Details of a Researcher - ITAGAKI Shiro

Papers - ITAGAKI Shiro

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Bicarbonate supplementation as a preventive way in statins-induced muscle damage

Masaki Kobayashi, Toshiki Kagawa, Katsuya Narumi, Shirou Itagaki, Takeshi Hirano, Ken Iseki

JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES ( CANADIAN SOC PHARMACEUTICAL SCIENCES ) 11 ( 1 ) 1 - 8 2008 [Refereed]

Purpose. The aim of this study was to evaluate the bicarbonate-induced improvement of statins, cerivastatin, simvastatin acid and lovastatin acid-induced apoptosis using rat myoblast cell line (L6) as a model of in vitro skeletal muscle and of cerivastatin-induced muscle damage in vivo study. Methods. Statin-induced reduction of cell viability and apoptosis was measured by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay and caspase assay. In vivo, we evaluated plasma creatine phosphokinase (CPK) level in cerivastatin-treated rat. Results. Bicarbonate prevented cerivastatin-, simvastatin-acid and lovastatin acid-induced reduction of cell viability, morphological change and caspase activation in L6 cells. Moreover, in the in vivo study, bicarbonate prevented cerivastatin-induced increase in CPK concentrations. Conclusions. These results from in vitro and in vivo studies support that bicarbonate supplementation prevented statin-induced muscle damage.

PubMed

Preventive effects of bicarbonate on cerivastatin-induced apoptosis

Masaki Kobayashi, Fumie Kaido, Toshiki Kagawa, Shirou Itagaki, Takeshi Hirano, Ken Iseki

INTERNATIONAL JOURNAL OF PHARMACEUTICS ( ELSEVIER SCIENCE BV ) 341 ( 1-2 ) 181 - 188 2007.08 [Refereed]

Although HMG-CoA reductase inhibitors such as statins are the most widely used cholesterol-lowering agents, there is a risk of myopathy or rhabdmyolysis occurring in patients taking these drugs. It has been reported that a number of lipophilic statins cause apoptosis in various cells, but it is still not clear whether intracellular acidification is involved in statin-induced apoptosis. There have been few studies aimed at identifying compounds that suppress statin-induced myotoxicity. In the present study, we examined the relationship between cerivastatin-induced apoptosis and intracellular acidification and the effect of bicarbonate on cerivastatin-induced apoptosis using an RD cell line as a model of in vitro skeletal muscle. Cerivastatin reduced the number of viable cells and caused dramatic morphological changes and DNA fragmentation in a concentration-dependent manner. Moreover, cerivastatin-induced apoptosis was associated with intracellular acidification and caspase-9 and -3/7 activation. On the other hand, bicarbonate suppressed cerivastatin-induced pH alteration, caspase activation, morphological change and reduction of cell viability. Accordingly, bicarbonate suppressed statin-induced apoptosis. The strategy to combine statins with bicarbonate can lead to reduction in the chance of the severe adverse events including myopathy or rhabdmyolysis. (c) 2007 Elsevier B.V. All rights reserved.

DOI PubMed

Effect of medium pH on the cytotoxicity of hydrophilic statins

Masaki Kobayashi, Toshiki Kagawa, Rumi Takano, Shirou Itagaki, Takeshi Hirano, Ken Iseki

JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES ( CANADIAN SOC PHARMACEUTICAL SCIENCES ) 10 ( 3 ) 332 - 339 2007.05 [Refereed]

Purpose. The aim of this study was to examine the mechanism of pravastatin- and rosuvastatin-induced cytotoxicity and the relationship between pravastatin- and rosuvastatin-induced cytotoxicity and medium pH using human prototypic embryonal rhabdomyosarcoma cell line (RD) and rat myoblast cell line (L6) as a model of in vitro skeletal muscle. Methods. Statin-induced reduction of cell viability and apoptosis was measured by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay and caspase assay. Intracellular accumulation of statins was determined using an HPLC system. Results. Rosuvastatin cytotoxicity, reduction of cell viability, morphological changes and caspase activation at acidic pH (pH 6.8) were significantly greater than those at neutral pH (pH 7.4). Rosuvastatin accumulation at acidic pH was greater than that at pH 7.4. On the other hand, medium pH had no effect on pravastatin accumulation. Conclusions. Rosuvastatin cytotoxicity at acidic pH is associated with increasing intracellular accumulation of rosuvastatin. On the other hand, medium pH had no effect on cytotoxicity of pravastatin.

PubMed

Functional and molecular analysis of D-serine transport in retinal Muller cells

Y. Dun, B. Mysona, S. Itagaki, A. Martin-Studdard, V. Ganapathy, S. B. Smith

EXPERIMENTAL EYE RESEARCH ( ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD ) 84 ( 1 ) 191 - 199 2007.01

D-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for D-serine in retinal Muller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for D-serine, is expressed in the rMC-1 Muller cell line and primary cultures of mouse Muller cells (1 degrees MCs). The relative contributions of different amino acid transport systems to D-serine uptake were determined based on differential substrate specificities and ion dependencies. D-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for D-serine in Muller cells. The Na+:substrate stoichiometry for the transport process was 1:1. D-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that D-serine transport in Muller cells occurs via ASCT2 rather than ASCT1 or ATB(0,+). The expression of mRNAs specific for ASCT1, ASCT2, and ATB(0,+) was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB(0,+), in Muller cells. Immunoblotting detected ASCT2 in neural retina and in 1 degrees MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of I'MCs. The efflux of D-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a Muller cell line and in 1 degrees MCs and suggest that D-serine, synthesized in Wiler cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons. (c) 2006 Published by Elsevier Ltd.

DOI

Down-regulation of intestinal multidrug resistance-associated protein 2 in long-evans Cinnamon rats

Makoto Chiba, Shirou Itagaki, Masaki Kobayashi, Takeshi Hirano, Ken Iseki

DRUG METABOLISM AND PHARMACOKINETICS ( JAP SOC STUDY XENOBIOTICS ) 22 ( 6 ) 450 - 455 2007 [Refereed]

Wilson's disease is an inherited, autosomal recessive disorder of copper accumulation and toxicity. Lifelong chelation therapy is essential in all Wilson's disease patients. Intestinal absorption of some compounds is limited partly because they are preferentially transported in the secretory direction. Several ATP-binding cassette (ABC) transporters are expressed in the apical membrane of the small intestine and secrete various drugs into the lumen. In this study, we investigated the characteristics of the intestinal efflux ABC transporters in LEC rats. We found that the expression of multidrug resistance-associated protein 2 (Mrp2) in the jejunum of Long-Evans Cinnamon (LEC) rats, an animal model for Wilson's disease, is decreased.

DOI CiNii

Efflux transport of n-monodesethylamiodarone by the human intestinal cell-line caco-2 cells

Emi Kimoto, Satoru Seki, Shirou Itagaki, Maya Matsuura, Masaki Kobayashi, Takeshi Hirano, Yoshikazu Goto, Koji Tadano, Ken Iseki

DRUG METABOLISM AND PHARMACOKINETICS ( JAP SOC STUDY XENOBIOTICS ) 22 ( 4 ) 307 - 312 2007 [Refereed]

Amiodarone (AMD) is a benzofurane derivative with class III antiarrhythmic activity that is effective in controlling intractable cardiac arrhythmias. One of the most common and serious drug interactions in clinical practice is the interaction between digoxin and an antiarrhythmic agent. It has been reported that AMD and N-monodesethylamiodarone (DEA), the active metabolite of AMD, inhibit the P-glycoprotein (P-gp/MDR1)-mediated digoxin transport. However, the intestinal transport processes of AMD and DEA have not been fully revealed. In this study, we focused on the intestinal transport mechanism of DEA and characterized the intestinal transport of DEA using Caco-2 cells. Basal-to-apical transport of DEA by Caco-2 cells was greater than apical-to-basal transport. The relationship between concentration and basal-to-apical flux rate appeared to approach saturation. The uptake of DEA by Caco-2 cells was increased in the presence of typical ATP-depletion compounds and thyroid hormones. On the other hand, substrates for P-gp, multidrug resistance-associated proteins (MRPs/ABCCs) and breast cancer resistance protein (BCRP/ABCG2) had no effect on the efflux of DEA. These results suggest that an ATP-binding cassette (ABC) transporter, which is different from P-gp, MRPs and BCRP, mediates the efflux of DEA across the apical membrane in Caco-2 cells and that thyroid hormone inhibits this transporter.

DOI

Characterization of hepatobiliary organic anion transporters in Long-Evans Cinnamon rats

Makoto Chiba, Shirou Itagaki, Masaki Kobayashi, Takeshi Hirano, Ken Iseki

DRUG METABOLISM AND PHARMACOKINETICS ( JAP SOC STUDY XENOBIOTICS ) 22 ( 5 ) 387 - 390 2007 [Refereed]

The liver plays important roles in the detoxification of xenobiotecs. Hepatobiliary transporters contribute to hepatic uptake and efflux processes of xenobiotecs. Expressions of these transporters may be modulated under the condition of hepatic failure. Long-Evans Cinnamon (LEC) rats provide a pertinent model for basic and clinical studies on hepatitis. However, only a few reports describing the properties of hepatobiliary transporters in LEC rats have appeared in the literature. We investigated the expression levels of hepatobiliary transporters in LEC rats by real-time RT-PCR. We found that hepatic expressions of three sinusoidal organic anion transporters, Ntcp, Oatp1a1 and Oatp1a4, were decreased in LEC rats. However, no significant difference of the expressions of Mrp2 and Bsep, organic anion transporters located on canalicular membrane, were found between Wistar rats and LEC rats.

DOI CiNii

Improvement in intestinal coenzyme q10 absorption by food intake

Akiko Ochiai, Shirou Itagaki, Toshimitsu Kurokawa, Masaki Kobayashi, Takeshi Hirano, Ken Iseki

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN ( PHARMACEUTICAL SOC JAPAN ) 127 ( 8 ) 1251 - 1254 2007 [Refereed]

Coenzyme Q10 (CoQ10) is widely consumed as a food supplement because of its recognition as an important nutrient in supporting human health. Absorption of compounds from the gastrointestinal tract is one of the important determinants of oral bioavailability. However, the absorption of dietary CoQ10 is slow and limited due to its hydrophobicity and large molecular weight. The absorption of orally applied compounds can be enhanced by interactions with food or food components. Thus, we investigated the effect of food intake on the absorption of CoQ10 after oral supplememation. In this study, we demonstrated that food intake enhanced the intestinal absorption of CoQ10. In order to improve intestinal absorption of CoQ10 after oral supplementation, we developed an emulsion formulation. Intestinal absorption of CoQ10 after administration of the emulsion formulation was also enhanced by food intake. Moreover, the peak concentration and the extent of absorption after administration of the emulsion formulation were greater than those after administration of a suspension formulation. It is possible that administration of CoQ10 in an emulsion formulation enhances the pharmacological effects of CoQ10.

DOI

Mechanism of the inhibitory effect of zwitterionic drugs (levofloxacin and grepafloxacin) on carnitine transporter (OCTN2) in Caco-2 cells

Takeshi Hirano, Satoru Yasuda, Yuki Osaka, Masaki Kobayashi, Shirou Itagaki, Ken Iseki

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES ( ELSEVIER SCIENCE BV ) 1758 ( 11 ) 1743 - 1750 2006.11 [Refereed]

L-Carnitine plays an important role in lipid metabolism by facilitating the transport of long-chain fatty acids across the mitocbondrial inner membrane followed by fatty acid beta-oxidation. It is known that L-carnitine exists as a zwitterion and that member of the OCTN family play an important role in its transport. The aims of this study were to characterize L-carnitine transport in the intestine by using Caco-2 cells and to elucidate the effects of levofloxacin (LVFX) and grepafloxacin (GPFX), which are zwitterionic drugs, on L-carnitine uptake. Kinetic analysis showed that the half-saturation Na+ concentration, Hill coefficient and K-m value of L-carnitine uptake in Caco-2 cells were 10.3 +/- 4.5 mM, 1.09 and 8.0 +/- 1.0 mu M, respectively, suggesting that OCTN2 mainly transports L-carnitine. LVFX and GPFX have two pK(a) values and the existence ratio of their zwitterionic forms is higher under a neutral condition than under an acidic condition. Experiments on the inhibitory effect of LVFX and GPFX on L-carnitine uptake showed that LVFX and GPFX inhibited L-carnitine uptake more strongly at pH 7.4 than at pH 5.5. It was concluded that the zwitterionic form of drugs plays an important role in inhibition of OCTN2 function. (c) 2006 Elsevier B.V. All rights reserved.

DOI

Antioxidant activity of a novel extract from bamboo grass (AHSS) against ischemia-reperfusion injury in rat small intestine

Toshimitsu Kurokawa, Shirou Itagaki, Toshihiko Yamaji, Chie Nakata, Toshihiro Noda, Takeshi Hirano, Ken Iseki

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 29 ( 11 ) 2301 - 2303 2006.11 [Refereed]

Production of free radical species in cells and body tissues is known to cause many pathological disorders. Therefore, free radical scavengers play an important role in the prevention of various human diseases. Bamboo grass, Sasa senanensis, is a native Japanese plant. Sasa has been used for medicine in Japan for many centuries. In this study, we investigated the antioxidative activity of Absolutely Hemicellulose Senanensis (AHSS), a novel extract from Sasa. In the first part of this study, we found that AHSS has antioxidant activities by the assay using superoxide anion-2-methyl-6-methoxyphenylethynylimidazopyrazynone (MPEC) reaction kit. We then confirmed its antioxidative activity using a rat ischemia and subsequent reperfusion (I/R) injury model. Breakdown of the intestinal wall caused by intestinal I/R was attenuated by pretreatment with AHSS. Moreover, AHSS inhibited the production of lipid peroxide by intestinal I/R. AHSS could be an important source of ingredients for use in functional foods and other applications.

DOI CiNii

Mechanism of the inhibitory effect of zwitterionic drugs (levofloxacin and grepafloxacin) on carnitine transporter (OCTN2) in Caco-2 cells.

Hirano T, Yasuda S, Osaka Y, Kobayashi M, Itagaki S, Iseki K

Biochimica et biophysica acta ( 11 ) 1758 ( 11 ) 1743 - 1750 2006.11 [Refereed]

DOI CiNii

Mechanism of the inhibitory effect of zwitterionic drugs (levofloxacin and grepafloxacin) on carnitine transporter (OCTN2) in Caco-2 cells.

Hirano T, Yasuda S, Osaka Y, Kobayashi M, Itagaki S, Iseki K

Biochimica et biophysica acta 1758 ( 11 ) 1743 - 1750 2006.11 [Refereed] [International journal]

L-Carnitine plays an important role in lipid metabolism by facilitating the transport of long-chain fatty acids across the mitochondrial inner membrane followed by fatty acid beta-oxidation. It is known that L-carnitine exists as a zwitterion and that member of the OCTN family play an important role in its transport. The aims of this study were to characterize L-carnitine transport in the intestine by using Caco-2 cells and to elucidate the effects of levofloxacin (LVFX) and grepafloxacin (GPFX), which are zwitterionic drugs, on L-carnitine uptake. Kinetic analysis showed that the half-saturation Na+ concentration, Hill coefficient and Km value of L-carnitine uptake in Caco-2 cells were 10.3 +/- 4.5 mM, 1.09 and 8.0 +/- 1.0 microM, respectively, suggesting that OCTN2 mainly transports L-carnitine. LVFX and GPFX have two pKa values and the existence ratio of their zwitterionic forms is higher under a neutral condition than under an acidic condition. Experiments on the inhibitory effect of LVFX and GPFX on L-carnitine uptake showed that LVFX and GPFX inhibited L-carnitine uptake more strongly at pH 7.4 than at pH 5.5. It was concluded that the zwitterionic form of drugs plays an important role in inhibition of OCTN2 function.

DOI PubMed

Characterization of the disposition of lutein after i.v. administration to rats

Shirou Itagaki, Wakako Ogura, Yuki Sato, Toshihiro Noda, Takeshi Hirano, Satoshi Mizuno, Ken Iseki

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 29 ( 10 ) 2123 - 2125 2006.10 [Refereed]

Lutein is a carotenoid that has antioxidant effects. Although lutein has received much attention recently due to its antioxidant activities, little information about the pharmacokinetic properties of lutein is available. The disposition of lutein after i.v. administration has not been investigated because lutein is now used as a supplement. The present study was undertaken to acquire additional data on the disposition of lutein after i.v. administration. After i.v. administration, lutein is preferentially distributed to the liver, spleen and lung. Intravenous administration of lutein may provide effective antioxidant activities in these tissues, not only the eye. The results of this study should provide valuable data for drug development.

DOI CiNii

Protective effect of lutein on ischemia-reperfusion injury in rat small intestine

Wakako Ogura, Shirou Itagaki, Toshirnitsu Kurokawa, Toshihiro Noda, Takeshi Hirano, Satoshi Mizuno, Ken Iseki

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 29 ( 8 ) 1764 - 1766 2006.08 [Refereed]

Lutein is a carotenoid and it has antioxidant effects. Lutein may have a protective effect on ischemia reperfusion (I/R) injury induced by free radical species. However, little is known about the protective effect of lutein on I/R injury in vivo. The present study was undertaken to clarify the protective effects of lutein on I/R injuries in the rat small intestine. Administration of lutein before intestinal I/R attenuated the damage to villi and deciduation of enterocytes and suppressed the increase in lipid peroxide.

DOI CiNii

Identity of SMCT1 (SLC5A8) as a neuron-specific Na+-coupled transporter for active uptake of L-lactate and ketone bodies in the brain

PM Martin, E Gopal, S Ananth, L Zhuang, S Itagaki, BM Prasad, SB Smith, PD Prasad, Ganapathy, V

JOURNAL OF NEUROCHEMISTRY ( BLACKWELL PUBLISHING ) 98 ( 1 ) 279 - 288 2006.07 [Refereed]

SMCT1 is a sodium-coupled (Na+-coupled) transporter for L-lactate and short-chain fatty acids. Here, we show that the ketone bodies, beta-D-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, alpha-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na+-coupled and electrogenic. The Michaelis constant is 1.4 +/- 0.1 mM for beta-D-hydroxybutyrate, 0.21 +/- 0.04 mM for acetoacetate and 0.21 +/- 0.03 mM for alpha-ketoisocaproate. The Na+ : substrate stoichiometry is 2 : 1. As L-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co-localizes with MCT2, a neuron-specific Na+-independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na+-coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of L-lactate and ketone bodies into neurons by a process driven by an electrochemical Na+ gradient and hence, contribute to the maintenance of the energy status and function of neurons.

DOI PubMed CiNii

Inhibitory effects of statins on human monocarboxylate transporter 4

Masaki Kobayashi, Yukio Otsuka, Shirou Itagaki, Takeshi Hirano, Ken Iseki

INTERNATIONAL JOURNAL OF PHARMACEUTICS ( ELSEVIER SCIENCE BV ) 317 ( 1 ) 19 - 25 2006.07 [Refereed]

Human MCT4 (SLC16A3) is responsible for the efflux Of L-lactic acid from skeletal muscle cells and is essential for muscle homeostasis. However, the effects of monocarboxylate drugs, such as statins on the MCT4-mediated transport Of L-lactic acid have not been elucidated. Inhibition Of L-lactic acid transport mediated by MCT4 might to lead to collapse of muscle homeostasis. The aim of this study was to establish an MCT4 transfected cell line and to clarify the transport mechanism Of L-lactic acid and the effects of statins on this transport system. Results of Western blot analyses and immunohistochemistry studies indicated that the expression of CD147 and MCT4-FLAG protein were observed and was displayed clear plasma membrane localization in CD147 and MCT4-FLAG co-transfected cell line (cm cells). Uptake of L-lactic acid in cm cells was significantly greater than that in cells transfected with a vector alone. L-lactic acid uptake was concentration-dependent with a K. value of 28.43 +/- 3.87 mM. The results of a previous study showing a K-m value of 28.5 mM in hMCT4-expressed oocytes. Lipophilic statins significantly inhibited [C-14] L-lactic acid uptake in a concentration-dependent manner. In contrast, the inhibitory effects of hydrophilic statins were very weak. (c) 2006 Elsevier B.V. All rights reserved.

DOI PubMed

Interaction of ibuprofen and other structurally related NSAIDs with the sodium-coupled monocarboxylate transporter SMCT1 (SLC5A8)

S Itagaki, E Gopal, LN Zhuang, YJ Fei, S Miyauchi, PD Prasad, Ganapathy, V

PHARMACEUTICAL RESEARCH ( SPRINGER/PLENUM PUBLISHERS ) 23 ( 6 ) 1209 - 1216 2006.06 [Refereed]

Purpose: Sodium-coupled monocarboxylate transporter 1 (SMCT1) is a Na+-coupled transporter for monocarboxylates. Many nonsteroidal anti-inflammatory drugs (NSAIDs) are monocarboxylates. Therefore, we investigated the interaction of these drugs with human SMCT1 (hSMCT1). Methods: We expressed hSMCT1 in a mammalian cell line and in Xenopus laevis oocytes and used the uptake of nicotinate and propionate-induced currents to monitor its transport function, respectively. We also used [C-14]-nicotinate and [H-3]-ibuprofen for direct measurements of uptake in oocytes. Results: In mammalian cells, hSMCT1-mediated nicotinate uptake was inhibited by ibuprofen and other structurally related NSAIDs. The inhibition was Na+ dependent. With ibuprofen, the concentration necessary for 50% inhibition was 64 +/- 16 mu M. In oocytes, the transport function of hSMCT1 was associated with inward currents in the presence of propionate. Under identical conditions, ibuprofen and other structurally related NSAIDs failed to induce inward currents. However, these compounds blocked propionate-induced currents. With ibuprofen, the blockade was dose dependent, Na+ dependent, and competitive. However, there was no uptake of [H-3]-ibuprofen into oocytes expressing hSMCT1, although the uptake of [C-14]-nicotinate was demonstrable under identical conditions. Conclusions: Ibuprofen and other structurally related NSAIDs interact with hSMCT1 as blockers of its transport function rather than as its transportable substrates.

DOI PubMed

Electrophysiological characterization and Modeling of the structure activity relationship of the human concentrative nucleoside transporter 3 (hCNT3)

HK Hu, CJ Endres, C Chang, NS Umapathy, EW Lee, YJ Fei, S Itagaki, PW Swaan, Ganapathy, V, JD Unadkat

MOLECULAR PHARMACOLOGY ( AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS ) 69 ( 5 ) 1542 - 1553 2006.05 [Refereed]

We characterized the electrophysiology, kinetics, and quantitative structure-activity relationship ( QSAR) of the human concentrative nucleoside transporter 3 (hCNT3) expressed in Xenopus laevis oocytes by measuring substrate-induced inward currents using a two-microelectrode voltage-clamp system. At membrane potentials between -30 and -150 mV, sodium activation of gemcitabine transport was sigmoidal, with a K-0.5 of 8.5 +/- 0.3 mM for Na+ and a Hill coefficient of 2.2 +/- 0.25 independent of membrane potential. We measured the I-max and K-0.5 for substrate at -50 mV for the nucleoside analog drugs gemcitabine (638 +/- 58 nA, 59.7 +/- 17.5 mu M), ribavirin (546 +/- 37 nA, 61.0 +/- 13.2 mu M), AZT (420 +/- 4 nA, 310 +/- 9 mu M), and 3-deazauridine (506 +/- 30 nA, 50.8 +/- 9.90 mu M). K-0.5 and I-max for substrate were dependent on membrane potential (both increasing as the membrane became more hyperpolarized) for all four drugs. hCNT3 also exhibited pre-steady-state currents. The quantitative structure-activity relationship (QSAR) was examined using comparative molecular field analysis and comparative molecular similarity indices analysis of the inward currents induced by 27 nucleoside analogs with substitutions at both the ribose and the nucleobase. Two statistically significant QSAR models identified electrostatic interaction as the major force in hCNT3 transport and attributed a critical role to the 3'-hydroxyl position of hCNT3 substrates. Steric hindrance at the 3- position and positive charge at the 5-position of the pyrimidine ring were favorable for transport. Two hCNT3 pharmacophore models revealed the minimal features required for hCNT3 transport as two hydrogen bond acceptors at 3'-OH and 5'-O and the hydrophobic center occupied by the base ring.

DOI PubMed

Purification by p-aminobenzoic acid (PABA)-affinity chromatography and the functional reconstitution of the nateglinide/H+ cotransport system in the rat intestinal brush-border membrane

Y Saito, S Itagaki, S Kubo, M Kobayashi, T Hirano, K Iseki

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 340 ( 3 ) 879 - 886 2006.02 [Refereed]

(-)-N-(trans-4-Isopropyleyclohexanecarbonyl)-D-phenylalanine (nateglinide) is a novel oral hypoglycemic agent possessing a peptide-type bond and a carboxyl group in its structure. Recently, we have shown that nateglinide transport occurs via the ceftibuten/H+ cotransport system, which is distinct from PepT1, and that the fluorescein/H+ cotransport system is involved in the Uptake of nateglinide. The aim Of this Study was to characterize the functional properties of the intestinal nateglinide transporter. In the first part of this Study, we demonstrated that the ceftibuten/H+ cotransport system is identical to the fluorescein/H+ cotransport system. We Succeeded in purification of the nateglinide transporter from brush-border membranes of the rat small intestine using p-aminobenzoic acid (PABA)-affinity chromatography. We then investigated the functional properties of the nateglinide transporter using proteoliposomes prepared from the PABA-affinity chromatography elute. We demonstrated that nateglinide, ceftibuten, and fluorescein are transported by the same transporter in the intestine. (c) 2005 Elsevier Inc. All rights reserved.

DOI PubMed CiNii

Characterization of secretory intestinal transport of the lactone form of CPT-11

Takemoto, I, S Itagaki, M Chiba, T Itoh, T Hirano, K Iseki

CANCER CHEMOTHERAPY AND PHARMACOLOGY ( SPRINGER ) 57 ( 1 ) 129 - 133 2006.01 [Refereed]

Purpose: It has been reported that a significant portion of the lactone form of 7-ethyl-10-(4-[1-piperidino]-1-piperidino)-carbonyloxy-camptothecin (CPT-11) is excreted into the gastrointestinal lumen via the intestinal membrane and that carboxylesterase activity, which converts CPT-11 to SN-38, was detected in the intestine. It is possible that a reduction in the excretion of CPT-11 lactone into the gastrointestinal lumen induces the gastrointestinal toxicity. The purpose of this study was to investigate the characteristics of transporter(s) that contribute to the jejunal efflux of the lactone form of CPT-11. Methods: The serosal-to-mucosal permeation rate of CPT-11 lactone was investigated in everted sac studies. Results: The secretory transport required metabolic energy and was diminished by sulfobromophthalein (BSP) and 1-naphthol, inhibitors of the ME3277 transport system. However, inhibitors of breast cancer resistance protein (Bcrp), multidrug resistance-associated protein 2 (Mrp2) and P-glycoprotein (P-gp) did not affect the secretion of CPT-11 lactone. Conclusions: The results suggest that a specific transport system, which is identical to the ME3277 transport system, plays a major role in the secretion of CPT-11 lactone.

DOI PubMed

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