Organic Anion Transporting Polypeptide 1A2 Research Articles

The Effects of Jabara Juice on the Intestinal Permeation of Fexofenadine.

Jabara juice and its component narirutin inhibit the activity of organic anion-transporting polypeptides (OATPs) 1A2 and OATP2B1, which are considered to play significant roles in the intestinal absorption of fexofenadine. In this study, we investigated the effects of jabara juice on the intestinal absorption of fexofenadine in mice and the inhibitory effects of jabara juice and narirutin on the permeation of fexofenadine using Caco-2 cell monolayers and LLC-GA5-COL300 cell monolayers. In the in vivo study, the area under the plasma concentration-time curve (AUC) of fexofenadine in mice was increased 1.8-fold by jabara juice. In the permeation study, 5% jabara juice significantly decreased the efflux ratio (ER) of fexofenadine for Caco-2 monolayers. Furthermore, the ERs of fexofenadine and digoxin, which is a typical substrate of P-glycoprotein (P-gp), for LLC-GA5-COL300 cell monolayers were decreased in a concentration-dependent manner by jabara juice extract, suggesting that jabara juice may increase the intestinal absorption of fexofenadine by inhibiting P-gp, rather than by narirutin inhibiting OATPs. The present study showed that jabara juice increases the intestinal absorption of fexofenadine both in vivo and in vitro. The intestinal absorption of fexofenadine may be altered by the co-administration of jabara juice in the clinical setting.

Thyroid hormone transport and metabolism are disturbed in the placental villi of miscarriage

BackgroundIt has been long known that thyroid hormone regulates placental villi development, which is associated with the occurrence of miscarriage. However, whether abnormal thyroid hormone metabolism and transport in placental villi are involved in miscarriage is still to be verified.MethodsPlacental villi of elective terminations of pregnancies (ETPs) and miscarriage were collected. Proliferative activity and apoptosis of villi trophoblasts and angiogenesis were detected by TUNEL and immunochemistry. The expressions of thyroid hormone receptors (THRs), transthyretin (TTR), monocarboxylate transporter 8 (MCT8), organic anion transporting polypeptides 1A1 (OATP1A1), deiodinase 2 (Dio2) and Dio3 were examined by RT-PCR, Western blot, immunohistochemistry and immunofluorescence. JEG3 cell was treated with iopanoic acid (IOP), an inhibitor of Dio2 activity, the expressions of Dio2, placenta growth factor (PLGF) and sFlt1 were detected by RT-PCR and Western blot.ResultsCell proliferation was suppressed and apoptosis was increased in placental villi cytotrophoblasts of miscarriage. CD34+ vessel number and vascular endothelial growth factor (VEGF) protein abundance were decreased in miscarriage. In miscarriage group, the gene expression of Dio2, Dio3, TTR and THRα, but not THRβ, MCT8 and OATP1A1, were downregulated. The protein abundances of TTR and THRα were downregulated in miscarriage group, but not THRβ. The protein abundance of Dio2 in miscarriage villi was decreased compared with that in ETP. In JEG3 cells, the gene expression of PLGF was decreased and the expression of sFlt1 was increased in IOP treatment; The protein abundance of Dio2 was downregulated but the gene expression of Dio2 was unaffected in IOP treatment.ConclusionThyroid hormone transport and metabolism in miscarriage were disturbed and may impaired angiogenesis of placental villi, which was associated with the occurrence of miscarriage.

The fluorescence-based competitive counterflow assay developed for organic anion transporting polypeptides 1A2, 1B1, 1B3 and 2B1 identifies pentamidine as a selective OATP1A2 substrate.

Organic anion transporting polypeptides OATP1A2, OATP1B1, OATP1B3 and OATP2B1 are Na+ - and ATP-independent exchangers of large, organic compounds, encompassing structurally diverse xenobiotics, including various drugs. These OATPs influence intestinal absorption (OATP2B1), hepatic clearance (OATP1B1/3) and blood to brain penetration (OATP1A2, OATP2B1) of their drug substrates. Consequently, OATP-mediated drug or food interactions may lead to altered pharmacokinetics and toxicity. During drug development, investigation of hepatic OATP1B1 and OATP1B3 is recommended by international regulatory agencies. Most frequently, OATP-drug interactions are investigated in an indirect assay, i.e., by examining uptake inhibition of a radioactive or fluorescent probe. However, indirect assays do not distinguish between transported substrates and non-transported OATP inhibitors. To fill this hiatus, a novel assay, termed competitive counterflow (CCF) has been developed and has since been applied for several OATPs to differentiate between substrates and non-transported inhibitors. However, previous OATP CCF assays, with the exception of that for OATP1B1, used radioactive probes. In the current study, we demonstrate that sulforhodamine 101 or pyranine can be used as fluorescent probes in a CCF assay to identify transported substrates of OATP1A2, or OATPs 1B1, 1B3 and 2B1, respectively. With the help of the newly developed fluorescence-based CCF method, we identify the FDA-approved anti-protozoal drug, pentamidine as a unique substrate of OATP1A2. Furthermore, we confirm the selective, OATP1A2-mediated uptake of pentamidine in a cytotoxicity assay. Based on our results, OATP1A2 may be an important determinant of pentamidine transport through the blood-brain barrier.

Oatp (Organic Anion Transporting Polypeptide)-Mediated Transport: A Mechanism for Atorvastatin Neuroprotection in Stroke.

Drug discovery for stroke is challenging as indicated by poor clinical translatability. In contrast, HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (ie, statins) improve poststroke neurological outcomes. This property requires transport across the blood-brain barrier via an endogenous uptake transporter (ie, Oatp1a4 [organic anion transporting polypeptide 1a4]). Our goal was to study Oatp1a4 as a drug delivery mechanism because the blood-brain barrier cannot be assumed to be completely open for all drugs in ischemic stroke. Male Sprague-Dawley rats (200-250 g) were subjected to middle cerebral artery occlusion (90 minutes) followed by reperfusion for up to 7 days. Atorvastatin (20 mg/kg, IV) was administered 2 hours following intraluminal suture removal. Involvement of Oatp-mediated transport was determined using fexofenadine (3.2 mg/kg, IV), a competitive Oatp inhibitor. Oatp1a4 transport activity was measured by in situ brain perfusion. Infarction volumes/brain edema ratios and neuronal nuclei expression were determined using 2,3,5-triphenyltetrazolium chloride-stained brain tissue slices and confocal microscopy, respectively. Poststroke functional outcomes were assessed via neurological deficit scores and rotarod analysis. At 2-hour post-middle cerebral artery occlusion, [3H]atorvastatin uptake was increased in ischemic brain tissue. A single dose of atorvastatin significantly reduced post-middle cerebral artery occlusion infarction volume, decreased brain edema ratio, increased caudoputamen neuronal nuclei expression, and improved functional neurological outcomes. All middle cerebral artery occlusion positive effects of atorvastatin were attenuated by fexofenadine coadministration (ie, an Oatp transport inhibitor). Our data demonstrate that neuroprotective effects of atorvastatin may require central nervous system delivery by Oatp-mediated transport at the blood-brain barrier, a mechanism that persists despite increased cerebrovascular permeability in ischemic stroke. These novel and translational findings support utility of blood-brain barrier transporters in drug delivery for neuroprotective agents.

Abstract TP209: Role Of Endoglin In The Regulation Of Organic Anion Transporting Polypeptide 1a4 At The Blood-brain Barrier: Relevance To The Treatment Of Ischemic Stroke

Objectives: We have shown that transforming growth factor-β (TGF-β)/activin-like kinase 1 (ALK1) signaling at the blood-brain barrier (BBB) can regulate organic anion transporting polypeptide 1a4 (Oatp1a4), a transporter involved in brain uptake of drugs relevant to stroke pharmacotherapy (i.e., 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins)). To date, the involvement of endoglin (ENG), a co-receptor that can modulate the TGF-β/ALK1 pathway, in regulation of Oatp1a4 expression/activity has not been elucidated. The objective of this study is to evaluate whether ENG plays a role in the control of BBB transport mechanisms for stroke therapeutics. Methods: Our studies were conducted using the immortalized mouse brain endothelial cell line bEnd.3. Endoglin knockdown mutants were generated through lentiviral transfection of ENG-shRNA and validated using confocal microscopy and western blot analysis. Cells were then treated with bone morphogenetic protein-9 (BMP-9; 1 nM), a known TGF-β/ALK1 agonist. Oatp1a4 protein expression and transport activity were measured using western blot analysis and in vitro uptake assays using the known Oatp substrate drug [ 3 H]atorvastatin. Additionally, ENG expression in bEnd.3 cells was assessed under normoxic and oxygen/glucose deprivation (OGD) conditions (i.e., 1% O 2 in glucose-free medium). Results: BMP-9 treatment in non-transfected bEnd.3 cells resulted in increased Oatp1a4 protein expression and increased cellular uptake of [ 3 H]atorvastatin. These enhancements in Oatp1a4 functional expression were attenuated in ENG-shRNA knockdown bEnd.3 cells treated with BMP-9. Confocal microscopy and western blot analysis confirmed decreased ENG protein expression following shRNA transfection. Under OGD conditions, expression of ENG in bEnd.3 cells was significantly increased. Conclusions: Our data provide the first evidence for involvement of ENG in regulation of Oatp1a4 by TGF-β/ALK1 signaling in brain microvessel endothelial cells. These observations have clear implications for CNS delivery of drugs relevant to treatment of stroke. Studies are ongoing in our laboratory to fully evaluate ENG regulation of BBB transport mechanisms in the setting of ischemic stroke.

Abstract TP205: Organic Anion Transporting Polypeptides: Endogenous Blood-brain Barrier Transporters That Are Required For Statins To Exert Neuroprotective Effects In Ischemic Stroke

Clinical development of new drugs for ischemic stroke has been challenging. In contrast, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (i.e., statins) are routinely administered to stroke patients because they improve neurological outcomes. This property of statins requires delivery across the blood-brain barrier (BBB). We have uncovered a BBB uptake transporter for statins: organic anion transporting polypeptide 1a4 (Oatp1a4). Our goal was to determine whether Oatp-mediated transport at the BBB is a required mechanism for a commonly prescribed statin (i.e., atorvastatin; ATV) to exert neuroprotective effects following ischemic stroke. Methods: Male Sprague-Dawley rats (200-250 g) were subjected to middle cerebral artery occlusion (MCAO; 90 min) followed by reperfusion. ATV (20 mg/kg, i.v.) was administered 2 h following intraluminal suture removal. Involvement of Oatp-mediated transport was determined using fexofenadine (FEX; 3.2 mg/kg, i.v.), a competitive Oatp inhibitor. Oatp1a4 transport activity was measured by in situ brain perfusion. Following MCAO/reperfusion, infarction volumes and brain edema ratios were calculated from TTC-stained brain tissue slices. Hippocampal neuronal cell numbers were assessed by immunofluorescence imaging of neuronal nuclei (NeuN) staining. Post-stroke functional outcomes were assessed via neurological deficit scores and rotarod analysis. At 2 h post-MCAO, ATV uptake was increased in ischemic brain tissue. FEX blocked blood-to-brain uptake of ATV, which confirmed involvement of an Oatp-mediated transport mechanism. ATV reduced post-MCAO infarction volume and brain edema ratio, increased hippocampal NeuN staining, and improved neurological outcomes. All positive effects of ATV were attenuated by co-administration of FEX. Conclusions: Our data demonstrate ATV requires functional expression of an endogenous BBB transporter (i.e., Oatp1a4) to exert neuroprotective effects and promote post-stroke recovery. These novel and translational findings provide mechanistic evidence on the critical role of BBB transporters in delivery of stroke drugs to the ischemic brain, information that can facilitate therapeutic advancement in clinical trials.

Comparison of the transport kinetics of fexofenadine and its pH dependency among OATP1A2 genetic variants

Little is known about the influence of non-synonymous genetic variations in the organic anion-transporting polypeptide (OATP) 1A2 on the transport kinetics of its substrate fexofenadine. Moreover, the pH-dependency of fexofenadine uptake also remains unclear. This study aimed to evaluate the effects of genetic variants (Ile13Thr, Asn128Tyr, Glu172Asp, Ala187Thr, and Thr668Ser) on the OATP1A2-mediated uptake of fexofenadine at pH 6.3 and 7.4 and compare the pH dependency of OATP1A2-mediated uptake of fexofenadine and estrone 3-sulfate. The uptake clearances of 0.3 μM and 300 μM fexofenadine were compared with those of 0.3 μM and 300 μM estrone 3-sulfate at pH 6.3 and 7.4. Among the six variants examined, the Thr668Ser variant showed the highest fexofenadine uptake clearance (Vmax/Km); i.e., 4.53- and 6.28-fold higher uptake clearance than the wild type at pH 6.3 and 7.4, respectively. All variants exhibited significantly higher fexofenadine uptake at pH 6.3 than at pH 7.4. Compared with estrone 3-sulfate uptake, the uptake of 0.3 μM fexofenadine was less sensitive to pH. Our findings suggest that genetic variations in OATP1A2 may lead to altered intestinal absorption of fexofenadine, such as increased absorption in subjects bearing the Thr668Ser variant, which showed higher uptake activity.

Comparison of the inhibitory properties of the fruit component naringenin and its glycosides against OATP1A2 genetic variants

Non-synonymous genetic variants of organic anion-transporting polypeptide (OATP) 1A2 with altered transport activity have been identified. Naringin and narirutin, which are found in grapefruit, and their aglycon naringenin inhibit OATP1A2. However, their inhibitory effects on OATP1A2 variants have not been investigated, nor has the influence of their molecular structure, such as the number of sugar moieties, on their inhibitory potency. This study aimed to investigate the inhibitory effects of naringenin, its monosaccharide glycoside prunin, and its disaccharide glycosides naringin and narirutin on fexofenadine (FEX) uptake by OATP1A2 variants (Ile13Thr, Asn128Tyr, Ala187Thr, and Thr668Ser).Naringin, narirutin, and prunin inhibited FEX (0.3 μM) uptake by all of the examined OATP1A2 variants in a concentration-dependent manner. Compared with those for the wild type, the inhibition constants (Ki) of naringin, narirutin, and prunin for the Ala187Thr variant were significantly increased by 3.36-fold, 7.55-fold, and 10.6-fold, respectively. Naringenin inhibited all of the OATP1A2 variants, except Ala187Thr, concentration-dependently. The order of inhibitory potency was as follows for all variants: aglycone > monosaccharide glycoside > disaccharide glycosides.These results suggest that the Ala187Thr variant is less vulnerable to inhibition by naringenin and its glycosides. Moreover, greater glycosylation of naringenin reduces its inhibitory potency against OATP1A2.

Regulation of Blood-Brain Barrier Transporters by Transforming Growth Factor-β/Activin Receptor-Like Kinase 1 Signaling: Relevance to the Brain Disposition of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors (i.e., Statins).

Our laboratory has shown that activation of transforming growth factor- β (TGF- β )/activin receptor-like kinase 1 (ALK1) signaling can increase protein expression and transport activity of organic anion transporting polypeptide 1a4 (Oatp1a4) at the blood-brain barrier (BBB). These results are relevant to treatment of ischemic stroke because Oatp transport substrates such as 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (i.e., statins) improve functional neurologic outcomes in patients. Advancement of our work requires determination if TGF- β /ALK1 signaling alters Oatp1a4 functional expression differently across brain regions and if such disparities affect central nervous system (CNS) statin disposition. Therefore, we studied regulation of Oatp1a4 by the TGF- β /ALK1 pathway, in vivo, in rat brain microvessels isolated from cerebral cortex, hippocampus, and cerebellum using the ALK1 agonist bone morphogenetic protein-9 (BMP-9) and the ALK1 inhibitor 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride 193189. We showed that Oatp1a4 protein expression and brain distribution of three currently marketed statin drugs (i.e., atorvastatin, pravastatin, and rosuvastatin) were increased in cortex relative to hippocampus and cerebellum. Additionally, BMP-9 treatment enhanced Oatp-mediated statin transport in cortical tissue but not in hippocampus or cerebellum. Although brain drug delivery is also dependent upon efflux transporters, such as P-glycoprotein and/or Breast Cancer Resistance Protein, our data showed that administration of BMP-9 did not alter the relative contribution of these transporters to CNS disposition of statins. Overall, this study provides evidence for differential regulation of Oatp1a4 by TGF- β /ALK1 signaling across brain regions, knowledge that is critical for development of therapeutic strategies to target Oatps at the BBB for CNS drug delivery. SIGNIFICANCE STATEMENT: Organic anion transporting polypeptides (Oatps) represent transporter targets for brain drug delivery. We have shown that Oatp1a4 statin uptake is higher in cortex versus hippocampus and cerebellum. Additionally, we report that the transforming growth factor- β /activin receptor-like kinase 1 agonist bone morphogenetic protein-9 increases Oatp1a4 functional expression, but not efflux transporters P-glycoprotein and Breast Cancer Resistance Protein, in cortical brain microvessels. Overall, this study provides critical data that will advance treatment for neurological diseases where drug development has been challenging.

Inhibitory Effects of Cranberry Juice and Its Components on Intestinal OATP1A2 and OATP2B1: Identification of Avicularin as a Novel Inhibitor.

Organic anion-transporting polypeptide (OATP) 1A2 and OATP2B1 mediate the intestinal absorption of drugs. This study aimed to identify fruit juices or fruit juice components that inhibit OATPs and assess the risk of associated food-drug interactions. Inhibitory potency was assessed by examining the uptake of [3H]estrone 3-sulfate and [3H]fexofenadine into HEK293 cells expressing OATP1A2 or OATP2B1. In vivo experiments were conducted using mice to evaluate the effects of cranberry juice on the pharmacokinetics of orally administered fexofenadine. Of eight examined fruit juices, cranberry juice inhibited the functions of both OATPs most potently. Avicularin, a component of cranberry juice, was identified as a novel OATP inhibitor. It exhibited IC50 values of 9.0 and 37 μM for the inhibition of estrone 3-sulfate uptake mediated by OATP1A2 and OATP2B1, respectively. A pharmacokinetic experiment revealed that fexofenadine exposure was significantly reduced (by 50%) by cranberry juice. Cranberry juice may cause drug interactions with OATP substrates.

Pharmacogenomics of celiprolol - evidence for a role of P-glycoprotein and organic anion transporting polypeptide 1A2 in celiprolol pharmacokinetics.

The aim of this study was to search for associations of genetic variants with celiprolol pharmacokinetics in a large set of pharmacokinetic genes, and, more specifically, in a set of previously identified candidate genes ABCB1, SLCO1A2, and SLCO2B1. To this end, we determined celiprolol single‐dose (200 mg) pharmacokinetics and sequenced 379 pharmacokinetic genes in 195 healthy volunteers. Analysis with 46,064 common sequence variants in the 379 genes did not identify any novel genes associated with celiprolol exposure. The candidate gene analysis showed that the ABCB1 c.3435T>C and c.2677T/G>A, and the SLCO1A2 c.516A>C variants were associated with reduced celiprolol area under the plasma concentration‐time curve (AUC0–∞). An alternative analysis with ABCB1 haplotypes showed that, in addition to SLCO1A2 c.516A>C, three ABCB1 haplotypes were associated with reduced celiprolol AUC0–∞. A genotype scoring system was developed based on these variants and applied to stratify the participants to low and high celiprolol exposure genotype groups. The mean AUC0–∞ of celiprolol in the low exposure genotype group was 55% of the mean AUC0–∞ in the high exposure group (p = 1.08 × 10−11). In addition, the results showed gene‐gene interactions in the effects of SLCO1A2 and ABCB1 variants on celiprolol AUC0–∞ (p < 5 × 10−6) suggesting an interplay between organic anion transporting polypeptide 1A2 and P‐glycoprotein in celiprolol absorption. Taken together, these data indicate that P‐glycoprotein and organic anion transporting polypeptide 1A2 play a role in celiprolol pharmacokinetics. Furthermore, patients with ABCB1 and SLCO1A2 genotypes associated with low celiprolol exposure may have an increased risk of poor blood‐pressure lowering response to celiprolol.

Role of Uptake Transporters OAT4, OATP2A1, and OATP1A2 in Human Placental Bio-disposition of Pravastatin

Pravastatin is currently under evaluation for prevention of preeclampsia. Factors contributing to placental disposition of pravastatin are important in assessment of potential undesirable fetal effects. The purpose of this study was to identify the uptake transporters that contribute to the placental disposition of pravastatin. Our data revealed the expression of organic anion transporting polypeptide 1A2 (OATP1A2) and OATP2A1 in the apical, and OATP2B1 and OATP5A1 in the basolateral membranes of the placenta, while organic anion transporter 4 (OAT4) exhibited higher expression in basolateral membrane but was detected in both membranes. Preloading placental membrane vesicles with glutarate increased the uptake of pravastatin suggesting involvement of glutarate-dependent transporters such as OAT4. In the HEK293 cells overexpressing individual uptake transporters, OATP2A1, OATP1A2 and OAT4 were determined to accept pravastatin as a substrate at physiological pH, while the uptake of pravastatin by OATP2B1 (known to interact with pravastatin at acidic pH) and OATP5A1 was not detected at pH 7.4. These findings led us to propose that OATP1A2 and OATP2A1 are responsible for the placental uptake of pravastatin from the maternal circulation, while OAT4 mediates the passage of the drug across placental basolateral membrane in the fetal-to-maternal direction.

Uptake of A\u03b2 by OATPs might be a new pathophysiological mechanism of Alzheimer disease

BackgroundThe accumulation of neurotoxic amyloid-beta (Aβ) in the brain is a characteristic of Alzheimer's disease (AD), at the same time, it is possible alterations of liver function could affect brain Aβ levels through changes in blood Aβ concentration. Over the last decade, a number of reports have shown that P-glycoprotein (encoded by ABC1B1) actively mediates the efflux transport of Aβ peptides. However, the mechanism by which Aβ peptides enter the cells is not clear. In the preliminary study, we found that the protein expression of organic anion transporting Polypeptide 1a4 (OATP1B1) in the liver tissue of mice with AD was significantly higher than that in the normal mice. In contrast, the protein expression of Oatp1a4 in the brain significantly decreased in mice with AD. OATP1B1, an important drug transporter might be related to the pathophysiology of AD.ResultsIn this study, we established an OATP1B1-GFP-HEK293T cell model to confirm the OATP1B1 mediated transport of Aβ1-42. Compared to the control group of GFP-HEK293Tcells, the uptake of Aβ1-42 protein in the OATP1B1-GFP-HEK293T group increased significantly with the increase in concentration of Aβ1-42, and also increased significantly with an increase in the duration of incubation. Similar results were observed in the flow cytometry experiment, and the uptake of Aβ1-42in HEK293T-OATP1B1 cells was almost twice that in the control group. These results indicate that OATPs may act as an important “carrier” for the transport of Aβ1-42 from the blood to the tissues, including liver and brain.ConclusionsThis is a novel and interesting finding and OATP1B1 can be investigated as a new treatment target for AD.

Constituents of Passiflora incarnata, but Not of Valeriana officinalis, Interact with the Organic Anion Transporting Polypeptides (OATP)2B1 and OATP1A2.

Herbal medication used in the treatment of sleep disorders and anxiety often contain extracts of Valeriana officinalis or Passiflora incarnata. Valerenic acid in V.officinalis and apigenin, orientin, and vitexin in P. incarnata are thought to contribute to their therapeutic effect. It was the aim of this study to test whether these constituents of herbal extracts are interacting with the uptake of estrone 3-sulfate, pregnenolone sulfate, and dehydroepiandrosterone sulfate mediated by the uptake transporters organic anion transporting polypeptide 2B1 (OATP2B1) or organic anion transporting polypeptide 1A2 (OATP1A2). Madin-Darby canine kidney cells overexpressing OATP2B1 or OATP1A2 were used to determine the influence of the constituents on the cellular accumulation of the sulfated steroids. Subsequently, competitive counterflow experiments were applied to test whether identified inhibitors are also substrates of the transporters. Valerenic acid only interacted with OATP2B1, whereas apigenin, orientin, and vitexin interacted with OATP2B1 and OATP1A2. Competitive counterflow revealed that orientin is a substrate of both transporters, while apigenin was transported by OATP1A2 and vitexin by OATP2B1. In a next step, commercially available P. incarnata preparations were assessed for their influence on the transporters, revealing inhibition of transporter-mediated estrone 3-sulfate uptake. HPLC-UV-MS analysis confirmed the presence of orientin and vitexin in these preparations, thereby suggesting that these constituents are involved in the interaction. Our data indicate that constituents of P. incarnata may alter the function of OATP2B1 and OATP1A2, which could affect the uptake of other compounds relying on uptake mediated by the transporters.

Citrus Fruit-Derived Flavanone Glycoside Narirutin is a Novel Potent Inhibitor of Organic Anion-Transporting Polypeptides.

Organic anion-transporting polypeptides (OATPs) 1A2 and OATP2B1 are expressed in the small intestine and are involved in drug absorption. We identified narirutin, which is present in grapefruit juice, as a novel OATP inhibitor. The citrus fruit jabara also contains high levels of narirutin; therefore, we investigated the inhibitory potency of jabara juice against OATPs. The inhibitory effects of various related compounds on the transport activity of OATPs were evaluated using OATP-expressing HEK293 cells. The IC50 values of narirutin for OATP1A2- and OATP2B1-mediated transport were 22.6 and 18.2 μM, respectively. Other flavanone derivatives from grapefruit juice also inhibited OATP1A2/OATP2B1-mediated transport (order of inhibitory potency: naringenin > narirutin > naringin). Five percent jabara juice significantly inhibited OATP1A2- and OATP2B1-mediated transport by 67 ± 11 and 81 ± 5.5%, respectively (p < 0.05). Based on their inhibitory potency and levels in grapefruit juice, the inhibition of OATPs by grapefruit juice is attributable to both naringin and narirutin. Citrus × jabara, which contains narirutin, potently inhibits OATP-mediated transport.

Transport Properties of Statins by Organic Anion Transporting Polypeptide 1A2 and Regulation by Transforming Growth Factor-β Signaling in Human Endothelial Cells.

Our in vivo rodent studies have shown that organic anion transporting polypeptide (Oatp) 1a4 is critical for blood-to-brain transport of statins, drugs that are effective neuroprotectants. Additionally, transforming growth factor-β (TGF-β) signaling via the activin receptor-like kinase 1 (ALK1) receptor regulates Oatp1a4 functional expression. The human ortholog of Oatp1a4 is OATP1A2. Therefore, the translational significance of our work requires demonstration that OATP1A2 can transport statins and is regulated by TGF-β/ALK1 signaling. Cellular uptake and monolayer permeability of atorvastatin, pravastatin, and rosuvastatin were investigated in vitro using human umbilical vein endothelial cells (HUVECs). Regulation of OATP1A2 by the TGF-β/ALK1 pathway was evaluated using bone morphogenetic protein 9 (BMP-9), a selective ALK1 agonist, and LDN193189, an ALK1 antagonist. We showed that statin accumulation in HUVECs requires OATP1A2-mediated uptake but is also affected by efflux transporters (i.e., P-glycoprotein, breast cancer resistance protein). Absorptive flux (i.e., apical-to-basolateral) for all statins was higher than secretory flux (i.e., basolateral-to-apical) and was decreased by an OATP inhibitor (i.e., estrone-3-sulfate). OATP1A2 protein expression, statin uptake, and cellular monolayer permeability were increased by BMP-9 treatment. This effect was attenuated in the presence of LDN193189. Apical-to-basolateral statin transport across human endothelial cellular monolayers requires functional expression of OATP1A2, which can be controlled by therapeutically targeting TGF-β/ALK1 signaling. Taken together with our previous work, the present data show that OATP-mediated drug transport is a critical mechanism in facilitating neuroprotective drug disposition across endothelial barriers of the blood-brain barrier. SIGNIFICANCE STATEMENT: Transporter data derived from rodent models requires validation in human models. Using human umbilical vein endothelial cells, this study has shown that statin transport is mediated by OATP1A2. Additionally, we demonstrated that OATP1A2 is regulated by transforming growth factor-β/activin receptor-like kinase 1 signaling. This work emphasizes the need to consider endothelial transporter kinetics and regulation during preclinical drug development. Furthermore, our forward-thinking approach can identify effective therapeutics for diseases for which drug development has been challenging (i.e., neurological diseases).

Inflammation-Induced Attenuation of Prostaglandin D2 Elimination across Rat Blood-Brain Barrier: Involvement of the Downregulation of Organic Anion Transporter 3 and Multidrug Resistance-Associated Protein 4.

Prostaglandin (PG) D2 is a lipid mediator, and in the brain, overproduction of PGD2 is reportedly involved in the progression and exacerbation of neuroinflammation. The objective of this study was to elucidate PGD2 efflux transport, under normal and inflammatory conditions, across the blood-brain barrier (BBB), which is formed by brain capillaries. Elimination of [3H]PGD2 across the BBB of normal and lipopolysaccharide (LPS)-induced inflammatory rats was examined by the intracerebral microinjection technique. After intracerebral injection, the percentage of [3H]PGD2 remaining in the ipsilateral cerebrum decreased with time, with a half-life of 13 min. This [3H]PGD2 elimination across the BBB was significantly inhibited by the co-administration of unlabeled PGD2, which suggests carrier-mediated PGD2 efflux transport at the BBB. In isolated rat brain capillaries, mRNA expression of organic anion transporter (Oat) 3, organic anion-transporting polypeptide (Oatp) 1a4, and multidrug resistance-associated protein (Mrp) 4 was observed. In addition, co-administration of substrates/inhibitors for Oat3, Oatp1a4, and/or Mrp4, such as benzylpenicillin and cefmetazole, reduced [3H]PGD2 elimination across the BBB. Data suggest that Oat3 and Mrp4, but not Oatp1a4 are involved in PGD2 elimination across the BBB, as Oatp1a4-expressing Xenopus (X.) oocytes did not show the significant [3H]PGD2 uptake compared with water-injected X. oocytes. In LPS-treated rats, [3H]PGD2 elimination across the BBB and mRNA expression levels of Oat3 and Mrp4 were significantly decreased. Our data suggest that Oat3- and Mrp4-mediated PGD2 elimination across the BBB is attenuated under inflammatory conditions.

Effect of Rumex Acetosa Extract, a Herbal Drug, on the Absorption of Fexofenadine.

Herbal drugs are widely used for the auxiliary treatment of diseases. The pharmacokinetics of a drug may be altered when it is coadministered with herbal drugs that can affect drug absorption. The effects of herbal drugs on absorption must be evaluated. In this study, we investigated the effects of Rumex acetosa (R. acetosa) extract on fexofenadine absorption. Fexofenadine was selected as a model drug that is a substrate of P-glycoprotein (P-gp) and organic anion transporting polypeptide 1A2 (OATP1A2). Emodine—the major component of R. acetosa extract—showed P-gp inhibition in vitro and in vivo. Uptake of fexofenadine via OATP1A2 was inhibited by R. acetosa extract in OATP1A2 transfected cells. A pharmacokinetic study showed that the area under the plasma concentration–time curve (AUC) of fexofenadine was smaller in the R. acetosa extract coadministered group than in the control group. R. acetosa extract also decreased aqueous solubility of fexofenadine HCl. The results of this study suggest that R. acetosa extract could inhibit the absorption of certain drugs via intervention in the aqueous solubility and the drug transporters. Therefore, R. acetosa extract may cause drug interactions when coadministered with substrates of drug transporters and poorly water-soluble drugs, although further clinical studies are needed.

PH-dependent transport kinetics of the human organic anion-transporting polypeptide 1A2

Organic anion-transporting polypeptide (OATP) 1A2 is expressed on the apical sides of intestinal and renal epithelial cells and considered to be involved in the intestinal absorption and renal reabsorption of drugs. Although the transport activity of OATP1A2 is considered to be pH-dependent, the effects of pH on its kinetic parameters and on the potency of OATP1A2 inhibitors are yet to be elucidated. Some OATP are known to have multiple binding sites (MBS), but it remains unclear whether OATP1A2 has MBS. In the present study, we evaluated the influence of pH on the OATP1A2-mediated uptake of estrone 3-sulfate using OATP1A2-expressing HEK293 cells. The uptake of 0.3 μM estrone 3-sulfate by HEK293-OATP1A2 cells was pH-dependent. OATP1A2 exhibited bimodal saturation kinetics at pH 6.3 and 7.4. Compared with that seen at pH 6.3 (5.62 μM), the Km value of the high-affinity site was 8-fold higher at pH 7.4 (43.2 μM). In addition, the influence of pH on the potency of inhibitors varied among the examined inhibitors. These results suggest that the transport properties of OATP1A2 under lower pH conditions, such as those found in the microenvironments of the small intestinal mucosa and distal tubules, differ from those seen under neutral pH conditions.

A novel fluorescence-based functional assay for human OATP1A2 and OATP1C1 identifies interaction between third-generation P-gp inhibitors and OATP1A2.

Organic anion-transporting polypeptide 1A2 (OATP1A2), expressed in the human blood-brain barrier, promotes drug uptake from the blood and hence can be exploited for central nervous system-targeted drug delivery. The thyroid transporter OATP1C1, expressed in the choroid plexus and in astrocytes, is also a potential pharmacological target. Based on their established pharmacological relevance, screening the drug interaction profile of OATP1A2 and OATP1C1 is highly desirable. However, drug interaction screens require suitable model systems and functional assays. In the current study, uptake of a set of cell-impermeable fluorescent dyes was screened in HEK-293 and A431 cell lines overexpressing OATP1A2 and OATP1C1. Based on the uptake of fluorescent dye substrates, a functional assay was developed, which was used to characterize OATP inhibitors/substrates. We identify Live/Dead Green (LDG), Live-or-Dye 488, and sulforhodamines 101, G, and B as novel fluorescent substrates of OATP1A2 and OATP1C1. We show that LDG uptake is proportional to OATP1A2/1C1 expression, allowing the isolation of cells expressing high transporter levels. Additionally, dye uptake can be used to characterize the drug interaction pattern of OATP1A2 and OATP1C1. We demonstrate that third-generation P-glycoprotein inhibitors elacridar, tariquidar, and zosuquidar inhibit OATP1A2 function. Increased toxicity of elacridar in OATP1A2-expressing cells suggests that OATP1A2 may modulate the distribution of this compound. The fluorescence-based assays developed in the current study are a good alternative of radioligand-based tests and pave the way toward high-throughput screens for OATP1A2/1C1 drug interaction studies.