of the symptoms. The main objective of this study was to investigate to what extent increased carbidopa doses (50 and 100 mg) increase the plasma levels of 100-mg immediate-release (IR) levodopa compared to a 25-mg carbidopa dose with and without co-administration of 200 mg entacapone. METHODS: A double-blind, placebo-controlled, randomized, crossover, phase I, pharmacokinetic study with 25 healthy volunteers was conducted. In addition, a semi-mechanistic pharmacokinetic model was built to theoretically evaluate the effect of inhibiting aromatic amino acid decarboxylase (AADC) and catechol-O-methyltransferase (COMT) mediated metabolism of levodopa on the exposure of levodopa. RESULTS: The effect of increased carbidopa doses 50 and 100 mg on the total exposure (AUC) of 100 mg IR levodopa was +29% and +36%, respectively, when entacapone was co-administered. Without entacapone, the corresponding increases were +13% and +17%. With entacapone co-administration, the increased carbidopa dose also clearly increased levodopa trough concentration. There was no significant effect on the peak concentrations of levodopa. CONCLUSIONS: Increasing carbidopa doses significantly increased the exposure and reduced the fluctuation of IR levodopa in plasma during simultaneous COMT inhibition with entacapone. Theoretical pharmacokinetic simulations suggested that the plasma profile of oral IR levodopa can be even further improved by optimizing AADC and COMT inhibition.

Functional in vitro characterization of SLCO1B1 variants and simulation of the clinical pharmacokinetic impact of impaired OATP1B1 function.

Organic Anion Transporting Polypeptide 1B1 is important to the hepatic elimination and distribution of many drugs. If OATP1B1 function is decreased, it can increase plasma exposure of e.g. several statins leading to increased risk of muscle toxicity. First, we examined the impact of three naturally occurring rare variants and the frequent SLCO1B1 c.388A>G variant on in vitro transport activity with cellular uptake assay using two substrates: 2', 7'-dichlorofluorescein (DCF) and rosuvastatin. Secondly, LC-MS/MS based quantitative targeted absolute proteomics measured the OATP1B1 protein abundance in crude membrane fractions of HEK293 cells over-expressing these single nucleotide variants. Additionally, we simulated the effect of impaired OATP1B1 function on rosuvastatin pharmacokinetics to estimate the need for genotype-guided dosing. R57Q impaired DCF and rosuvastatin transport significantly yet did not change protein expression considerably, while N130D and N151S did not alter activity but increased protein expression. R253Q did not change protein expression but reduced DCF uptake and increased rosuvastatin Km. Based on pharmacokinetic simulations, doses of 30 mg (with 50% OATP1B1 function) and 20 mg (with 0% OATP1B1 function) result in plasma exposure similar to 40 mg dose (with 100% OATP1B1 function). Therefore dose reductions might be considered to avoid increased plasma exposure caused by function-impairing OATP1B1 genetic variants, such as R57Q.

Regulation of Drug Transport Proteins-From Mechanisms to Clinical Impact: A White Paper on Behalf of the International Transporter Consortium.

Membrane transport proteins are involved in the absorption, disposition, efficacy, and/or toxicity of many drugs. Numerous mechanisms (e.g., nuclear receptors, epigenetic gene regulation, microRNAs, alternative splicing, post-translational modifications, and trafficking) regulate transport protein levels, localization, and function. Various factors associated with disease, medications, and dietary constituents, for example, may alter the regulation and activity of transport proteins in the intestine, liver, kidneys, brain, lungs, placenta, and other important sites, such as tumor tissue. This white paper reviews key mechanisms and regulatory factors that alter the function of clinically relevant transport proteins involved in drug disposition. Current considerations with in vitro and in vivo models that are used to investigate transporter regulation are discussed, including strengths, limitations, and the inherent challenges in predicting the impact of changes due to regulation of one transporter on compensatory pathways and overall drug disposition. In addition, translation and scaling of in vitro observations to in vivo outcomes are considered. The importance of incorporating altered transporter regulation in modeling and simulation approaches to predict the clinical impact on drug disposition is also discussed. Regulation of transporters is highly complex and, therefore, identification of knowledge gaps will aid in directing future research to expand our understanding of clinically relevant molecular mechanisms of transporter regulation. This information is critical to the development of tools and approaches to improve therapeutic outcomes by predicting more accurately the impact of regulation-mediated changes in transporter function on drug disposition and response.

Identification of Key Amino Acids that Impact Organic Solute Transporter α/ß (OSTα/ß).

Organic solute transporter α/ß (OSTα/ß) is a bidirectional bile acid transporter localized on the basolateral membrane of hepatic, intestinal, and renal epithelial cells. OSTα/ß plays a critical role in intestinal bile acid reabsorption and is upregulated in hepatic diseases characterized by elevated bile acids, whereas genetic variants in SLC51A/B have been associated with clinical cholestasis. OSTα/ß also transports and is inhibited by commonly used medications. However, there is currently no high-resolution structure of OSTα/ß, and structure-function data for OSTα, the proposed substrate-binding subunit, are lacking. The present study addressed this knowledge gap and identified amino acids in OSTα that are important for bile acid transport. This was accomplished using computational modeling and site-directed mutagenesis of the OSTα subunit to generate OSTα/ß mutant cell lines. Out of the 10 OSTα/ß mutants investigated, four (S228K, T229S, Q269E, Q269K) exhibited decreased [3H]-taurocholate (TCA) uptake (ratio of geometric means relative to OSTα/ß wild type (WT) of 0.76, 0.75, 0.79, and 0.13, respectively). Three OSTα/ß mutants (S228K, Q269K, E305A) had reduced [3H]-TCA efflux % (ratio of geometric means relative to OSTα/ß WT of 0.86, 0.65, and 0.79, respectively). Additionally, several OSTα/ß mutants demonstrated altered expression and cellular localization when compared with OSTα/ß WT. In summary, we identified OSTα residues (Ser228, Thr229, Gln269, Glu305) in predicted transmembrane domains that affect expression of OSTα/ß and may influence OSTα/ß-mediated bile acid transport. These data advance our understanding of OSTα/ß structure/function and can inform future studies designed to gain further insight into OSTα/ß structure or to identify additional OSTα/ß substrates and inhibitors. SIGNIFICANCE STATEMENT: OSTα/ß is a clinically important transporter involved in enterohepatic bile acid recycling with currently no high-resolution protein structure and limited structure-function data. This study identified four OSTα amino acids (Ser228, Thr229, Gln269, Glu305) that affect expression of OSTα/ß and may influence OSTα/ß-mediated bile acid transport. These data can be utilized to inform future investigation of OSTα/ß structure and refine molecular modeling approaches to facilitate the identification of substrates and/or inhibitors of OSTα/ß.

The Role of Uptake and Efflux Transporters in the Disposition of Glucuronide and Sulfate Conjugates.

Glucuronidation and sulfation are the most typical phase II metabolic reactions of drugs. The resulting glucuronide and sulfate conjugates are generally considered inactive and safe. They may, however, be the most prominent drug-related material in the circulation and excreta of humans. The glucuronide and sulfate metabolites of drugs typically have limited cell membrane permeability and subsequently, their distribution and excretion from the human body requires transport proteins. Uptake transporters, such as organic anion transporters (OATs and OATPs), mediate the uptake of conjugates into the liver and kidney, while efflux transporters, such as multidrug resistance proteins (MRPs) and breast cancer resistance protein (BCRP), mediate expulsion of conjugates into bile, urine and the intestinal lumen. Understanding the active transport of conjugated drug metabolites is important for predicting the fate of a drug in the body and its safety and efficacy. The aim of this review is to compile the understanding of transporter-mediated disposition of phase II conjugates. We review the literature on hepatic, intestinal and renal uptake transporters participating in the transport of glucuronide and sulfate metabolites of drugs, other xenobiotics and endobiotics. In addition, we provide an update on the involvement of efflux transporters in the disposition of glucuronide and sulfate metabolites. Finally, we discuss the interplay between uptake and efflux transport in the intestine, liver and kidneys as well as the role of transporters in glucuronide and sulfate conjugate toxicity, drug interactions, pharmacogenetics and species differences.

Physiologically-Based Pharmacokinetic Model of Morphine and Morphine-3-Glucuronide in Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH), the progressive form of nonalcoholic fatty liver disease, is increasing in prevalence. NASH-related alterations in hepatic protein expression (e.g., transporters) and in overall physiology may affect drug exposure by altering drug disposition and elimination. The aim of this study was to build a physiologically-based pharmacokinetic (PBPK) model to predict drug exposure in NASH by incorporating NASH-related changes in hepatic transporters. Morphine and morphine-3-glucuronide (M3G) were used as model compounds. A PBPK model of morphine with permeability-limited hepatic disposition was extended to include M3G disposition and enterohepatic recycling (EHR). The model captured the area under the plasma concentration-time curve (AUC) of morphine and M3G after intravenous morphine administration within 0.82-fold and 1.94-fold of observed values from 3 independent clinical studies for healthy adult subjects (6, 10, and 14 individuals). When NASH-related changes in multidrug resistance-associated protein 2 (MRP2) and MRP3 were incorporated into the model, the predicted M3G mean AUC in NASH was 1.34-fold higher compared to healthy subjects, which is slightly lower than the observed value (1.63-fold). Exploratory simulations on other physiological changes occurring in NASH (e.g., moderate decreases in glomerular filtration rate and portal vein blood flow) revealed that the effect of transporter changes was most prominent. Additionally, NASH-related transporter changes resulted in decreased morphine EHR, which could be important for drugs with extensive EHR. This study is an important first step to predict drug disposition in complex diseases such as NASH using PBPK modeling.

Role of Organic Solute Transporter Alpha/Beta in Hepatotoxic Bile Acid Transport and Drug Interactions.

Organic solute transporter (OST) α/ß is a key bile acid transporter expressed in various organs, including the liver under cholestatic conditions. However, little is known about the involvement of OSTα/ß in bile acid-mediated drug-induced liver injury (DILI), a major safety concern in drug development. This study investigated whether OSTα/ß preferentially transports more hepatotoxic, conjugated, primary bile acids and to what extent xenobiotics inhibit this transport. Kinetic studies with OSTα/ß-overexpressing cells revealed that OSTα/ß preferentially transported bile acids in the following order: taurochenodeoxycholate > glycochenodeoxycholate > taurocholate > glycocholate. The apparent half-maximal inhibitory concentrations for OSTα/ß-mediated bile acid (5 µM) transport inhibition by fidaxomicin, troglitazone sulfate, and ethinyl estradiol were: 210, 334, and 1050 µM, respectively, for taurochenodeoxycholate; 97.6, 333, and 337 µM, respectively, for glycochenodeoxycholate; 140, 265, and 527 µM, respectively, for taurocholate; 59.8, 102, and 117 µM, respectively, for glycocholate. The potential role of OSTα/ß in hepatocellular glycine-conjugated bile acid accumulation and cholestatic DILI was evaluated using sandwich-cultured human hepatocytes (SCHH). Treatment of SCHH with the farnesoid X receptor agonist chenodeoxycholate (100 µM) resulted in substantial OSTα/ß induction, among other proteomic alterations, reducing glycochenodeoxycholate and glycocholate accumulation in cells+bile 4.0- and 4.5-fold, respectively. Treatment of SCHH with troglitazone and fidaxomicin together under cholestatic conditions resulted in increased hepatocellular toxicity compared with either compound alone, suggesting that OSTα/ß inhibition may accentuate DILI. In conclusion, this study provides insights into the role of OSTα/ß in preferential disposition of bile acids associated with hepatotoxicity, the impact of xenobiotics on OSTα/ß-mediated bile acid transport, and the role of this transporter in SCHH and cholestatic DILI.

Hepatic Transporter Alterations by Nuclear Receptor Agonist T0901317 in Sandwich-Cultured Human Hepatocytes: Proteomic Analysis and PBPK Modeling to Evaluate Drug-Drug Interaction Risk.

In vitro approaches for predicting drug-drug interactions (DDIs) caused by alterations in transporter protein regulation are not well established. However, reports of transporter regulation via nuclear receptor (NR) modulation by drugs are increasing. This study examined alterations in transporter protein levels in sandwich-cultured human hepatocytes (SCHH; n = 3 donors) measured by liquid chromatography-tandem mass spectrometry-based proteomic analysis after treatment with N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide (T0901317), the first described synthetic liver X receptor agonist. T0901317 treatment (10 µM, 48 hours) decreased the levels of organic cation transporter (OCT) 1 (0.22-, 0.43-, and 0.71-fold of control) and organic anion transporter (OAT) 2 (0.38-, 0.38-, and 0.53-fold of control) and increased multidrug resistance protein (MDR) 1 (1.37-, 1.48-, and 1.59-fold of control). The induction of NR downstream gene expression supports the hypothesis that T0901317 off-target effects on farnesoid X receptor and pregnane X receptor activation are responsible for the unexpected changes in OCT1, OAT2, and MDR1. Uptake of the OCT1 substrate metformin in SCHH was decreased by T0901317 treatment. Effects of decreased OCT1 levels on metformin were simulated using a physiologically-based pharmacokinetic (PBPK) model. Simulations showed a clear decrease in metformin hepatic exposure resulting in a decreased pharmacodynamic effect. This DDI would not be predicted by the modest changes in simulated metformin plasma concentrations. Altogether, the current study demonstrated that an approach combining SCHH, proteomic analysis, and PBPK modeling is useful for revealing tissue concentration-based DDIs caused by unexpected regulation of hepatic transporters by NR modulators. SIGNIFICANCE STATEMENT: This study utilized an approach combining sandwich-cultured human hepatocytes, proteomic analysis, and physiologically based pharmacokinetic modeling to evaluate alterations in pharmacokinetics (PK) and pharmacodynamics (PD) caused by transporter regulation by nuclear receptor modulators. The importance of this approach from a mechanistic and clinically relevant perspective is that it can reveal drug-drug interactions (DDIs) caused by unexpected regulation of hepatic transporters and enable prediction of altered PK and PD changes, especially for tissue concentration-based DDIs.

Mechanistic Modeling of the Hepatic Disposition of Estradiol-17ß-Glucuronide in Sandwich-Cultured Human Hepatocytes.

Estradiol-17ß-glucuronide (E217G) is an estrogen metabolite that has cholestatic properties. In humans, circulating E217G is transported into hepatocytes by organic anion transporting polypeptides (OATPs) and is excreted into bile by multidrug-resistance associated protein 2 (MRP2). E217G is also a substrate of the basolateral efflux transporters MRP3 and MRP4, which translocate E217G from hepatocytes to blood. However, the contribution of basolateral efflux to hepatocyte disposition of E217G has not been evaluated previously. To address this question, E217G disposition was studied in sandwich-cultured human hepatocytes and mechanistic modeling was applied to calculate clearance values (mean ± S.D.) for uptake, intrinsic biliary excretion (CLint,bile) and intrinsic basolateral efflux (CLint,BL). The biliary excretion index of E217G was 45% ± 6%. The CLint,BL of E217G [0.18 ± 0.03 (ml/min)/g liver) was 1.6-fold higher than CLint,bile [0.11 ± 0.06 (ml/min)/g liver]. Simulations were performed to study the effects of increased CLint,BL and a concomitant decrease in CLint,bile on hepatic E217G exposure. Results demonstrated that increased CLint,BL can effectively reduce hepatocellular and biliary exposure to this potent cholestatic agent. Simulations also revealed that basolateral efflux can compensate for impaired biliary excretion and, vice versa, to avoid accumulation of E217G in hepatocytes. However, when both clearance processes are impaired by 90%, hepatocyte E217G exposure increases up to 10-fold. These data highlight the contribution of basolateral efflux transport, in addition to MRP2-mediated biliary excretion, to E217G disposition in human hepatocytes. This elimination route could be important, especially in cases where basolateral efflux is induced, such as cholestasis. SIGNIFICANCE STATEMENT: The disposition of the cholestatic estrogen metabolite estradiol-17ß-glucuronide (E217G) was characterized in sandwich-cultured human hepatocytes. The intrinsic basolateral efflux clearance was estimated to be 1.6-fold higher than the intrinsic biliary excretion clearance, emphasizing the contribution of basolateral elimination in addition to biliary excretion. Simulations highlight how hepatocytes can effectively cope with increased E217G through the regulation of both basolateral and biliary transporters.

Efflux transport of nicotine, cotinine and trans-3'-hydroxycotinine glucuronides by human hepatic transporters.

Nicotine is the addiction causing alkaloid in tobacco, and it is used in smoking cessation therapies. Although the metabolic pathways of nicotine are well known and mainly occur in the liver, the transport of nicotine and its metabolites is poorly characterized. The highly hydrophilic nature and urinary excretion of nicotine glucuronide metabolites indicate that hepatic basolateral efflux transporters mediate their excretion. We aimed here to find the transporters responsible for the hepatic excretion of nicotine, cotinine and trans-3'-hydroxycotinine (OH-cotinine) glucuronides. To this end, we tested their transport by multidrug resistance-associated proteins 1 (MRP1, ABCC1) and MRP3-6 (ABCC3-6), which are located on the basolateral membranes of hepatocytes, as well as MRP2 (ABCC2), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MDR1, P-gp, ABCB1) that are expressed in the apical membranes of these cells. ATP-dependent transport of these glucuronides was evaluated in inside-out membrane vesicles expressing the transporter of interest. In addition, potential interactions of both the glucuronides and parent compounds with selected transporters were tested by inhibition assays. Considerable ATP-dependent transport was observed only for OH-cotinine glucuronide by MRP3. The kinetics of this transport activity was characterized, resulting in an estimated Km value of 895 µmol/L. No significant transport was found for nicotine or cotinine glucuronides by any of the tested transporters at either 5 or 50 µmol/L substrate concentration. Furthermore, neither nicotine, cotinine nor OH-cotinine inhibited MRP2-4, BCRP or MDR1. In this study, we directly examined, for the first time, efflux transport of the three hydrophilic nicotine glucuronide metabolites by the major human hepatic efflux transporters. Despite multiple transporters studied here, our results indicate that an unknown transporter may be responsible for the hepatic excretion of nicotine and cotinine glucuronides.

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-17ß-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 ±â€¯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.

Novel in Vitro Method Reveals Drugs That Inhibit Organic Solute Transporter Alpha/Beta (OSTα/ß).

Drug interactions with the organic solute transporter alpha/beta (OSTα/ß) are understudied even though OSTα/ß is an important transporter that is expressed in multiple human tissues including the intestine, kidneys, and liver. In this study, an in vitro method to identify novel OSTα/ß inhibitors was first developed using OSTα/ß-overexpressing Flp-In 293 cells. Incubation conditions were optimized using previously reported OSTα/ß inhibitors. A method including a 10 min preincubation step with the test compound was used to screen for OSTα/ß inhibition by 77 structurally diverse compounds and fixed-dose combinations. Seven compounds and one fixed-dose combination (100 µM final concentration) inhibited OSTα/ß-mediated dehydroepiandrosterone sulfate (DHEAS) uptake by >25%. Concentration-dependent OSTα/ß inhibition was evaluated for all putative inhibitors (atorvastatin, ethinylestradiol, fidaxomicin, glycochenodeoxycholate, norgestimate, troglitazone, and troglitazone sulfate). Ethinylestradiol, fidaxomicin, and troglitazone sulfate yielded a clear concentration-inhibition response with IC50 values <200 µM. Among all tested compounds, there was no clear association between physicochemical properties, the severity of hepatotoxicity, and the degree of OSTα/ß inhibition. This study utilized a novel in vitro method to identify OSTα/ß inhibitors and, for the first time, provided IC50 values for OSTα/ß inhibition. These data provide evidence that several drugs, some of which are associated with cholestatic drug-induced liver injury, may impair the function of the OSTα/ß transporter.

Interaction of Food Additives with Intestinal Efflux Transporters.

Breast cancer resistance protein (BCRP), multidrug resistance associated protein 2 (MRP2) and P-glycoprotein (P-gp) are ABC transporters that are expressed in the intestine, where they are involved in the efflux of many drugs from enterocytes back into the intestinal lumen. The inhibition of BCRP, MRP2, and P-gp can result in enhanced absorption and exposure of substrate drugs. Food additives are widely used by the food industry to improve the stability, flavor, and consistency of food products. Although they are considered safe for consumption, their interactions with intestinal transporters are poorly characterized. Therefore, in this study, selected food additives, including preservatives, colorants, and sweeteners, were studied in vitro for their inhibitory effects on intestinal ABC transporters. Among the studied compounds, several colorants were able to inhibit BCRP and MRP2, whereas P-gp was fairly insensitive to inhibition. Additionally, one sweetener was identified as a potent inhibitor of BCRP. Dose-response studies revealed that the IC50 values of the inhibitors were lower than the estimated intestinal concentrations after the consumption of beverages containing food colorants. This suggests that there is potential for previously unrecognized transporter-mediated food additive-drug interactions.

Transmembrane Domain Single-Nucleotide Polymorphisms Impair Expression and Transport Activity of ABC Transporter ABCG2.

PURPOSE: To study the function and expression of nine naturally occurring single-nucleotide polymorphisms (G406R, F431L, S441N, P480L, F489L, M515R, L525R, A528T and T542A) that are predicted to reside in the transmembrane regions of the ABC transporter ABCG2. METHODS: The transport activity of the variants was tested in inside-out membrane vesicles from Sf9 insect and human derived HEK293 cells overexpressing ABCG2. Lucifer Yellow and estrone sulfate were used as probe substrates of activity. The expression levels and cellular localization of the variants was compared to the wild-type ABCG2 by western blotting and immunofluorescence microscopy. RESULTS: All studied variants of ABCG2 displayed markedly decreased transport in both Sf9-ABCG2 and HEK293-ABCG2 vesicles. Impaired transport could be explained for some variants by altered expression levels and cellular localization. Moreover, the destructive effect on transport activity of variants G406R, P480L, M515R and T542A is, to our knowledge, reported for the first time. CONCLUSIONS: These results indicate that the transmembrane region of ABCG2 is sensitive to amino acid substitution and that patients harboring these ABCG2 variant forms could suffer from unexpected pharmacokinetic events of ABCG2 substrate drugs or have an increased risk for diseases such as gout where ABCG2 is implicated.

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.