The Effects of N-Glycosylation on the Expression and Transport Activity of OATP1A2 and OATP2B1.

Organic anion transporting polypeptide (OATP)1A2 and OATP2B1 have potential N-glycosylation sites, but their influence remains unclear. This study aimed to identify the N-glycosylation sites of OATP1A2/2B1 and investigate their impact on the expression and function of OATP1A2/2B1. Human embryonic kidney cells expressing OATP1A2 or OATP2B1 (HEK293-OATP1A2/2B1) were exposed to tunicamycin, an N-glycosylation inhibitor, and a plasma membrane fraction (PMF) Western blot assay and an estrone 3-sulfate (E3S) uptake study were conducted. HEK293-OATP1A2/OATP2B1 cell lines with mutation(s) at potential N-glycosylation sites were established, and the Western blotting and uptake study were repeated. Tunicamycin reduced the PMF levels and E3S uptake of OATP1A2/OATP2B1. The Asn124Gln, Asn135Gln, and Asn492Gln mutations in OATP1A2 and Asn176Gln and Asn538Gln mutations in OATP2B1 reduced the molecular weights of the OATP molecules and their PMF levels. The PMF levels of OATP1A2 Asn124/135Gln, OATP1A2 Asn124/135/492Gln, and OATP2B1 Asn176/538Gln were further reduced. The maximum transport velocities of OATP1A2 Asn124Gln, OATP1A2 Asn135Gln, and OATP2B1 Asn176/538Gln were markedly reduced to 10 %, 4 %, and 10 % of the wild-type level, respectively. In conclusion, the N-glycans at Asn124 and Asn135 of OATP1A2 and those at Asn176 and Asn538 of OATP2B1 are essential for the plasma membrane expression of these molecules and also affect their transport function.

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.

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.

Fullerene derivatives as dual inhibitors of HIV-1 reverse transcriptase and protease.

In the present study, we newly synthesized three types of novel fullerene derivatives: pyridinium-type derivatives trans-3a and 4a-5b, piperidinium-type derivative 9, and proline-type derivatives 10a-12. Among the assessed compounds, 5a, 10e, 10f, 10i, 11a-d, and 12 were found to inhibit both HIV reverse transcriptase and HIV protease (HIV-PR), with IC50 values in the low micromolar range being observed. Regarding HIV-PR inhibition activity, proline-type derivatives 11a-11d and 12, bearing an alkyl chain between the hydroxylmethylcarbonyl (HMC) moiety and pyrrolidine ring, were more potent than other derivatives. This result might indicate that connecting HMC moieties with proline-type fullerene derivatives through properly sized alkyl chain leads to improved HIV-PR inhibitory activity.

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.

Novel fullerene derivatives as dual inhibitors of Hepatitis C virus NS5B polymerase and NS3/4A protease.

We evaluated the Hepatitis C virus (HCV) NS5B polymerase and HCV NS3/4A protease inhibition activities of a new set of proline-type fullerene derivatives. All of the compounds had the potential to inhibit both the enzymes, indicating that the fullerene derivatives may be dual inhibitors against NS5B and NS3/4A and could be novel lead compounds for the treatment of HCV infections.

Unmetabolized fenofibrate, but not fenofibric acid, activates AMPK and inhibits the expression of phosphoenolpyruvate carboxykinase in hepatocytes.

AIMS: A lipid-lowering agent, fenofibrate, has been reported to reduce hepatic glucose production and the expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme for gluconeogenesis in the liver. However, the precise mechanisms of these effects have remained unclear. MAIN METHODS: Rat hepatoma-derived H4IIE cells and murine myoblast-derived C2C12 cells were incubated with the ester form of fenofibrate and fenofibric acid, a metabolite of fenofibrate ester, and the phosphorylation of AMPK, acetyl-CoA carboxylase (ACC), PEPCK mRNA expression and glucose production were assessed. KEY FINDINGS: Incubation of H4IIE hepatoma cells with the ester form of fenofibrate increased the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) and decreased PEPCK mRNA expression and glucose production. Fenofibrate-induced reductions in PEPCK expression and glucose production were abrogated by compound C, a specific AMPK inhibitor. Fenofibric acid, a metabolite of fenofibrate ester, had no effects on AMPK phosphorylation, PEPCK gene expression, or glucose production in H4IIE cells. Fenofibrate-treated mice exhibited increases in AMPK phosphorylation and a decrease in PEPCK expression in the liver but not in skeletal muscles, suggesting that unmetabolized fenofibrate accumulated and affected AMPK only in the liver. SIGNIFICANCE: These results demonstrate that fenofibrate inhibits PEPCK gene expression and hepatic glucose production in the liver via AMPK activation, even though the metabolite loses its effects on AMPK and does not work in vivo in myocytes. This novel feature of fenofibrate may provide additional benefit for the treatment of patients with disorders of both lipid and glucose metabolism.

Angiotensin II type 1 receptor blockers prevent tumor necrosis factor-alpha-mediated endothelial nitric oxide synthase reduction and superoxide production in human umbilical vein endothelial cells.

Decrease in endothelial nitric oxide synthase (eNOS) expression is one of the adverse outcomes of endothelial dysfunction. Tumor necrosis factor-alpha (TNF-alpha) is known to decrease eNOS expression and is an important mediator of endothelial dysfunction. We hypothesized that an angiotensin II type 1 (AT1) receptor blocker would improve endothelial function via not only inhibition of the angiotensin II signaling but also inhibition of the TNF-alpha-mediated signaling. Therefore we investigated whether an AT1 receptor blocker would restore the TNF-alpha-induced decrease in eNOS expression in cultured human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with an antioxidant (superoxide dismutase, alpha-tocopherol) or AT1 receptor blockers (olmesartan or candesartan) restored the TNF-alpha-dependent reduction of eNOS. The AT1 receptor blocker decreased the TNF-alpha-dependent increase of 8-isoprostane. The superoxide dismutase activities in HUVEC were stable during AT1 receptor blocker treatment, and the AT1 receptor blocker did not scavenge superoxide directly. The AT1 receptor blocker also decreased TNF-alpha-induced phosphorylation of I kappaB alpha and cell death. These results suggest that AT1 receptor blockers are able to ameliorate TNF-alpha-dependent eNOS reduction or cell injury by inhibiting superoxide production or nuclear factor-kappaB activation.

The effects of endothelial nitric oxide synthase gene polymorphisms on endothelial function and metabolic risk factors in healthy subjects: the significance of plasma adiponectin levels.

OBJECTIVE: Genetic variants of the endothelial nitric oxide synthase (eNOS) gene, Glu298Asp and T-786C, have been reported to be associated with cardiovascular disease. Adiponectin is an adipocyte-derived plasma protein with insulin-sensitizing and vascular protective effects; its levels are typically low in metabolic syndrome. Therefore, eNOS gene polymorphisms may also be associated with specific metabolic profiles, including plasma adiponectin levels and atherogenic lipids. METHODS: We evaluated the functional significance of eNOS gene Glu298Asp and T-786C polymorphisms on endothelial function and metabolic profiles in 101 healthy young men (mean age 30.3 years) before the progression of atherosclerotic lesions. RESULTS: No linkage disequilibrium was found between the two genotypes. The Asp298 allele carriers of the eNOS gene presented significantly higher plasma low density lipoprotein (LDL) cholesterol, LDL particle size, malondialdehyde-modified LDL (MDA-LDL), and fasting insulin levels and lower plasma high density lipoprotein (HDL) cholesterol, apolipoprotein A-I levels, and endothelium-dependent vasodilation when compared with noncarriers. In spite of higher MDA-LDL levels, Asp298 carriers had significantly larger LDL particle size. By contrast, in C-786 allele carriers, systolic blood pressure was significantly higher, and plasma high-molecular-weight adiponectin levels and endothelium-dependent vasodilation were significantly lower than those in non-carriers. CONCLUSIONS: Although both eNOS polymorphisms induced endothelial dysfunction, the eNOS T-786C polymorphism may be associated with adiponectin levels, whereas the Glu298Asp polymorphism may be associated with atherogenic lipid levels.

Association of gene polymorphisms with coronary artery disease in low- or high-risk subjects defined by conventional risk factors.

OBJECTIVES: The aim of the study was to identify genes that confer susceptibility to coronary artery disease (CAD) in low- or high-risk men or women separately and thereby to assess the genetic risk of CAD in such individuals. BACKGROUND: The prevention of CAD would be facilitated by the identification of genes that confer susceptibility to this condition independently in low- or high-risk individuals, as defined by conventional risk factors. METHODS: The study population comprised 1661 unrelated Japanese individuals, including 1011 patients with CAD and 650 control subjects. Among all study subjects, 601 individuals (high-risk subjects) had hypertension, diabetes mellitus, and hypercholesterolemia, and 1060 individuals (low-risk subjects) had none of these risk factors for CAD. The genotypes for 37 polymorphisms of 31 candidate genes were determined by a fluorescence- or colorimetry-based allele-specific DNA primer-probe assay system. RESULTS: Multivariate logistic regression analysis, with adjustment for age, body mass index, and the prevalence of smoking and hyperuricemia, revealed that the -219G-->T polymorphism of the apolipoprotein E gene in low-risk men, the -1171/5A-->6A polymorphism of the stromelysin-1 gene in low-risk women, the 1019C-->T polymorphism of the connexin 37 gene in high-risk men, and the 3932T-->C polymorphism of the apolipoprotein E gene in high-risk women were significantly associated with CAD. A stepwise forward selection procedure revealed that the effects of these polymorphisms on CAD were statistically independent of age or conventional risk factors. CONCLUSIONS: Genotyping of these polymorphisms may prove informative for assessment of the genetic risk of CAD in low- or high-risk men or women.

Diet and lung cancer risk from a 14-year population-based prospective study in Japan: with special reference to fish consumption.

N-3 polyunsaturated fatty acids in fish oil exhibit a variety of health benefits, and there is evidence that they can inhibit the development of human lung mucoepidermoid and other carcinomas. To examine the hypothesis that fish consumption reduces the risk of lung cancer, we conducted a population-based prospective study, following 5,885 residents for 14 yr. Person-years were used to calculate the relative risk (RR) by the Cox proportional hazards model, with adjustment for potential confounding factors. A total of 51 incident lung cancer cases were observed, and we found linearly decreasing RRs for lung cancer with increased frequency of consumption of fish and shellfish (RRs = 1.00, 0.99, and 0.32, P for trend = 0.003) but not with intake of dried/salted fish. Decreased RRs were apparent with both broiling and boiling cooking methods, but reduction with raw and deep-fried fish consumption was not statistically significant. We conclude that frequent fresh fish consumption, irrespective of the cooking method, may reduce the risk of lung cancer.