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.

Determination of single-molecule transport activity of OATP2B1 by measuring the number of transporter molecules using electrophysiological approach.

Transporter-mediated clearance is determined by two factors, its single-molecule clearance, and expression level. However, no reliable method has been developed to evaluate them separately. This study aimed to develop a reliable method for evaluating the single-molecule activity of membrane transporters, such as organic anion transporting polypeptide (OATP) 2B1. HEK293 cells that co-expressed large conductance calcium-activated potassium (BK) channel and OATP2B1 were established and used for the following experiments. i) BK channel-mediated whole-cell conductance was measured using patch-clamp technique and divided by its unitary conductance to estimate the number of channels on plasma membrane (QI). ii) Using plasma membrane fraction, quantitative targeted absolute proteomics determined the stoichiometric ratio (ρ) of OATP2B1 to BK channel. iii) The uptake of estrone 3-sulfate was evaluated to calculate the Michaelis constant and uptake clearance (CL) per cell. Single-molecule clearance (CLint) was calculated by dividing CL by QI·ρ. QI and ρ values were estimated to be 916 and 2.16, respectively, yielding CLint of 5.23 fL/min/molecule. We successfully developed a novel method to reliably measure the single-molecule activity of a transporter, which could be used to evaluate the influences of factors such as genetic variations and post-translational modifications on the intrinsic activity of transporters.

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.

Rubella Virus Triggers Type I Interferon Antiviral Response in Cultured Human Neural Cells: Involvement in the Control of Viral Gene Expression and Infectious Progeny Production.

The type I interferon (IFN) response is one of the primary defense systems against various pathogens. Although rubella virus (RuV) infection is known to cause dysfunction of various organs and systems, including the central nervous system, little is known about how human neural cells evoke protective immunity against RuV infection, leading to controlling RuV replication. Using cultured human neural cells experimentally infected with RuV RA27/3 strain, we characterized the type I IFN immune response against the virus. RuV infected cultured human neural cell lines and induced IFN-ß production, leading to the activation of signal transducer and activator of transcription 1 (STAT1) and the increased expression of IFN-stimulated genes (ISGs). Melanoma-differentiation-associated gene 5 (MDA5), one of the cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors, is required for the RuV-triggered IFN-ß mRNA induction in U373MG cells. We also showed that upregulation of RuV-triggered ISGs was attenuated by blocking IFN-α/ß receptor subunit 2 (IFNAR2) using an IFNAR2-specific neutralizing antibody or by repressing mitochondrial antiviral signaling protein (MAVS) expression using MAVS-targeting short hairpin RNA (shRNA). Furthermore, treating RuV-infected cells with BX-795, a TANK-binding kinase 1 (TBK1)/I kappa B kinase ε (IKKε) inhibitor, robustly reduced STAT1 phosphorylation and expression of ISGs, enhancing viral gene expression and infectious virion production. Overall, our findings suggest that the RuV-triggered type I IFN-mediated antiviral response is essential in controlling RuV gene expression and viral replication in human neural cells.

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.

Suppressive Role of Lactoferrin in Overweight-Related Female Fertility Problems.

The secretory glycoprotein lactoferrin (LF) is suggested to ameliorate overweight regardless of non-genetic or genetic mechanisms. Although maternal overweight represents a key predictor of offspring growth, the efficacy of LF on fertility problems in overweight and obese mothers remains unknown. To address this issue, we examined the effect of LF ingestion by analyzing overweight mice (Institute of Cancer Research (ICR) mice with high-fat diets; HF mice) and obese mice (leptin-deficient mice with type II diabetes; ob/ob mice). Plasma insulin, leptin, glucose, and cholesterol levels were measured, and thermal imaging and histological analysis were employed. The litter size of HF females was reduced due to miscarriage, which was reversed by LF ingestion. In addition, LF ingestion suppressed overweight prevalence in their offspring. The component analysis of the maternal blood demonstrated that glucose concentration in both HF females and their offspring was normalized by LF ingestion, which further standardized the concentration of insulin, but not leptin. LF ingestion was unable to reverse female infertility in ob/ob mice, although their obesity and uterine function were partially improved. Our results indicate that LF upregulates female fertility by reinforcing ovarian and uterine functions in females that are overweight due to caloric surplus.

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.

Dual kinetics of OATP2B1: Inhibitory potency and pH-dependence of OATP2B1 inhibitors.

Organic anion transporting polypeptide (OATP) 2B1 is expressed in the intestine and liver, and OATP2B1-mediated transport of estrone 3-sulfate is pH-dependent and consists of: the high-affinity component (Hc) and low-affinity component (Lc). This study aimed to evaluate the influence of pH on the transport kinetics of each component, along with the inhibitory nature of ten OATP2B1 inhibitors. The Michaelis constants (Km) were 4-fold and 1.5-fold lower at pH 6.3 than at pH 7.4, for Hc and Lc respectively. The inhibitory potencies of diclofenac, indomethacin, and ibuprofen towards Hc were 1.5-4.3 fold lower at pH 6.3 than at pH 7.4. Contrastingly, inhibitory potencies towards Lc were 9.0-52 fold lower at pH 7.4. Similarly, the inhibitory effect of naproxen was stronger towards Hc at pH 6.3 and towards Lc at pH 7.4. On the other hand, celecoxib selectively inhibited Lc transport at pH 7.4. Rifampicin inhibited both components at pH 6.3 and 7.4 to a similar extent, while bromosulphophthalein, naringin, and gefitinib selectively inhibited Hc irrespective of pH. Fexofenadine inhibited neither component. In conclusion, the transport affinities of both Hc and Lc were enhanced under acidic conditions. The influence of pH on the inhibitory potency towards each component varied among the inhibitors.

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.

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.