Conversion of Olmesartan to Olmesartan Medoxomil, A Prodrug that Improves Intestinal Absorption, Confers Substrate Recognition by OATP2B1.

PURPOSE: Olmesartan medoxomil (olmesartan-MX), an ester-type prodrug of the angiotensin II receptor blocker (ARB) olmesartan, is predominantly anionic at intestinal pH. Human organic anion transporting polypeptide 2B1 (OATP2B1) is expressed in the small intestine and is involved in the absorption of various acidic drugs. This study was designed to test the hypothesis that OATP2B1-mediated uptake contributes to the enhanced intestinal absorption of olmesartan-MX, even though olmesartan itself is not a substrate of OATP2B1. METHODS: Tetracycline-inducible human OATP2B1- and rat Oatp2b1-overexpressing HEK 293 cell lines (hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293, respectively) were established to characterize OATP2B1-mediated uptake. Rat jejunal permeability was measured using Ussing chambers. ARBs were quantified by liquid chromatography-tandem mass spectrometry. RESULTS: Significant olmesartan-MX uptake was observed in hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293 cells, whereas olmesartan uptake was undetectable or much lower than olmesartan-MX uptake, respectively. Furthermore, olmesartan-MX exhibited several-fold higher uptake in Caco-2 cells and greater permeability in rat jejunum compared to olmesartan. Olmesartan-MX uptake in hOATP2B1/T-REx-293 cells and in Caco-2 cells was significantly decreased by OATP2B1 substrates/inhibitors such as 1 mM estrone-3-sulfate, 100 µM rifamycin SV, and 100 µM fluvastatin. Rat Oatp2b1-mediated uptake and rat jejunal permeability of olmesartan-MX were significantly decreased by 50 µM naringin, an OATP2B1 inhibitor. Oral administration of olmesartan-MX with 50 µM naringin to rats significantly reduced the area under the plasma concentration-time curve of olmesartan to 76.9%. CONCLUSION: Olmesartan-MX is a substrate for OATP2B1, and the naringin-sensitive transport system contributes to the improved intestinal absorption of olmesartan-MX compared with its parent drug, olmesartan.

Differential effects of OATP2B1 on statin accumulation and toxicity in a beta cell model.

An increased risk of new-onset diabetes mellitus has been recently reported for statin therapy, and experimental studies have shown reduced glucose-stimulated insulin secretion (GSIS) and mitochondrial dysfunction in beta cells with effects differing among agents. Organic anion transporting polypeptide (OATP) 2B1 contributes to hepatic uptake of rosuvastatin, atorvastatin and pravastatin, three known substrates. Since OATP2B1 is present in beta cells of the human pancreas, we investigated if OATP2B1 facilitates the local accumulation of statins in a rat beta cell model INS-1 832/13 (INS-1) thereby amplifying statin-induced toxicity. OATP2B1 overexpression in INS-1 cells via adenoviral transduction showed 2.5-, 1.8- and 1.4-fold higher cellular retention of rosuvastatin, atorvastatin and pravastatin, respectively, relative to LacZ control, while absolute intracellular concentration was about twice as high for the lipophilic atorvastatin compared to the more hydrophilic rosuvastatin and pravastatin. After 24 h statin treatment at high concentrations, OATP2B1 enhanced statin toxicity involving activation of intrinsic apoptosis (caspase 3/7 activation) and mitochondrial dysfunction (NADH dehydrogenase activity) following rosuvastatin and atorvastatin, which was partly reversed by isoprenoids. OATP2B1 had no effect on statin-induced reduction in GSIS, mitochondrial electron transport chain complex expression or caspase 9 activation. We confirmed a dose-dependent reduction in insulin secretion by rosuvastatin and atorvastatin in native INS-1 with a modest change in cellular ATP. Collectively, our results indicate a role of OATP2B1, which is abundant in human beta cells, in statin accumulation and statin-induced toxicity but not insulin secretion of rosuvastatin and atorvastatin in INS-1 cells.

The influence of OATP2B1 and atorvastatin on coproporphyrin isomers in rats.

Coproporphyrin I (CPI) and III (CPIII) are discussed as biomarkers for organic anion transporting polypeptides (OATPs). We report on CPI and CPIII levels in wildtype, rSlco2b1-knockout, and SLCO2B1-humanized rats at baseline and after administration of atorvastatin, an inhibitor of the CPIII-specific rOATP2B1/hOATP2B1 and the CPI/CPIII-transporting rOATP1B2. OATP-inhibition by atorvastatin leads to significantly increased CPI and CPIII serum levels. However, basal CP serum levels in rSlco2b1-knockout animals were significantly lower (CPI), or unaffected (CPIII). In the presence of atorvastatin, this genotype effect was abolished. In conclusion, our results indicate an unexpected impact of OATP2B1 on CP serum levels in rats.

Pulmonary inflammation caused by cigarette smoke combined with lipopolysaccharide up-regulated OATP2B1 in rat lung tissue and pulmonary epithelial cells.

Organic anion transport polypeptide 2B1 (OATP2B1), as an uptake transporter, is involved in the transport of many related substrate drugs and endogenous substances in the lungs. A large amount of data shows that cigarette smoke plays an important role in the occurrence and development of lung diseases such as chronic obstructive pulmonary disease (COPD), asthma and bronchitis. However, the effect of cigarette smoke combined with lipopolysaccharide-induced pulmonary inflammation on the expression of OATP2B1 is not clear. In this study, we used cigarette smoke combined with lipopolysaccharide to establish a lung inflammation model in vivo and in vitro to explore the effect of inflammation on the expression of OATP2B1. Our study found that cigarette smoke combined with lipopolysaccharide-induced pulmonary inflammation upregulated the mRNA and protein expression of OATP2B1 and related inflammatory factors, and the expression level of related proteins was higher with the aggravation of inflammation. The experimental results of animals in vivo were consistent with those of cells in vitro. In summary, these findings provide a model and basis for a follow-up study of the mechanism of OATP2B1 in pulmonary inflammation.

Recent advances in drug transporter sciences: highlights from the year 2020.

Drug Metabolism Reviews has an impressive track record of providing scientific reviews in the area of xenobiotic biotransformation over 47 years. It has consistently proved to be resourceful to many scientists from pharmaceutical industry, academia, regulatory agencies working in diverse areas including enzymology, pharmacology, pharmacokinetics, and toxicology. Over the last 5 years Drug metabolism Reviews has annually published an industry commentary aimed to highlight novel insights and approaches that have made significant impacts on the field of biotransformation (led by Cyrus Khojasteh). We hope to continue this tradition by providing an overview of advances made in the field of drug transporters during 2020. The field of drug transporters is rapidly evolving as they play an essential role in drug absorption, distribution, clearance, and elimination. In this review, we have selected outstanding drug transporter articles that have significantly contributed to moving forward the field of transporter science with respect to translation and improved understanding of diverse aspects including uptake clearance, clinical biomarkers, induction, proteomics, emerging transporters, and tissue targeting. The theme of this review consists of a synopsis that summarizes each article followed by our commentary. The objective of this work is not to provide a comprehensive review but rather to exemplify novel insights and state-of-the-art highlights of recent research that have advanced our understanding of drug transporters in drug disposition. We are hopeful that this effort will prove useful to the scientific community and as such request feedback, and further extend an invitation to anyone interested in contributing to future reviews.

Uptake Pathway of Apple-derived Nanoparticle by Intestinal Cells to Deliver its Cargo.

PURPOSE: Food-derived nanoparticles exert cytoprotective effects on intestinal cells by delivering their cargo, which includes macromolecules such as microRNAs and proteins, as well as low-molecular weight compounds. We previously reported that apple-derived nanoparticles (APNPs) downregulate the expression of human intestinal transporter OATP2B1/SLCO2B1 mRNA. To verify the involvement of the cargo of APNPs in affecting the expression of transporters, we characterized the uptake mechanism of APNPs in intestinal cells. METHODS: The uptake of fluorescent PKH26-labeled APNPs (PKH-APNPs) into Caco-2, LS180, and HT-29MTX cells was evaluated by confocal microscopy and flow cytometry. RESULTS: The uptake of PKH-APNPs was prevented in the presence of clathrin-dependent endocytosis inhibitors, chlorpromazine and Pitstop2. Furthermore, PKH-APNPs were incorporated by the HT29-MTX cells, despite the disturbance of the mucus layer. Additionally, the decrease in SLCO2B1 mRNA by APNPs was reversed by Pitstop 2 in Caco-2 cells, indicating that APNPs decrease SLCO2B1 by being incorporated via clathrin-dependent endocytosis. CONCLUSIONS: We demonstrated that clathrin-dependent endocytosis was mainly involved in the uptake of APNPs by intestinal cells, and that the cargo in the APNPs downregulate the mRNA expression of SLCO2B1. Therefore, APNPs could be a useful tool to deliver large molecules such as microRNAs to intestinal cells.

Design, synthesis and biological evaluation of novel estrone phosphonates as high affinity organic anion-transporting polypeptide 2B1 (OATP2B1) inhibitors.

Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific membrane transporter mediating the cellular uptake of various exo- and endobiotics, including drugs and steroid hormones. Increased uptake of steroid hormones by OATP2B1 may increase tumor proliferation. Therefore, understanding OATP2B1's substrate/inhibitor recognition and inhibition of its function, e.g., in hormone-dependent tumors, would be highly desirable. To identify the crucial structural features that correlate with OATP2B1 inhibition, here we designed modifications at four positions of the estrane skeleton. 13α- or 13ß-estrone phosphonates modified at ring A or ring D were synthesized. Hirao and Cu(I)-catalyzed azide-alkyne click reactions served in the syntheses as key steps. 13ß-Derivatives displayed outstanding OATP2B1 inhibitory action with IC50 values in the nanomolar range (41-87 nM). A BODIPY-13α-estrone conjugate was additionally synthesized, modified at C-3-O of the steroid, containing a four-carbon linker between the triazole moiety and the BODIPY core. The fluorescent conjugate displayed efficient, submicromolar OATP2B1 inhibitory potency. The newly identified inhibitors and the structure-activity relationships specified here promote our understanding about drug recognition of OATP2B1.

Zebrafish (Danio rerio) Oatp2b1 as a functional ortholog of the human OATP2B1 transporter.

OATP2B1 belongs to a highly conserved organic anion transporting polypeptide (OATP) family of transporters, involved in the cellular uptake of both endogenous and exogenous compounds. The reported substrates of human OATP2B1 include steroid conjugates, bile salts, and thyroid hormones, as well as pharmaceuticals. Human OATP2B1 has orthologous genes in other vertebrate species, including zebrafish (Danio rerio), a widely used model organism in biomedical and environmental research. Our previous studies showed that zebrafish Oatp2b1 was phylogenetically closest to mammalian OATP2B1/Oatp2b1 and that it shares a similar tissue expression pattern. In this study, we aimed at discovering whether zebrafish Oatp2b1 could be a functional ortholog of human and rodent OATP2B1. To test this hypothesis, our primary goal was to obtain the first in vitro and in silico insights related to the structure and potential substrate preferences of zebrafish Oatp2b1. We generated cells transiently and stably transfected with zebrafish Oatp2b1 cloned from zebrafish liver, constructed an Oatp2b1 homology model, developed transport activity assays with model fluorescent substrate Lucifer yellow, and finally utilized this assay to analyze the interaction of zebrafish Oatp2b1 with both physiological and xenobiotic substances. Apart from structure similarities, our data revealed the strongest interaction of zebrafish Oatp2b1 with bile acids, steroid sulfates, thyroid hormones, and bilirubin, as well as xenobiotics bromosulfophthalein and sulfasalazine, which indicates its functional orthology with human OATP2B1.

Regulation of Organic Anion Transporting Polypeptide 2B1 Expression by MicroRNA in the Human Liver.

Organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) is an uptake transporter expressed in several tissues, including the liver, intestine, brain, kidney, and skeletal muscle. Hepatocyte nuclear factor 4 alpha (HNF4α) is known as an important transcriptional factor of OATP2B1 in the liver. It has been reported that there are large interindividual differences in OATP2B1 mRNA and protein expressions in human livers. The mechanism causing the interindividual differences in OATP2B1 expression is still unclear. MicroRNAs (miRNAs) control gene expression by leading translational repression and/or degradation of the target mRNA. There is no significant correlation between OATP2B1 mRNA and protein expression, suggesting that post-transcriptional regulating mechanisms, such as miRNAs, play an important role in the interindividual differences in OATP2B1 expression. In this study, we hypothesized that certain miRNAs cause the interindividual differences in OATP2B1 expression in the human liver. In silico analysis showed that miR-24 was a candidate miRNA regulating OATP2B1 expression. It has been reported that miR-24 degrades HNF4α mRNA expression. We revealed that the miR-24 expression level was negatively correlated with OATP2B1 mRNA, protein, and HNF4α mRNA expression levels in human livers. Transfection by the miR-24 precursor decreased the luciferase activity in the transfected cells with the vector containing the OATP2B1 3' untranslated region (3'UTR) or SLCO2B1 promoter region. In HepaRG cells, miR-24 decreased the OATP2B1 and HNF4α expression levels. These results suggest that miR-24 represses not only the translation of OATP2B1 but also the transcription of OATP2B1 by HNF4α mRNA degradation.

Food Additives as Inhibitors of Intestinal Drug Transporter OATP2B1.

Food additives are compounds that are added to food and beverage to improve the taste, color, preservation, or composition. Generally, food additives are considered safe for human use due to safety evaluations conducted by food safety authorities and high safety margins applied to permitted usage levels. However, the interaction potential of food additives with simultaneously administered medication has not received much attention. Even though many food additives are poorly absorbed into systemic circulation, high concentrations could exist in the intestinal lumen, making intestinal drug transporters, such as the uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1), a possible site of food additive-drug interactions. In the present work, we aimed to characterize the interaction of a selection of 25 food additives including colorants, preservatives, and sweeteners with OATP2B1 in vitro. In human embryonic kidney 293 (HEK293) cells transiently overexpressing OATP2B1 or control, uptake of dibromofluorescein was studied with and without 50 µM food additive at pH 7.4. As OATP2B1 displays substrate- and pH-dependent transport functions and the intraluminal pH varies along the gastrointestinal tract, we performed the studies also at pH 5.5 using estrone sulfate as an OATP2B1 substrate. Food additives that inhibited OATP2B1-mediated substrate transport by ≥50% were subjected to dose-response studies. Six colorants were identified and validated as OATP2B1 inhibitors at pH 5.5, but only three of these were categorized as inhibitors at pH 7.4. One sweetener was validated as an inhibitor under both assay conditions, whereas none of the preservatives exhibited ≥50% inhibition of OATP2B1-mediated transport. Extrapolation of computed inhibitory constants (Ki values) to estimations of intestinal food additive concentrations implies that selected colorants could inhibit intestinal OATP2B1 also in vivo. These results suggest that food additives, especially colorants, could alter the pharmacokinetics of orally administered OATP2B1 substrate drugs, although further in vivo studies are warranted to understand the overall clinical consequences of the findings.

OATP1A2 and OATP2B1 Are Interacting with Dopamine-Receptor Agonists and Antagonists.

Interaction with the dopaminergic system in the central nervous system is either therapeutically intended or it is a side effect. In both cases, dopamine-receptor agonists (DRA) like the ergoline derivative bromocriptine and dopamine-receptor antagonists (DRAn) like metoclopramide have to cross the blood-brain barrier (BBB). The organic anion transporting polypeptides (OATP) 1A2 and 2B1 are cellular uptake carriers for a variety of endogenous and xenobiotic compounds. As both transporters are expressed in endothelial cells of the BBB, the aim of the present study was to determine whether the DRA bromocriptine, cabergoline, and pergolide and the DRAn metoclopramide and domperidone are interacting with OATP1A2 and 2B1 and could therefore be candidate genes modifying wanted and unwanted effects of these drugs. Localization of both transporters in the brain was confirmed using LC-MS/MS and immunofluorescence stainings. For the functional studies, MDCKII cells stably expressing OATP1A2 or 2B1 were used. Initial interaction studies with the well-characterized transporter substrate estrone 3-sulfate revealed that all tested compounds except pergolide inhibit the transport function of both proteins with the most potent effect for bromocriptine (IC50 = 2.2 µM (OATP1A2) and IC50 = 2.5 µM (OATP2B1)). Further studies using the indirect competitive counterflow method identified bromocriptine, cabergoline, and domperidone as substrates of both transporters, whereas metoclopramide was only transported by OATP1A2. These findings were verified for domperidone by direct measurements using its tritium-labeled form as a tracer. Moreover, the transporter-mediated uptake of this compound was sensitive to the OATP1A2 and OATP2B1 inhibitor naringin. In conclusion, this study suggests that OATP1A2 and 2B1 may play a role in the uptake of DR agonists and antagonists into the brain.

Investigation of Fluorescent Substrates and Substrate-Dependent Interactions of a Drug Transporter Organic Anion Transporting Polypeptide 2B1 (OATP2B1).

PURPOSE: In this study, we investigated organic anion transporting polypeptide 2B1 (OATP2B1)-mediated uptake of fluorescent anions to better identify fluorescent substrates for in vitro OATP2B1 assays. The OATP2B1 is involved in the intestinal absorption and one of the pharmacokinetic determinants of orally administered drugs. METHODS: A microplate reader was used to determine the cellular accumulation of the fluorescent compounds into the OATP2B1 or the empty vector-transfected HEK293 cells. RESULTS: Two types of derivatives were found to be OATP2B1 substrates: heavy halogenated derivatives, such as 4',5'-dibromofluorescein (DBF), and carboxylated derivatives, such as 5-carboxyfluorescein (5-CF). The DBF and 5-CF were transported in a time and concentration-dependent manner. The DBF was transported at a broad pH (pH 6.5-8.0) while 5-CF was transported at an acidic pH (pH 5.5-6.5). The Km values were 0.818 ± 0.067 µM at pH 7.4 for DBF and 8.56 ± 0.41 µM at pH 5.5 for 5-CF. The OATP2B1 inhibitors, including atorvastatin, bromosulfophthalein, glibenclamide, sulfasalazine, talinolol, and estrone 3-sulfate, inhibited the DBF and the 5-CF transport. Contrastively, testosterone, dehydroepiandrosterone sulfate, and progesterone inhibited the DBF transport but stimulated the 5-CF transport. Natural flavonoid aglycones, such as naringenin and baicalein, also exhibited substrate-dependent effects in this manner. CONCLUSION: We found two fluorescein analogs, DBF and 5-CF as the OATP2B1 substrates that exhibited substrate-dependent interactions.

Organic anion transporting polypeptide 2B1 (OATP2B1), an expanded substrate profile, does it align with OATP2B1's hypothesized function?

An expanded view of the substrate landscape of organic anion transporting polypeptide (OATP) 2B1 was pursued with the goal of understanding if the identification of novel in vitro substrates could shed additional light on the impact of OATP2B1 on intestinal absorption and brain penetration.To examine this hypothesis, a series of experiments measured the cellular accumulation of a diverse array of compounds. Representative angiotensin II receptor blockers (ARBs) and other compounds of interest were subsequently investigated for inhibition, time dependence, and kinetics.The study identified ARBs as a class of OATP2B1 substrates and found balsalazide, olsalzine, and gavestinel to be novel substrates of OATP2B1 too. Some compounds previously reported to be OATP2B1 substrates in the literature, aliskiren, erlotinib, montelukast, fexofenadine, and taurocholate could not be confirmed as substrates.Literature describing in vivo outcomes for OATP2B1 substrates, coproporphyrin III, ARBs, balsalazide, olsalzine, and gavestinel highlight the absence of a substantial impact of OATP2B1 on the oral absorption and/or brain penetration of OATP2B1 substrates. Suggestions of including OATP2B1 assessment as part of the drug approval process are likely premature and further mechanistic work with more robust OATP2B1 substrates, which may include some of those described here, is desirable.

ß-Glucuronidase- and OATP2B1-mediated drug interaction of scutellarin in Dengzhan Xixin Injection: A formulation aspect.

Scutellarin is the major and active constituent of Dengzhan Xixin Injection (DZXX), a traditional Chinese medicine prepared from the aqueous extract of Erigeron breviscapus and widely used for the treatment of various cerebrovascular diseases in clinic. In present study, the possible pharmacokinetic differences of scutellarin after intravenous administration of scutellarin alone or DZXX were explored. Additional, the potential roles of ß-glucuronidase (GLU) and OATP2B1 in drug-drug interaction (DDI) between scutellarin and constituents of DZXX were further evaluated in vitro. The plasma concentration, urinary and biliary excretion of scutellarin in rats after administration of DZXX, were significantly higher than those received scutellarin, while pharmacokinetic profile of Apigenin 7-O-glucuronide (AG) in rats was similar no matter AG or DZXX group. Furthermore, higher concentration in brain and plasma, however, lower level of scutellarin in intestine were observed after intravenous administration of DZXX. Finally, AG and caffeoylquinic acid esters were found to significantly inhibit GLU and OATP2B1 in vitro, which might explain, at least in part, the pharmacokinetic DDI between scutellarin and other chemical constituents in DZXX. The findings provided deep insight into the prescription-formulating principle in DZXX for treating the cerebrovascular diseases.

Establishment and Validation of Competitive Counterflow as a Method To Detect Substrates of the Organic Anion Transporting Polypeptide 2B1.

The organic anion transporting polypeptide (OATP) 2B1 is ubiquitously expressed and known to facilitate cellular entry. It is widely accepted that transport proteins play a pivotal role in pharmacokinetics. Consequently, testing for interaction with drug transporters became an important part in the assessment of new molecular entities in order to predict and prevent drug-drug interactions. Recently, competitive counterflow (CCF), an indirect method allowing the identification of substrates, was successfully applied to the organic cation transporter 2. It was the aim of this study to test whether CCF can be used to identify substrates of OATP2B1. A protocol for CCF experiments using estrone 3-sulfate (E1S) as the driven compound in expression-verified MDCKII-OATP2B1 cells was established. The protocol was tested using a substance library, which was prior screened for inhibition of OATP2B1-mediated transport accounting for both E1S-binding sites. In CCF experiments, all previously reported OATP2B1 substrates significantly reduced the amount of E1S in equilibrium, classifying them as substrates. In addition, we identified and verified novel substrates of OATP2B1, namely, astemizole and domperidone. Results of the CCF were complemented with cytotoxicity assays or cell-based reporter gene assays to validate the finding of etoposide and teniposide or hyperforin being substrates of OATP2B1, respectively. Our study indicates that the method of CCF can be used to identify substrates of OATP2B1, irrespective, whether interacting with binding site A or A and B, but is limited by solubility issues or the amount of transporter that is expressed in the used cellular system.

Effect of Rifampicin on the Distribution of [11C]Erlotinib to the Liver, a Translational PET Study in Humans and in Mice.

Organic anion-transporting polypeptides (OATPs) mediate the uptake of various drugs from blood into the liver in the basolateral membrane of hepatocytes. Positron emission tomography (PET) is a potentially powerful tool to assess the activity of hepatic OATPs in vivo, but its utility critically depends on the availability of transporter-selective probe substrates. We have shown before that among the three OATPs expressed in hepatocytes (OATP1B1, OATP1B3, and OATP2B1), [11C]erlotinib is selectively transported by OATP2B1. In contrast to OATP1B1 and OATP1B3, OATP2B1 has not been thoroughly explored yet, and no specific probe substrates are currently available. To assess if the prototypical OATP inhibitor rifampicin can inhibit liver uptake of [11C]erlotinib in vivo, we performed [11C]erlotinib PET scans in six healthy volunteers without and with intravenous pretreatment with rifampicin (600 mg). In addition, FVB mice underwent [11C]erlotinib PET scans without and with concurrent intravenous infusion of high-dose rifampicin (100 mg/kg). Rifampicin caused a moderate reduction in the liver distribution of [11C]erlotinib in humans, while a more pronounced effect of rifampicin was observed in mice, in which rifampicin plasma concentrations were higher than in humans. In vitro uptake experiments in an OATP2B1-overexpressing cell line indicated that rifampicin inhibited OATP2B1 transport of [11C]erlotinib in a concentration-dependent manner with a half-maximum inhibitory concentration of 72.0 ± 1.4 µM. Our results suggest that rifampicin-inhibitable uptake transporter(s) contributed to the liver distribution of [11C]erlotinib in humans and mice and that [11C]erlotinib PET in combination with rifampicin may be used to measure the activity of this/these uptake transporter(s) in vivo. Furthermore, our data suggest that a standard clinical dose of rifampicin may exert in vivo a moderate inhibitory effect on hepatic OATP2B1.

Pd-Catalyzed microwave-assisted synthesis of phosphonated 13α-estrones as potential OATP2B1, 17ß-HSD1 and/or STS inhibitors.

Novel 2- or 4-phosphonated 13α-estrone derivatives were synthesized via the Hirao reaction. Bromo regioisomers (2- or 4-) of 13α-estrone and its 3-benzyl or 3-methyl ether were reacted with diethyl phosphite or diphenylphosphine oxide using Pd(PPh3)4 as catalyst under microwave irradiation. The influence of the new compounds on the transport function of the organic anion transporting polypeptide OATP2B1 was investigated by measuring Cascade Blue uptake. Derivatives bearing a 3-benzyl ether function displayed substantial submicromolar OATP2B1 inhibitory activity. The inhibitory effects of the compounds on human placental steroid sulfatase (STS) and 17ß-hydroxysteroid dehydrogenase type 1 isozyme (17ß-HSD1) were investigated by in vitro radiosubstrate incubation methods. None of the test compounds inhibited the STS markedly. The structure-activity relationship evaluation revealed that 2-substituted 3-hydroxy derivatives are able to inhibit the 17ß-HSD1 enzyme with submicromolar IC50 values. Dual OATP2B1 and 17ß-HSD1 inhibitors have been identified.

Prioritizing pharmacokinetic drug interaction precipitants in natural products: application to OATP inhibitors in grapefruit juice.

Natural products, including botanical dietary supplements and exotic drinks, represent an ever-increasing share of the health-care market. The parallel ever-increasing popularity of self-medicating with natural products increases the likelihood of co-consumption with conventional drugs, raising concerns for unwanted natural product-drug interactions. Assessing the drug interaction liability of natural products is challenging due to the complex and variable chemical composition inherent to these products, necessitating a streamlined preclinical testing approach to prioritize precipitant individual constituents for further investigation. Such an approach was evaluated in the current work to prioritize constituents in the model natural product, grapefruit juice, as inhibitors of intestinal organic anion-transporting peptide (OATP)-mediated uptake. Using OATP2B1-expressing MDCKII cells (Madin-Darby canine kidney type II) and the probe substrate estrone 3-sulfate, IC50s were determined for constituents representative of the flavanone (naringin, naringenin, hesperidin), furanocoumarin (bergamottin, 6',7'-dihydroxybergamottin) and polymethoxyflavone (nobiletin and tangeretin) classes contained in grapefruit juice. Nobiletin was the most potent (IC50 , 3.7 µm); 6',7'-dihydroxybergamottin, naringin, naringenin and tangeretin were moderately potent (IC50 , 20-50 µm); and bergamottin and hesperidin were the least potent (IC50 , >300 µm) OATP2B1 inhibitors. Intestinal absorption simulations based on physiochemical properties were used to determine the ratios of unbound concentration to IC50 for each constituent within enterocytes and to prioritize in order of pre-defined cut-off values. This streamlined approach could be applied to other natural products that contain multiple precipitants of natural product-drug interactions. Copyright © 2016 John Wiley & Sons, Ltd.

Tryptophan Residue Located at the Middle of Putative Transmembrane Domain 11 Is Critical for the Function of Organic Anion Transporting Polypeptide 2B1.

Organic anion transporting polypeptide 2B1 (OATP2B1), which is highly expressed in enterocytes and hepatocytes could be a key determinant for the intestinal absorption and hepatic uptake of its substrates, most of which are amphipathic organic anions. Tryptophan residues may possess a multitude of functions for a transport protein through aromatic interactions, such as maintaining the proper protein structure, guiding the depth of membrane insertion, or interacting directly with substrates. There are totally six tryptophan residues in OATP2B1. However, little is known about their role in the function and expression of OATP2B1. Our results show that, while W272, W276, and W277 located at the border of extracellular loop 3 and transmembrane domain 6 exhibit a moderate effect on the surface expression of OATP2B1, W611 located at the middle of transmembrane domain 11 plays a critical role in the function of OATP2B1. The tryptophan-to-alanine mutation of W611 changes the kinetic characteristics of OATP2B1-mediated estrone-3-sulfate (E3S) transport radically, from a monophasic saturation curve (with Km and Vmax values being of 7.1 ± 1.1 µM and 182 ± 7 pmol/normalized mg/min, respectively) to a linear curve. Replacing alanine with a phenylalanine will rescue most of OATP2B1's function, suggesting that the aromatic side chain of residue 611 is very important. However, hydrogen-bond forming and positively charged groups at this position are not favorable. The important role of W611 is not substrate-dependent. Molecular modeling indicates that the side chain of W611 faces toward the substrate translocation pathway and might interact with substrates directly. Taken together, our findings reveal that W611 is critical for the function of OATP2B1.

Quantitative Atlas of Cytochrome P450, UDP-Glucuronosyltransferase, and Transporter Proteins in Jejunum of Morbidly Obese Subjects.

Protein expression levels of drug-metabolizing enzymes and transporters in human jejunal tissues excised from morbidly obese subjects during gastric bypass surgery were evaluated using quantitative targeted absolute proteomics. Protein expression levels of 15 cytochrome P450 (CYP) enzymes, 10 UDP-glucuronosyltransferase (UGT) enzymes, and NADPH-P450 reductase (P450R) in microsomal fractions from 28 subjects and 49 transporters in plasma membrane fractions from 24 of the same subjects were determined using liquid chromatography-tandem mass spectrometry. Based on average values, UGT1A1, UGT2B15, UGT2B17, SGLT1, and GLUT2 exhibited high expression levels (over 10 fmol/µg protein), though UGT2B15 expression was detected at a high level in only one subject. CYP2C9, CYP2D6, CYP3A5, UGT1A6, P450R, ABCG2, GLUT5, PEPT1, MCT1, 4F2 cell-surface antigen heavy chain (4F2hc), LAT2, OSTα, and OSTß showed intermediate levels (1-10 fmol/µg protein), and CYP1A1, CYP1A2, CYP1B1, CYP2C18, CYP2C19, CYP2J2, CYP3A7, CYP4A11, CYP51A1, UGT1A3, UGT1A4, UGT1A8, UGT2B4, ABCC1, ABCC4, ABCC5, ABCC6, ABCG8, TAUT, OATP2A1, OATP2B1, OATP3A1, OATP4A1, OCTN1, CNT2, PCFT, MCT4, GLUT4, and SLC22A18 showed low levels (less than 1 fmol/µg protein). The greatest interindividual difference (364-fold) was detected for UGT2B17. However, differences in expression levels of other quantified UGTs (except UGT2B15 and UGT2B17), CYPs (except CYP1A1 and CYP3A5), and P450R, and all quantified transporters, were within 10-fold. Expression levels of CYP1A2 and GLUT4 were significantly correlated with body-mass index. The levels of 4F2hc showed significant gender differences. Smokers showed increased levels of UGT1A1 and UGT1A3. These findings provide a basis for understanding the changes in molecular mechanisms of jejunal metabolism and transport, as well as their interindividual variability, in morbidly obese patients.