Details of a Researcher - ITAGAKI Shiro

Papers - ITAGAKI Shiro

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Exploration of candidate odorants derived from patient skin lesions using gas chromatography-mass spectrometry

Ryosuke IIZAWA, Masami HORIGUCHI, Hisashi UHARA, Yuji KAN, Kunihiro TOGASHI, Makoto SUZUKI, Takafumi NOMURA, Shiro ITAGAKI, Tomoko UNO, Madoka NAKAMURA

Journal of Japan Association on Odor Environment ( Japan Association on Odor Environment ) 55 ( 5 ) 315 - 318 2024.09 [Refereed]

DOI

A simple, dual direct expression plasmid system in prokaryotic and mammalian cells.

Manabu Murakami, Agnieszka M Murakami, Manabu Yonekura, Ichiro Miyoshi, Shirou Itagaki, Yasutaka Niwa

PNAS nexus 2 ( 5 ) pgad139 2023.05 [International journal]

We introduce a simple, dual direct cloning plasmid system (pgMAX-II) for gene expression analysis in both prokaryotic (Escherichia coli) and mammalian cells. This system, which uses a prokaryotic expression unit adapted from the pgMAX system and a mammalian promoter, is effective for subcloning using the DNA topoisomerase II toxin CcdB. Given that molecular biological cloning systems broadly rely on E. coli for rapid growth, the proposed concept may have wide applicability beyond mammalian cells.

DOI PubMed

Rapid method for plasmid DNA recombination (Murakami-system).

Agnieszka M Murakami, Manabu Yonekura, Katsuhiro Nagatomo, Yasutaka Niwa, Shirou Itagaki, Manabu Murakami

MethodsX 10 102167 - 102167 2023 [International journal]

DNA recombination is a useful technology for cloning and subsequent functional analysis, while standard techniques for plasmid DNA recombination have remained unchanged. In the present study, we introduced rapid method for plasmid DNA recombination, which we named "Murakami-system", to complete the experiments in under 33 h. For this purpose, we selected the following: PCR amplification with 25 cycles and E. coli strain with rapid growth (incubation time of 6-8 h). In addition, we selected rapid plasmid DNA purification (mini-prep; ∼10 min) and rapid restriction enzyme incubation (20 min). This recombination system enabled rapid plasmid DNA recombination within 24-33 h, which could be useful in various fields. We also established a 1-day method for competent cell preparation. Our rapid recombination system allowed several sessions of plasmid DNA recombination to be performed every week, which improves the functional analysis of various genes.•"Rapid method for plasmid DNA recombination (Murakami-system).•E. coli strain with rapid growth (incubation time of 6-8 h).•Combination of rapid protocols (PCR, electrophoresis, DNA purification, ligation, and mini-prep) enabled plasmid DNA recombination within 24-33 h.

DOI PubMed

Enhanced β-adrenergic response in mice with dominant-negative expression of the PKD2L1 channel

Manabu Murakami, Agnieszka M. Murakami, Takayuki Nemoto, Takayoshi Ohba, Manabu Yonekura, Yuichi Toyama, Hirofumi Tomita, Yasushi Matsuzaki, Daisuke Sawamura, Kazuyoshi Hirota, Shirou Itagaki, Yujiro Asada, Ichiro Miyoshi

PLOS ONE ( Public Library of Science (PLoS) ) 17 ( 1 ) e0261668 - e0261668 2022

Polycystic kidney disease (PKD) is the most common genetic cause of kidney failure in humans. Among the various PKD-related molecules, PKD2L1 forms cation channels, but its physiological importance is obscure. In the present study, we established a transgenic mouse line by overexpressing the dominant-negative form of the mouse PKD2L1 gene (i.e., lacking the pore-forming domain). The resulting PKD2L1del-Tg mice exhibited supraventricular premature contraction, as well as enhanced sensitivity to β-adrenergic stimulation and unstable R-R intervals in electrocardiography. During spontaneous atrial contraction, PKD2L1del-Tg atria showed enhanced sensitivity to isoproterenol, norepinephrine, and epinephrine. Action potential recording revealed a shortened action potential duration in PKD2L1del-Tg atria in response to isoproterenol. These findings indicated increased adrenergic sensitivity in PKD2L1del-Tg mice, suggesting that PKD2L1 is involved in sympathetic regulation.

DOI

Attenuated β-adrenergic response in calcium/calmodulin-dependent protein kinase IV-knockout mice.

Manabu Murakami, Agnieszka M Murakami, Yasushi Matsuzaki, Daisuke Sawamura, Takayoshi Ohba, Ichirou Miyoshi, Shirou Itagaki, Hiroyuki Sakagami

PloS one 16 ( 4 ) e0249932 2021 [Refereed] [International journal]

In the present study, we examined the importance of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) in the regulation of cardiac function using genetically modified CaMKIV-null mice. RT-PCR analysis revealed decreased expression of voltage-dependent calcium channels in the cardiac myocytes of CaMKIV-null mice compared with wild-type mice. CaMKIV-null mice showed shortened QT time on electrocardiograms. Pharmacological analysis revealed decreased responsiveness to the β-adrenergic blocker propranolol in CaMKIV-null mice, whereas the plasma norepinephrine level was not affected. CaMKIV-null mice showed decreased baroreflex on electrocardiograms. Heart rate variability analysis showed unstable R-R intervals, a decreased low frequency power/high frequency power (LF/HF) ratio, and increased standard deviation of the normal to normal R-R intervals (SDNN) in CaMKIV-null mice, suggesting decreased responsiveness to β-adrenergic stimulation in CaMKIV-null mice. Atrial contraction analysis and cardiac action potential recording showed a decreased response to the β-adrenoceptor agonist isoproterenol in CaMKIV-null mice. Furthermore, fluorescence imaging in a CRE-hrGFP assay revealed a decreased response to isoproterenol in CaMKIV-null cardiac myocytes. Taken together, our data strongly suggest a significant effect of CaMKIV gene ablation on cardiac β-adrenergic signal transduction.

DOI PubMed

Establishment of cardiac action potential recording using a membrane potential indicator in the mouse sinoatrial node.

Toyama Y, Yonekura M, Arakawa Y, Higuchi S, Tomita H, Murakami AM, Itagaki S, Murakami M

Hirosaki Med J 70 163 - 171 2020 [Refereed]

Molecular cloning of medaka orexin and orexin receptor and decreased spontaneous movement in response to an orexin inhibitor.

Niisawa T, Naruta N, Murakami AM, Yonekura M, Hirota K, Itagaki S, Murakami M

Hirosaki Med. J. 71 55 - 64 2020 [Refereed]

Decreased cardiac pacemaking and attenuated β-adrenergic response in TRIC-A knockout mice.

Manabu Murakami, Yuichi Toyama, Manabu Yonekura, Takayoshi Ohba, Yasushi Matsuzaki, Daisuke Sawamura, Agnieszka M Murakami, Miyuki Nishi, Shirou Itagaki, Hirofumi Tomita, Hiroshi Takeshima

PloS one 15 ( 12 ) e0244254 2020 [Refereed] [International journal]

Changes in intracellular calcium levels in the sinus node modulate cardiac pacemaking (the calcium clock). Trimeric intracellular cation (TRIC) channels are counterion channels on the surface of the sarcoplasmic reticulum and compensate for calcium release from ryanodine receptors, which play a major role in calcium-induced calcium release (CICR) and the calcium clock. TRIC channels are expected to affect the calcium clock in the sinus node. However, their physiological importance in cardiac rhythm formation remains unclear. We evaluated the importance of TRIC channels on cardiac pacemaking using TRIC-A-null (TRIC-A-/-) as well as TRIC-B+/-mice. Although systolic blood pressure (SBP) was not significantly different between wild-type (WT), TRIC-B+/-, and TRIC-A-/-mice, heart rate (HR) was significantly lower in TRIC-A-/-mice than other lines. Interestingly, HR and SBP showed a positive correlation in WT and TRIC-B+/-mice, while no such correlation was observed in TRIC-A-/-mice, suggesting modification of the blood pressure regulatory system in these mice. Isoproterenol (0.3 mg/kg) increased the HR in WT mice (98.8 ± 15.1 bpm), whereas a decreased response in HR was observed in TRIC-A-/-mice (23.8 ± 5.8 bpm), suggesting decreased sympathetic responses in TRIC-A-/-mice. Electrocardiography revealed unstable R-R intervals in TRIC-A-/-mice. Furthermore, TRIC-A-/-mice sometimes showed sinus pauses, suggesting a significant role of TRIC-A channels in cardiac pacemaking. In isolated atrium contraction or action potential recording, TRIC-A-/-mice showed decreased response to a β-adrenergic sympathetic nerve agonist (isoproterenol, 100 nM), indicating decreased sympathetic responses. In summary, TRIC-A-/-mice showed decreased cardiac pacemaking in the sinus node and attenuated responses to β-adrenergic stimulation, indicating the involvement of TRIC-A channels in cardiac rhythm formation and decreased sympathetic responses.

DOI PubMed

A simple and dual expression plasmid system in prokaryotic (E. coli) and mammalian cells.

Murakami M, Ohba T, Murakami A.M, Han C, Yonekura M, Kuwasako K, Itagaki S

Plos One, 14 e0216169 2019.05 [Refereed]

入院患者の健康食品・サプリメントと医薬品の相互作用リスクに関する実態調査

金森未侑, 冨澤登志子, 板垣史郎

日本老年薬学会雑誌 2 9 - 18 2019.04 [Refereed]

Medaka as a model for ECG analysis and the effect of verapamil

Manabu Yonekura, Nami Kondoh, Chong Han, Yuichi Toyama, Takayoshi Ohba, Kyoichi Ono, Shirou Itagaki, Hirofumi Tomita, Manabu Murakami

Journal of Pharmacological Sciences ( Japanese Pharmacological Society ) 137 ( 1 ) 55 - 60 2018.05 [Refereed]

The heart of the medaka, a small fish native to East Asia, has electrophysiological aspects similar to mammalian hearts. We found that the heart rates of medaka were more similar to humans than mice or rats. Medaka exhibited similar electrocardiogram patterns to those of humans, suggesting a similarity in cardiac impulse formation and propagation. Their hearts also exhibited similar responsiveness to verapamil, a calcium channel antagonist atropine, a parasympathetic nerve blocker propranolol, a sympathetic β-adrenergic blocker and isoproterenol, a sympathetic β-adrenergic agonist. We successfully analyzed action potentials and cardiac contractile forces in vivo. Verapamil affected action potential duration and reduced heart rate, suggesting the importance of voltage-dependent calcium channels in determining the heart rhythm of medaka. We also analyzed the expression of the voltage-dependent calcium channel β2 subunit, which participates in channel formation in cardiac myocytes, and found that splice variant type-2 was the only major transcript in the heart. Our results indicate that medaka could be an appropriate animal model for studying cardiovascular pharmacology.

DOI

Modified parasympathetic nervous system in Goto-Kakizaki diabetic rats.

Han C, Inaba W, Yonekura M, Ogata Y, Ohba T, Kushikata T, Niwa H, Yanagisawa T, Tomita H, Itagaki S, Hirota K, Akai H, Mizukami H, Murakami M

Hirosaki Med. J., ( 弘前大学大学院医学研究科・弘前医学会 ) 68 ( 1 ) 33 - 43 2017.10 [Refereed]

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Storage stability of serum formulations containing ofloxacin for autologous serum eardrop therapy

N. Hashimoto, K. Togami, K. Encou, H. Onodera, H. Tanimoto, S. Itagaki, S. Chono

PHARMAZIE ( GOVI-VERLAG PHARMAZEUTISCHER VERLAG GMBH ) 72 ( 3 ) 139 - 142 2017.03 [Refereed]

Objective: The storage stability of serum formulations containing ofloxacin for autologous serum eardrop therapy was evaluated for microbiological quality and component stability. Methods: Sterile serum formulations were prepared by mixing human serum and ofloxacin otic solution (1:1, v/v). To simulate eardrop contamination with external ear surface substances, prepared serum formulations were contaminated with a cotton swab that was rubbed sufficiently on the human external ear. Formulations were stored at 4 degrees C or room temperature in the dark. Colony forming units (CFUs), ofloxacin, and basic fibroblast growth factor (bFGF) concentrations in the stored serum formulations were determined. Results: The growth of microorganisms derived from the external ear was not detected in serum formulations after storage for 14 days, regardless of temperature. However, microbial growth was detected in serum formulations stored without ofloxacin, indicating that this is necessary for storage. In addition, concentrations of ofloxacin and bFGF did not decrease over 14 days, indicating that ofloxacin and bFGF in serum formulations are stable for this time period. Conclusion: The present study indicates that the efficacy and safety of serum formulations used as a therapy for perforated eardrums are stable and safe for at least 14 days.

DOI

Effects of halothane anesthesia on electrocardiogram parameters in mice.

Nozaki Shuhei, Ogata Yoshiki, Yonekura Manabu, Han Chong, Niwa Hidetoshi, Kushikata Tetsuya, Hirota Kazuyoshi, Matsuzaki Yasushi, Tomita Hirofumi, Imaizumi Tadaatsu, Itagaki Shirou, Sawamura Daisuke, Murakami Manabu

Hirosaki Med. J. ( 弘前大学大学院医学研究科・弘前医学会 ) 67 ( 2 ) 129 - 135 2017.02 [Refereed]

We analyzed the effects of halothane as an inhalational anesthetic agent on electrocardiogram （ECG）parameters in C57/BL6 mice. Following induction of anesthesia using 2% halothane, the ECGs showed a regularpattern and the heart rate （HR） was within an acceptable range （~500 bpm）. The HR decreased with increasinghalothane concentration in a concentration-dependent fashion. The frequency domain analysis showed that the high-frequency （HF） component decreased and the lowfrequency（LF） component increased in a concentration-dependent fashion. Therefore, the LF/HF ratio increasedwith increasing halothane concentration, suggesting effects on the autonomic nervous system. We analyzed the pharmacological response to sympathetic blockade with propranolol, a typical adrenergicβ-blocker, under halothane anesthesia. Propranolol administration resulted in a decreased HR; interestingly,intraperitoneal injection of propranolol （120 μg/kg body weight） resulted in arrhythmia （sick sinus syndrome）during anesthesia with 3% halothane. Our results indicate the importance of selecting a suitable anesthetic agent for C57/BL6 mice in pharmacological studies

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Aerosolized liposomes with dipalmitoyl phosphatidylcholine enhance pulmonary absorption of encapsulated insulin compared with co-administered insulin

Sumio Chono, Kohei Togami, Shirou Itagaki

DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY ( TAYLOR & FRANCIS LTD ) 43 ( 11 ) 1892 - 1898 2017 [Refereed]

Objective: We have previously shown that aerosolized liposomes with dipalmitoyl phosphatidylcholine (DPPC) enhance the pulmonary absorption of encapsulated insulin. In this study, we aimed to compare insulin encapsulated into the liposomes versus co-administration of empty liposomes and unencapsulated free insulin, where the DPCC liposomes would serve as absorption enhancer. Significance: The present study provides the useful information for development of noninvasive treatment of diabetes. Methods: Co-administration of empty DPPC liposomes and unencapsulated free insulin was investigated in vivo to assess the potential enhancement in protein pulmonary absorption. Co-administration was compared to DPPC liposomes encapsulating insulin, and free insulin. Results: DPPC liposomes enhanced the pulmonary absorption of unencapsulated free insulin; however, the enhancing effect was lower than that of the DPPC liposomes encapsulating insulin. The mechanism of the pulmonary absorption of unencapsulated free insulin by DPPC liposomes involved the opening of epithelial cell space in alveolar mucosa, and not mucosal cell damage, similar to that of the DPPC liposomes encapsulating insulin. In an in vitro stability test, insulin in the alveolar mucus layer that covers epithelial cells was stable. These findings suggest that, although unencapsulated free insulin spreads throughout the alveolar mucus layer, the concentration of insulin released near the absorption surface is increased by the encapsulation of insulin into DPPC liposomes and the absorption efficiency is also increased. Conclusion: We revealed that the encapsulation of insulin into DPPC liposomes is more effective for pulmonary insulin absorption than co-administration of DPPC liposomes and unencapsulated free insulin.

DOI PubMed

Sustained distribution of aerosolized PEGylated liposomes in epithelial lining fluids on alveolar surfaces

Keita Kaneko, Kohei Togami, Eri Yamamoto, Shujun Wang, Kazuhiro Morimoto, Shirou Itagaki, Sumio Chono

DRUG DELIVERY AND TRANSLATIONAL RESEARCH ( SPRINGER HEIDELBERG ) 6 ( 5 ) 565 - 571 2016.10 [Refereed]

The distribution characteristics of aerosolized PEGylated liposomes in alveolar epithelial lining fluid (ELF) were examined in rats, and the ensuing mechanisms were investigated in the in vitro uptake and protein adsorption experiments. Nonmodified or PEGylated liposomes (particle size 100 nm) were aerosolized into rat lungs. PEGylated liposomes were distributed more sustainably in ELFs than nonmodified liposomes. Furthermore, the uptake of PEGylated liposomes by alveolar macrophages (AMs) was less than that of nonmodified liposomes. In further in vitro uptake experiments, nonmodified and PEGylated liposomes were opsonized with rat ELF components and then added to NR8383 cells as cultured rat AMs. The uptake of opsonized PEGylated liposomes by NR8383 cells was lower than that of opsonized nonmodified liposomes. Moreover, the protein absorption levels in opsonized PEGylated liposomes were lower than those in opsonized nonmodified liposomes. These findings suggest that sustained distributions of aerosolized PEGylated liposomes in ELFs reflect evasion of liposomal opsonization with surfactant proteins and consequent reductions in uptake by AMs. These data indicate the potential of PEGylated liposomes as aerosol-based drug delivery system that target ELF for the treatment of respiratory diseases.

DOI PubMed

「感染性心内膜炎薬物治療の手引き」を用いた抗菌薬適正使用への貢献

小笠原真帆, 太田, 岡村祐嗣, 津山博匡, 板垣史郎, 萱場広之, 早狩誠

日本病院薬剤師会雑誌 ( (一社)日本病院薬剤師会 ) 52 ( 10 ) 1287 - 1292 2016.10 [Refereed]

感染防止対策加算連携施設間における抗菌薬使用量サーベイランスの評価

岡村祐嗣, 倉内寿孝, 津山博匡, 板垣史郎, 萱場広之, 早狩誠

日本環境感染学会誌 ( (一社)日本環境感染学会 ) 31 ( 5 ) 326 - 334 2016.09 [Refereed]

The present study used antimicrobial consumption surveillance to assess the resistance rate transition of Pseudomonas aeruginosa in facilities cooperating in infection prevention medical fees using the antimicrobial use density (AUD) and days of therapy (DOT) as parameters, and compared the surveillance results in 2014 to 2012. In our hospital, the change in DOT of first-generation cephalosporins, third-generation cephalosporins, fourth-generation cephalosporins, carbapenems, aminoglycosides, glycopeptides was greater than that of AUD. In stroke center, the change in DOT of penicillins, antipseudomonal penicillins, carbapenems was greater than that of AUD. The surveillance comparing the AUD transition with DOT transition revealed the influence of antimicrobial dose and duration on the change of AUD was more accurately evaluated by surveillance using the AUD transition with DOT transition. In both facilities, the antimicrobial dose and duration were not associated with change in the resistance rate of P. aeruginosa. The present results suggest that the effects of change in antimicrobial dose and duration transition on the change in resistance rate of P. aeruginosa are limited.

DOI CiNii

ヒト官能試験による口腔内崩壊錠の服用性の実証

丁野純男, 谷本裕幸, 松井萌, 中村勝貴, 佐藤卓馬, 中川麻奈, 山田恵乃, 金子圭太, 山佳織, 佐藤恵亮, 山下美妃, 丹保好子, 郡修徳, 森本一洋, 板垣史郎, 葛西亮也

臨床と研究 93 ( 5 ) 171 - 174 2016.05

膵臓がん組織の抗がん剤反応性を予測するバイオマーカーの探索

板垣史郎

医療の広場, 55 13 - 16 2015.04 [Invited]

THE USEFULNESS OF ANTICANCER DRUG SENSITIVITY TEST (HDRA) FOR THE ADJUVANT CHEMOTHERAPY AND PROFILING IN PANCREATIC CANCER TISSUE AFTER HDRA

照井一史, 川崎仁司, 細井一広, 中川潤一, 板垣史郎, 津山博匡, 下山律子, 袴田健一, 早狩誠

The Hirosaki medical journal ( Hirosaki University ) 65 ( 2 ) 173 - 181 2014.09 [Refereed]

約10種の腫瘍組織（10種）についてHDRA 法による抗がん剤の感受性試験を行った．その結果，5-FU はすべての腫瘍組織に対し陽性を示し，CDDP も5-FU と同様に多くの腫瘍組織で陽性を示したが，抑制率はやや低下していた．術前に5-FU が投与されていない食道癌群における感受性の抑制率は71.9±11.6%（n = 6）であり，術前に5-FU が投与された食道癌群の抑制率に有意な差は認められず，測定した症例では耐性が生じていない可能性が示唆された．その後S-1 投与患者において，5-FU に感受性を示した群は，陰性群に比べ生存日数の延長が認められた.膵癌組織での5-FU およびGEM に対する感受性結果から，5-FU またはS-1 が膵癌への薬物療法で有用な薬剤である可能性が示唆された．膵癌組織に対する質量分析によるプロファイリングを行った結果，5-FU に対する感受性が陰性の試料に出現が増加するシグナルが，5-FU 無効例のバイオマーカーになる可能性が示唆された．

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ラット肝化学発癌過程におけるCyclophilin Bの発現変化

細井一広, 照井一史, 中川潤一, 下山律子, 津山博匡, 板垣史郎, 土田成紀, 早狩誠

弘前医学 164 - 172 2014.09 [Refereed]

PROFILING OF RAT BRAIN PEPTIDES TREATED WITH CENTRALLY ACTIVE ACE INHIBITOR

金澤佐知子, 細井一広, 照井一史, 下山律子, 中川潤一, 板垣史郎, 早狩誠

The Hirosaki medical journal ( Hirosaki University ) 65 ( 1 ) 95 - 103 2014.03 [Refereed]

本研究は，中枢移行性ACE 阻害剤（ACEI）による記憶保持亢進の機序を解明することである．中枢移行性ACEI（captopril），非中枢移行性ACEI （imidapril）およびARB（losartan）投与ラットでの脳内ペプチドの発現変化をHPLC 法およびTOF-MS 法を用いて検索した．その結果，TOF-MS 法では，captopril 投与群で特異的に発現が亢進するペプチドを多数検出できたが，その多くは質量数3,000以下であった．なお，これらの質量数はすべて一価イオンとして検出されたことから，すべて物質固有の質量数を反映していた．検出した質量数は，ACE が分解する脳内ペプチド（LH-RH,substance P, β-neoendorphin, neuromedin B, LVV-hemorphin-7, amyloid β-protein）やinsulin-regulated aminopeptidase（IRAP）の基質と考えられているvasopressin とは異なる値を示した．脳内にはACE やIRAP 以外にも活性中心にZn2+を有するメタロプロテアーゼが存在することから，今回得られた多くの質量数は，キレート形成能を有するcaptoprilにより阻害されたメタロプロテアーゼの内在性基質の可能性がある．

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Rapid Stimulating Effect of the Antiarrhythmic Agent Amiodarone on Absorption of Organic Anion Compounds

Masahiro Segawa, Jiro Ogura, Satoru Seki, Shirou Itagaki, Natsuko Takahashi, Masaki Kobayashi, Takeshi Hirano, Hiroaki Yamaguchi, Ken Iseki

DRUG METABOLISM AND PHARMACOKINETICS ( JAPANESE SOC STUDY XENOBIOTICS ) 28 ( 3 ) 178 - 186 2013.06 [Refereed]

In a clinical setting, changes in pharmacokinetics due to drug-drug interactions can often directly affect the therapeutic safety and efficacy of drugs. Recently, interest has been shown in drug-drug interactions in the intestine. It is now recognized that changes in the functions of drug transporters substantially influence the absorption of administered drugs from the intestine. Amiodarone (AMD) is a potent drug used in the treatment of serious supraventricular and ventricular tachyarrhythmias. Despite its potent pharmacological effects, its wide clinical use is precluded by drug-drug interactions. In this study, we characterized the transporter function between AMD and various compounds in human intestinal model Caco-2 cells. AMD significantly and rapidly increased the uptake of [H-3]estrone-3-sulfate (E-3-S) for 5 min. The apical-to-basal transport of [H-3]E-3-S was significantly increased by AMD. The AMD-stimulated [H-3]E-3-S uptake was inhibited by organic anion transporting polypeptide (OATP) substrates. Caco-2 cells treated with AMD showed increased OATP2B1 expression on the cell surface. AMD also increased the absorption of sulfobromophthalein (ESP), which is a typical organic anion compound, and the expression level of Oatp2b1 at the membrane in in vivo experiments. The results indicate that AMD induces OATP2B1/Oatp2b1 expression at the membrane in the intestine and enhances absorption of organic anion compounds.

DOI PubMed

アンジオテンシン変換酵素に焦点を当てた認知症患者への食事支援に関する基礎研究

内山道子, 板垣史郎, 金澤佐知子, 細井一広, 照井一史, 下山律子, 早狩誠

弘前医学 64 41 - 49 2013.05 [Refereed]

IDENTIFICATION OF THE ANGIOTENSIN CONVERTING ENZYME INHIBITORS IN THE LOCAL PRODUCT OF AOMORI PREFECTURE

板垣史郎, 内山道子, 金澤佐知子, 細井一広, 照井一史, 下山律子

The Hirosaki medical journal ( Hirosaki University ) 64 ( 1 ) 50 - 57 2013.05 [Refereed]

本研究では，食事が多様な食品機能成分の摂取経路という一面を有し，疾病予防法を構築する上で有用な切り口であることを鑑み，アンジオテンシンＩから強力な血圧上昇を有するアンジオテンシンIIへの変換を司るアンジオテンシン変換酵素（ACE）を標的として，青森県特産の農産物及び水産物に含まれるACE阻害物質の探索を行った．青森県特産食材より蒸留水で抽出した抽出液について，ACE阻害活性の評価を行った．ACE阻害活性を示した抽出液から各種カラム分画法にてACE阻害活性物質を単離・精製し，質量分析法により，ACE阻害成分を同定した．その結果，カボチャ，ニンニク，大豆，大鰐モヤシの抽出液に特に強いACE阻害活性を見出した．その中から，カボチャを対象としてACE阻害活性成分の特定を試み，分子量303のムギネ酸前駆物質，ニコチアナミンがその機能実体である可能性を強く示唆する結果を得た．

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STUDY ON IMPROVEMENT OF THE ACCURACY AND PRECISION OF PREDICTED PLASMA CONCENTRATION OF VANCOMYCIN IN THE ADMINISTRATION PLANNING SUPPORT FOR CHILDREN

板垣史郎, 工藤正純, 新岡丈典, 内山道子, 金澤佐知子, 細井一広, 照井一史, 小島佳也, 保嶋実, 早狩誠

The Hirosaki medical journal ( Hirosaki University ) 64 ( 1 ) 58 - 64 2013.05 [Refereed]

小児バンコマイシン（VCM）投与設計支援時に散見される，血中濃度予測値と実測値のずれの原因を特定し，予測系の正確度精度向上を図った．VCM投与時にTDMが実施された16歳以下の患者のうち，クレアチニンクリアランス（CLcr）値が推定可能であった25名を解析対象とした．初回VCM血中濃度ならびにVCMクリアランスを成人母集団パラメータ（A-PPK）および小児母集団パラメータ（P-PPK）を用いて予測し，実測値との差（残差）を基に予測系の正確度精度を評価した．また，初回VCM血中濃度について残差と患者背景因子との相関性を評価した．初回VCM血中濃度，VCMクリアランス共にA-PPK使用系の正確度精度が良好であった．A-PPK使用系の初回VCM血中濃度は年齢，体重，体表面積と有意に相関しており，年齢に関するカットオフ値は6歳と推定された．小児においてCLcrが推定できる場合は，6歳以上を目安にCLcrが組み込まれているA-PPKを用いることが推奨される．

CiNii

処方せんチェックシートを用いたlevofloxacinの用法・用量チェックシステムの効果

岡村祐嗣, 高橋志織, 小原信一, 板垣史郎, 早狩誠

医薬品相互作用研究 36 1 - 6 2013.05 [Refereed]

Electrophysiological Characterization of the Polyspecific Organic Cation Transporter Plasma Membrane Monoamine Transporter

Shiro Itagaki, Vadivel Ganapathy, Horace T. B. Ho, Mingyan Zhou, Ellappan Babu, Joanne Wang

DRUG METABOLISM AND DISPOSITION ( AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS ) 40 ( 6 ) 1138 - 1143 2012.06 [Refereed]

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation (OC) transporter that transports a variety of endogenous biogenic amines and xenobiotic cations. Previous radiotracer uptake studies showed that PMAT-mediated OC transport is sensitive to changes in membrane potential and extracellular pH, but the precise role of membrane potential and protons on PMAT-mediated OC transport is unknown. Here, we characterized the electrophysiological properties of PMAT in Xenopus laevis oocytes using a two-microelectrode voltage-clamp approach. PMAT-mediated histamine uptake is associated with inward currents under voltage-clamp conditions, and the currents increased in magnitude as the holding membrane potential became more negative. A similar effect was also observed for another cation, nicotine. Substrate-induced currents were largely independent of Na+ but showed strong dependence on membrane potential and pH of the perfusate. Detailed kinetic analysis of histamine uptake revealed that the energizing effect of membrane potentials on PMAT transport is mainly due to an augmentation of I-max with little effect on K-0.5. At most holding membrane potentials, I-max at pH 6.0 is approximately 3- to 4-fold higher than that at pH 7.5, whereas K-0.5 is not dependent on pH. Together, these data unequivocally demonstrate PMAT as an electrogenic transporter and establish the physiological inside-negative membrane potential as a driving force for PMAT-mediated OC transport. The important role of membrane potential and pH in modulating the transport activity of PMAT toward OCs suggests that the in vivo activity of PMAT could be regulated by pathophysiological processes that alter physiological pH or membrane potential.

DOI PubMed

効果発現パターンにより抗がん薬を分類するための基礎的検討

高橋夏子, 小林正紀, 板垣史郎, 平野剛, 武隈洋, 菅原満, 井関健

薬学雑誌 ( (公社)日本薬学会 ) 132 ( 6 ) 777 - 783 2012.06 [Refereed]

ヒト肺癌由来株化細胞A549およびヒト乳癌由来株化細胞MCF-7を用い、作用発現が明確な4種の薬物(シスプラチン(CDDP)、カルボプラチン(CBDCA)、パクリタキセル(PTX)、ゲムシタビン(GEM))を使用し、より簡便な抗癌薬の効果の判定と、投与設計への寄与を目的とし、in vitroでの抗癌薬の分類について基礎的検討を行った。CDDP、CBDCAは濃度依存的に抗腫瘍効果を発現する薬物として分類されることが推測された。一方、PTX、GEMについては細胞生存曲線の低濃度側へのシフトおよびIC 50値の著しい低下がみられことから、時間依存的に抗腫瘍効果を発揮する薬物として、分類可能であることが示唆された。更にCDDP、CBDCAの濃度依存性をより確実なものとするため、IC 50値を基準として前後10倍の濃度を設定し、濃度依存性を評価した結果、薬物濃度に比例した細胞生存率の低下がみられたことから、CDDP、CBDCAは濃度依存的に抗腫瘍効果を発揮する傾向の強い薬物であることが判明した。また、納所依存性の検討においても、用いた2種の細胞間で各抗癌薬の効果発現の様式に同様な傾向がみられた。研究結果より、細胞生存曲線及びIC 50値の変化を指標とし、作用の異なる抗癌薬を濃度依存的薬物および時間依存的薬物に分類できる可能性が示唆された。

Regulation of human monocarboxylate transporter 4 in skeletal muscle cells: The role of protein kinase C (PKC)

Katsuya Narumi, Masaki Kobayashi, Sho Otake, Ayako Furugen, Natsuko Takahashi, Jiro Ogura, Shirou Itagaki, Takeshi Hirano, Hiroaki Yamaguchi, Ken Iseki

INTERNATIONAL JOURNAL OF PHARMACEUTICS ( ELSEVIER SCIENCE BV ) 428 ( 1-2 ) 25 - 32 2012.05 [Refereed]

In the present study, to clarify the role of protein kinase C (PKC) in the regulation of monocarboxylate transporter 4 (MCT4) expression, we examined the regulation mechanism of MCT4 expression in human rhabdomyosarcoma (RD) cells, an in vitro skeletal muscle model. Exposure of RD cells to PMA, a PKC activator, for 24 h resulted in a two-fold increase in the amount of lactic acid in the growth medium. In parallel to an increase in lactic acid release from RD cells, the level of MCT4 mRNA and protein were also significantly increased in RD cells. A PKC inhibitory study indicated that PMA-induced stimulation of MCT4 expression can be mediated through a novel PKC isoform, especially PKC delta. Moreover, rottlerin, a selective PKC delta inhibitor, decreased PMA-induced MCT4 promoter activity. Deletion and mutational analysis suggested that the potential hypoxia-response elements (HREs) played a major role in the observed modulation of MCT4 expression by PMA. Furthermore, we found that small interfering RNA (siRNA)-mediated knockdown of hypoxia-inducible factor 1 alpha (HIF-1 alpha) significantly inhibited PMA-induced MCT4 promoter activity. Our results show that the effects of PMA on MCT4 expression are mediated through an indirect pathway partially involving PKC delta and HIF-1 alpha transcription factor. (C) 2012 Published by Elsevier B.V.

DOI PubMed

Effects of Acidification and Alkalinization Agent on Statins-induced Muscle Toxicity

Masaki Kobayashi, Kazuhiro Hidaka, Ikumi Chisaki, Natsuko Takahashi, Jiro Ogura, Shirou Itagaki, Takeshi Hirano, Hiroaki Yamaguchi, Ken Iseki

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN ( PHARMACEUTICAL SOC JAPAN ) 132 ( 5 ) 609 - 615 2012.05 [Refereed]

The aim of this study was to determine the effects of alpha-cyano-4-hydroxycinnamic acid (CHC), a lactate efflux inhibitor, and citrate, an alkaline reagent, on statin-induced muscle injury using a human prototypic embryonal rhabdomyosarcoma cell line (RD) as a model of in vitro skeletal muscle and on statin-induced muscle damage in an in vivo study. Statin-induced reduction of cell viability and apoptosis was measured by the 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay and caspase assay. In an in vivo study, plasma creatine phosphokinase (CPK) level was examined in cerivastatin-treated rats. CHC increased growth inhibition of RD cells induced by cerivastatin, a lipophilic statin, but not these induced by pravastatin, a hydrophilic statin. On the other hand, citrate suppressed cerivastatin-, simvastatin- and atorvastatin-induced reduction of cell viability and caspase activation in RD cells. Moreover, citrate prevented cerivastatin-induced increase in CPK concentration in a concentration-dependent manner. This is first study to evaluate CHC or citrate-induced exacerbation or improvement of statin-induced muscle damage.

DOI PubMed

Intestinal Ischemia-Reperfusion Increases Efflux for Uric Acid Via Paracellular Route in the Intestine, but Decreases that Via Transcellular Route Mediated by BCRP

Jiro Ogura, Kaori Kuwayama, Atsushi Takaya, Yusuke Terada, Takashi Tsujimoto, Takahiro Koizumi, Hajime Maruyama, Asuka Fujikawa, Natsuko Takahashi, Masaki Kobayashi, Shirou Itagaki, Takeshi Hirano, Hiroaki Yamaguchi, Ken Iseki

JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES ( CANADIAN SOC PHARMACEUTICAL SCIENCES ) 15 ( 2 ) 295 - 304 2012 [Refereed]

Purpose. Uric acid is thought to be one of the most important antioxidants in human biological fluids. Intestinal ischemia-reperfusion (I/R) is an important factor associated with high rates of morbidity and mortality. Reactive oxygen species (ROS) are responsible for intestinal I/R injury. The aim of this study was to clarify the efflux for uric acid from the intestine after intestinal I/R. Methods. We used intestinal ischemia-reperfusion (I/R) model rats. Serosal to mucosal flux for [C-14]-uric acid was assessed by using Ussing-type diffusion chambers. BCRP/Bcrp expression was assessed by Western blot analysis. Caco-2 cells were used for a model of the intestinal epithelium, and rotenone was used as a mitochondrial dysfunction inducer. Results. Serosal to mucosal flux for uric acid was increased after intestinal I/R, and that for mannitol was also increased. Ko143, which is a BCRP inhibitor, did not affect the uric acid transport. The decreasing uric acid transport mediated by Bcrp was caused by decrease in the level of Bcrp homodimer, bridged by an S-S bond. The suppression of Bcrp S-S bond formation was associated with mitochondrial dysfunction. Moreover, BCRP S-S bond formation activity was decreased by rotenone in Caco-2 cells. Conclusions. Serosal to mucosal flux for uric acid is significantly increased via the paracelluler route, but that via the transcellular route mediated by Bcrp is decreased after intestinal I/R. The decreasing uric acid flux mediated by Bcrp is caused by suppression of Bcrp S-S bond formation. This suppression of Bcrp S-S bond formation may be related to mitochondrial dysfunction.

PubMed

Involvement of Cholesterol Membrane Transporter Niemann-Pick C1-Like 1 in the Intestinal Absorption of Lutein

Yuki Sato, Risa Suzuki, Masaki Kobayashi, Shirou Itagaki, Takeshi Hirano, Toshihiro Noda, Satoshi Mizuno, Mitsuru Sugawara, Ken Iseki

JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES ( CANADIAN SOC PHARMACEUTICAL SCIENCES ) 15 ( 2 ) 256 - 264 2012 [Refereed]

PURPOSE. Lutein is a carotenoid mainly found in green leafy vegetables and is located in the macula lutea in the human eye. Since humans cannot synthesize lutein de novo, it must be digested as food. The physiological importance of an orally administered compound depends on its interaction with target tissues. It is therefore important to clarify the absorption mechanism in the intestine. Cholesterol membrane transporters Niemann-Pick C1 Like 1 (NPC1L1) and scavenger receptor class B type 1 (SR-B1) are involved in the intestinal absorption of highly lipophilic compounds including cholesterol. Ezetimibe, a selective inhibitor of intestinal NPC1L1, is the widespread lipid-lowering agent. It is important to investigate the possibility of food-drug interactions in order to prevent undesirable and harmful clinical consequences. The aim of this work was to determine whether NPC1L1, SR-B1 and other transporters are involved in absorption of lutein. METHODS. Caco-2 cells were used for accumulation and permeability study of lutein. Lutein concentration was determined by an HPLC system. The cDNA of transporters was isolated from total RNA of Caco-2 cells, and the expression of these transporters was confirmed by RT-PCR (reverse transcription - polymerase chain reaction). RESULTS. Ezetimibe inhibited up to 40% of lutein accumulation by Caco-2 cell monolayers. Block lipid transport 1 (BLT-1), a selective chemical inhibitor of SR-B1, also inhibited lutein accumulation by Caco-2 cells. On the other hand, ATP-depletion reagents (sodium fluoride and sodium azide or carbonyl cyanide m-chlorophenylhydrazone) did not influence the accumulation or permeation of lutein significantly. CONCLUSIONS. The results show that lutein absorption is, at least in part, mediated by influx transporters NPC1L1 and SR-B1 rather than mediated by efflux transporters such as ABC (ATP-binding cassette) transporters.

PubMed

The Decrease in Farnesoid X Receptor, Pregnane X Receptor and Constitutive Androstane Receptor in the Liver after Intestinal Ischemia-Reperfusion

Jiro Ogura, Yusuke Terada, Takashi Tsujimoto, Takahiro Koizumi, Kaori Kuwayama, Hajime Maruyama, Asuka Fujikawa, Atsushi Takaya, Masaki Kobayashi, Shirou Itagaki, Natsuko Takahashi, Takeshi Hirano, Hiroaki Yamaguchi, Ken Iseki

JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES ( CANADIAN SOC PHARMACEUTICAL SCIENCES ) 15 ( 5 ) 616 - 631 2012 [Refereed]

Purpose. Intestinal ischemia-reperfusion (I/R) damages remote organs, including the liver, and promotes multi-organ failure (MOF). However, the molecular mechanisms underlying acute liver injury after intestinal I/R have not been completely elucidated. Farnesoid X receptor (FXR), pregnane X receptor (PXR) and constitutive androstane receptor (CAR) regulate metabolizing enzymes and transporters, and coordinately prevent hepatotoxicity reflecting an inability of appropriate excretion of endogenous toxic compounds. In this study, we assessed FXR, PXR and CAR expression levels and their localization levels in nuclei in the liver after intestinal I/R. We also investigated the effect of IL-6 on FXR, PXR and CAR expression levels and their localization levels in nuclei in in vitro experiments. Methods. We used intestinal I/R model rats. Moreover, HepG2 cells were used in in vitro study. Real-time PCR and Western blotting were used to assess mRNA and protein expression levels. Nuclear receptor localization in nuclei was analyzed by Western blotting using nuclear extracts. Results. FXR and PXR expression levels began to be decreased at 3 h, and FXR, PXR and CAR expression levels were decreased at 6 h after intestinal I/R. The localization levels of FXR, PXR and CAR in nuclei began to be decreased at 3 h, and all of them were decreased at 6 h after intestinal I/R. In HepG2 cells, FXR, PXR and CAR expression levels were decreased by 0.5-1 ng/mL, 0.5-100 ng/mL and 100 ng/mL IL-6 treatment for 24 h, respectively. FXR, PXR and CAR localization levels in nuclei were suppressed by 0.5-10 ng/mL, 10-100 ng/mL and 10-100 ng/mL IL-6 treatment for 24 h, respectively. Conclusions. FXR, PXR and CAR expression levels are decreased in the liver after intestinal I/R. IL-6 is one of main causes the decreases in expressions of these receptors.

PubMed

Protective Effect of Soy Isoflavone Genistein on Ischemia-Reperfusion in the Rat Small Intestine

Yuki Sato, Shirou Itagaki, Setsu Oikawa, Jiro Ogura, Masaki Kobayashi, Takeshi Hirano, Mitsuru Sugawara, Ken Iseki

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 34 ( 9 ) 1448 - 1454 2011.09 [Refereed]

Ischemia-reperfusion (I/R) injury of the intestine is an important factor associated with high rates of morbidity and mortality. Intestinal I/R is a common clinical problem in the settings of severe burns, circulatory shock and strangulation ileus. Intestinal I/R damages remote organs and promotes multi-organ failure. It has been shown that enteral feeding before ischemic insults is beneficial for reducing organ injury and improving survival after intestinal I/R. In that study, the authors used a standard complex enteral diet and they suggested that it is important to find new nutrient formulas. Since reactive oxygen species are responsible for intestinal I/R injury, we focused on a dietary polyphenol, the soy isoflavone genistein. Genistein has a wide spectrum of biochemical and pharmacological activities. However, the possibility of a protective effect of genistein as enteral nutrition on I/R injury has not been investigated. We therefore investigated the protective effect of genistein on oxidative injury using intestinal I/R model rats. We found that genistein, which has combined antioxidant activity from radical scavenging, xanthine oxidase inhibition and chain-breaking effects, exhibits a protective effect on intestinal I/R injury. The results suggest that genistein, a soy isoflavone, has the possibility as a new nutrient formula of enteral feeding.

DOI PubMed CiNii

Protective effect of lutein after ischemia-reperfusion in the small intestine

Yuki Sato, Masaki Kobayashi, Shirou Itagaki, Takeshi Hirano, Toshihiro Noda, Satoshi Mizuno, Mitsuru Sugawara, Ken Iseki

FOOD CHEMISTRY ( ELSEVIER SCI LTD ) 127 ( 3 ) 893 - 898 2011.08 [Refereed]

Lutein is a carotenoid mainly found in green leafy vegetables and is located in the macula lutea in the human eye. Since humans cannot synthesise lutein de novo, it must be digested as food. The physiological importance of an orally administered compound depends on its interaction with target tissues. There is little information about the effects of intake of lutein in tissues other than the eyes. The aim of this study was to clarify the protective effect of lutein against oxidative injury using ischemia-reperfusion (I/R) model rats and to determine the relationship between pharmacokinetics and antioxidant activity of lutein. Intestinal I/R was induced by 30-min occlusion of the superior mesenteric artery followed by 60-min reperfusion. After 60 min of reperfusion, intestinal tissue was Used for analysis of Evans blue dye extravasation, lipid peroxidation and myeloperoxidase activity. Lutein administered before I/R had a significant protective effect against oxidative injury. (C) 2011 Elsevier Ltd. All rights reserved.

DOI CiNii

Regulation mechanism of ABCA1 expression by statins in hepatocytes

Masaki Kobayashi, Keisuke Gouda, Ikumi Chisaki, Manami Ochiai, Shirou Itagaki, Ken Iseki

EUROPEAN JOURNAL OF PHARMACOLOGY ( ELSEVIER SCIENCE BV ) 662 ( 1-3 ) 9 - 14 2011.07 [Refereed]

ATP-binding cassette transporter A1 (ABCA1) is predicted to be involved in the control of apolipoprotein AI-mediated cholesterol efflux: biosynthesis of high-density lipoprotein (HDL). However, the effects of HMG-CoA reductase inhibitors (statins) on ABCA1 in the liver and the precise mechanisms of their actions have been obscure. The aims of this study were to determine whether statins (atorvastatin (Ato) and pitavastatin (Pit)) affect hepatic ABCA1 expression and to clarify the mechanisms of their actions using HepG2 cells and the rat liver. We examined alterations in mRNA and protein levels of ABCA1 and peroxisome proliferator-activated receptors (PPARs) by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. In vitro and in vivo studies suggested that Pit increases ABCA1 mRNA level, but not Ato. Pit greatly increased Abca1 mRNA level and also increased the amount of plasma HDL and the mRNA level of PPAR alpha. Clofibrate (PPAR alpha agonist) increased ABCA1 expression in HepG2 cells and rat primary hepatocytes more than did PPAR beta/delta and gamma agonists. Pit-induced ABCA1 expression alteration was blocked by GW6471 (PPAR alpha antagonist) and by PPAR alpha knockdown. In this study, we demonstrated that Pit affect ABCA1 expression via PPAR alpha in hepatocytes. The strategy to target a PPAR alpha agonist in the liver can lead to increases in ABCA1 expression and HDL level. (C) 2011 Elsevier B.V. All rights reserved.

DOI

Pharmacokinetic properties of lutein emulsion after oral administration to rats and effect of food intake on plasma concentration of lutein

Yuki Sato, Masaki Kobayashi, Shirou Itagaki, Takeshi Hirano, Toshihiro Noda, Satoshi Mizuno, Mitsuru Sugawara, Ken Iseki

BIOPHARMACEUTICS & DRUG DISPOSITION ( WILEY-BLACKWELL ) 32 ( 3 ) 151 - 158 2011.04 [Refereed]

Lutein is a carotenoid found mainly in green leafy vegetables and is located in the macula lutea in the human eye. An intake of lutein as food is needed since humans cannot synthesize it de novo. Although lutein has received much attention recently due to its antioxidant activities, little information about the pharmacokinetic properties of lutein is available. Lutein emulsion formulation was used and the pharmacokinetics of lutein emulsion after oral administration to rats was investigated. The bioavailability of lutein using this formulation was calculated to be 5.20%. It was found that a large amount of lutein was accumulated in the intestinal mucosa. The absorption of orally administered compounds in the intestine can be enhanced by interaction with food or food components. Thus, the effect of food intake on the intestinal absorption of lutein was investigated. The plasma concentration of lutein after oral administration of the emulsion formulation was improved significantly by food intake. It is possible that the absorption of lutein in the intestine is improved significantly by some food components. Bile acids may also play important roles in the intestinal absorption of lutein since the absorption of lipophilic compounds such as cholesterol is related to bile acids. The results of these studies should contribute to an improvement of lutein absorption and provide important information for obtaining more effective pharmacological effects of lutein. Copyright (C) 2011 John Wiley & Sons, Ltd.

DOI PubMed

Alteration of P-gp Expression after Intestinal Ischemia-reperfusion Following 16-h Fasting in Rats

Jiro Ogura, Asuka Fujikawa, Hajime Maruyama, Masaki Kobayashi, Shirou Itagaki, Ken Iseki

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN ( PHARMACEUTICAL SOC JAPAN ) 131 ( 3 ) 453 - 462 2011.03 [Refereed]

Alteration of P-glycoprotein (P-gp) expression influences the pharmacokinetics of P-gp substrates after intestinal ischemia-reperfusion (I/R). Fasting before intestinal I/R affects intestinal I/R injury. However, the effect of fasting on the alteration of P-gp expression after intestinal I/R has not been clarified. We previously reported that P-gp expression was altered after intestinal I/R following feeding. In the present study, we investigated the expression of P-gp after intestinal I/R following 16-h fasting. The mdr1a levels were decreased at 6 h in the jejunum, at I h in the ileum, at 6 and 24 h in the liver and at 6 h in the kidney. The mdr1b levels were decreased at 6 h in the ileum and at 1 and 6 h in the kidney. The mdr1b level in the liver was increased at 1 and 6 h. The expression of P-gp was decreased at 6 h in the jejunum, at 1, 6 and 24 h in the ileum, and at 6 h in the kidney. These alterations were different to those after intestinal I/R following feeding. In particular, the decrease in P-gp expression in the 16-h fasting I/R rats occurred at an early time during reperfusion compared with that in the feeding I/R rats. In conclusion, feeding condition affects the alteration of P-gp expression after intestinal I/R. Therefore, it is important to understand the patient's feeding condition for safe drug therapy.

DOI CiNii

In vitro and in vivo antioxidant properties of chlorogenic acid and caffeic acid

Yuki Sato, Shirou Itagaki, Toshimitsu Kurokawa, Jiro Ogura, Masaki Kobayashi, Takeshi Hirano, Mitsuru Sugawara, Ken Iseki

INTERNATIONAL JOURNAL OF PHARMACEUTICS ( ELSEVIER SCIENCE BV ) 403 ( 1-2 ) 136 - 138 2011.01 [Refereed]

Dietary polyphenols are thought to be beneficial for human health as antioxidants. Coffee beans contain a common polyphenol, chlorogenic acid. Chlorogenic acid is the ester of caffeic acid and quinic acid. Although these polyphenols have received much attention, there is little evidence indicating a relationship between the effect and the rate of absorption. In this study, we focused on the beneficial effects of chlorogenic acid and caffeic acid, a major metabolite of chlorogenic acid. We carried out in vitro and in vivo experiments. In the in vitro study, caffeic acid had stronger antioxidant activity than that of chlorogenic acid. The uptake of chlorogenic acid by Caco-2 cells was much less than that of caffeic acid. The physiological importance of an orally administered compound depends on its availability for intestinal absorption and subsequent interaction with target tissues. We then used an intestinal ischemia-reperfusion model to evaluate antioxidant activities in vivo. We found that both chlorogenic acid and caffeic acid had effects on intestinal ischemia-reperfusion injury. Since caffeic acid has a stronger antioxidant activity than that of chlorogenic acid and chlorogenic acid is hydrolyzed into caffeic acid in the intestine, it is possible that caffeic acid plays a major role in the protective effect of chlorogenic acid against ischemia-reperfusion injury. (C) 2010 Elsevier B.V. All rights reserved.

DOI PubMed

AMP-activated protein kinase regulates the expression of monocarboxylate transporter 4 in skeletal muscle

Ayako Furugen, Masaki Kobayashi, Katsuya Narumi, Meguho Watanabe, Sho Otake, Shirou Itagaki, Ken Iseki

LIFE SCIENCES ( PERGAMON-ELSEVIER SCIENCE LTD ) 88 ( 3-4 ) 163 - 168 2011.01 [Refereed]

Aims: The aim of this study was to determine the effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, on monocarboxylate transporter 4 (MCT4) expression in rat skeletal muscle and a prototypic embryonal rhabdomyosarcoma cell line (RD cells). Main methods: We examined the alteration in Glucose transporter 4 (GLUT4) and MCT4 mRNA levels by quantitative real-time PCR. Alteration in GLUT4 and MCT4 protein levels was examined by Western blotting. Key findings: In an in vivo study. AICAR increased MCT4 mRNA and protein levels in a fiber-type specific manner. In an in vitro study, AICAR increased MCT4 mRNA and protein levels. Moreover, AICAR-induced MCT4 expression was blocked by Compound C, an AMPK inhibitor. Significance: In this study, we found that AMPK activation induced expression of MCT4 in RD cells and rat skeletal muscle in a fiber-type specific manner. These results indicate the possible involvement of an AMPK-mediated pathway associated with MCT4 expression in skeletal muscle. (C) 2010 Elsevier Inc. All rights reserved.

DOI PubMed CiNii

有機アニオントランスポータOATP2B1の臨床的有用性

板垣史郎

医薬品相互作用研究 35 ( 1 ) 1 - 10 2011 [Refereed] [Invited]

Regulation of Monocarboxylate Transporter 1 in Skeletal Muscle Cells by Intracellular Signaling Pathways

Katsuya Narumi, Ayako Furugen, Masaki Kobayashi, Sho Otake, Shirou Itagaki, Ken Iseki

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 33 ( 9 ) 1568 - 1573 2010.09 [Refereed]

Skeletal muscle is the major producer of lactic acid in the body, but its oxidative fibers also use lactic acid as a respiratory fuel. Monocarboxylate transporter (MCT) 1 has been suggested to play a major role in influx of L-lactic acid for oxidation. The regulation mechanism of MCT1 was characterized utilizing rhabdomyosarcoma cells as an in vitro skeletal muscle model. The uptake of L-lactic acid via MCT1 was studied in the presence of various intracellular regulatory pathways, including pathways mediated by protein kinases A, C and G (PKA, PKC and PKG), protein tyrosine kinase (PTK), and Ca2+/calmodulin modulators. The results showed that PKG-, PTK-, and Ca2+/calmodulin-mediated regulatory pathways play no role in the regulation of L-lactic acid uptake, but a role for PKC- and PKA-mediated pathways was apparent. Uptake of L-lactic acid appeared to be stimulated by phorbol 12-myristate 13-acetate (PMA, a PKC activator) via an increase in V-max of transport processes with no alteration in K-m. In parallel, PMA treatment also resulted in an increase in the level of MCT1 expression. On the other hand, exposure to 8-Br-cAMP, a cAMP analog, and to forskolin, an adenylyl cyclase activator, resulted in a significant decrease in L-lactic acid uptake. Additionally, 8-Br-cAMP reduced V-max but not K-m values. Parallel to the decrease in V-max of L-lactic acid uptake, the level of MCT1 expression was decreased in response to incubation with 8-Br-cAMP. These results indicate the possible involvement of a PKC- and PKA-mediated pathway associated with expression of MCT1 and lactate transport.

DOI CiNii

放射線治療に伴う口腔粘膜障害に対する予防・軽減効果が期待される物質の探索

板垣史郎, 中田千絵, 平野剛, 鷹野瑠美, 笠師久美子, 菅原満, 高橋夏子, 小林正紀, 井関健

医療薬学 ( (一社)日本医療薬学会 ) 36 ( 9 ) 696 - 702 2010.09 [Refereed]

放射線台療に伴う口腔粘膜障害に対する予防・軽減効果が期待される物質を探索した。16Gyの放射線照射を行った後、5日間培養したHO-1-N-1細胞を用いて、種々の化合物の障害抑制効果について検討した。抗酸化作用を有するN-アセチルシステイン(NAC)およびカテキン類に着目し、その障害抑制効果について検討した。高濃度のNACの添加により、放射線照射による細胞生存率の低下が有意に抑制された。カテキン類の添加により、放射線照射による細胞生存率の低下は有意に抑制された。NACおよびカテキン類の添加により、放射線照射による蛍光強度の増大は抑制された。HO-1-N-1細胞に放射線を照射したところ、アポトーシスに特徴的なDNAの断片化が観察され、アポトーシスが誘導されていることが示された。一方、NACおよびカテキン類の添加により、DNAの断片化は顕著に抑制された。

DOI

Pharmacokinetics of Oral and Intravenous Administration of Digoxin after Intestinal Ischemia-Reperfusion

Jiro Ogura, Hajime Maruyama, Masaki Kobayashi, Shirou Itagaki, Ken Iseki

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 33 ( 5 ) 922 - 925 2010.05 [Refereed]

Intestinal ischemia-reperfusion (I/R) causes gut dysfunction characterized by decreased basement membrane integrity and decreased barrier function. Indeed, it has been reported that the absorption of several drugs is altered after intestinal I/R. Intestinal I/R also promotes multi-organ failure (MOF). The liver and kidney can be affected by MOF after intestinal I/R. However, little is known about the alteration of pharmacokinetics after intravenous administration in intestinal I/R injury. In the present study, we investigated pharmacokinetics of digoxin after oral administration and intravenous administration in intestinal I/R injury. Plasma digoxin concentration in I/R rats after oral administration was not significantly altered at any time compared with that in sham-operated rats. Plasma digoxin concentration in rats reperfused for 1 h after intravenous administration was significantly higher than that in sham-operated rats. Plasma digoxin concentrations in rats reperfused for 6 and 24 h were the same as those in sham-operated rats. The area under the concentration time curve after intravenous administraion (AUC(i.s.)) and total clearance (CLtot) in rats reperfused for 1 h was 1.89- and 0.57-fold higher than that in sham-operated rats. However, elimination rate (k(e)) and half-life (t(1/2)) in rats reperfused for 1 h were not altered. Distribution volume (V-d) in rats reperfused for 1 h was decreased than that in sham-operated rats, but there was not statistical difference. These results suggest that intestinal I/R affected the V-d of digoxin, and plasma concentration of digoxin was increased. The present study suggests that understanding pharmacokinetics of drug after intravenous administration in intestinal I/R injury is important to provide valuable information for safe drug therapy for intestinal I/R patients.

DOI

Grapefruit juice enhance the uptake of coenzyme Q10 in the human intestinal cell-line Caco-2

Shirou Itagaki, Akiko Ochiai, Masaki Kobayashi, Mitsuru Sugawara, Takeshi Hirano, Ken Iseki

FOOD CHEMISTRY ( ELSEVIER SCI LTD ) 120 ( 2 ) 552 - 555 2010.05 [Refereed]

Coenzyme Q10 (CoQ10) is very widely consumed by humans as a food supplement. However, CoQ10 is taken up from the intestine into the circulation at a low rate. The absorption of compounds from the gastrointestinal tract is one of the important determinants for oral bioavailability. Secretory transport limits the Oral bioavailability of compounds. It has been reported that efflux transport of CoQ10 is mediated by P-glycoprotein (P-gp) in Caco-2 cells. We tried to improve intestinal absorption of CoQ10 by modulating P-gp. Since grapefruit juice (GFJ) is reported to inhibit P-gp function, we investigated the effect of GFJ on the transport of CoQ10 by Caco-2 cells. In the presence of GFJ, the basal-to-apical transport of CoQ10 was decreased and the uptake of CoQ10 was increased. These findings suggest that the combined administration of CoQ10 and GFJ could enhance CoQ10 absorption. (C) 2009 Elsevier Ltd. All rights reserved.

DOI CiNii

In vitro実験系による肺がん化学療法レジメンの抗腫瘍効果の評価

斎藤由起子, 平野剛, 沖洋充, 笠師久美子, 菅原満, 小林正紀, 高橋夏子, 板垣史郎, 井関健

医療薬学 ( (一社)日本医療薬学会 ) 36 ( 4 ) 220 - 226 2010.04 [Refereed]

In vitro実験系による肺がん化学療法レジメンの抗腫瘍効果について検討した。肺がんのモデルとしてヒト肺扁平上皮がん由来A549細胞を使用した。CDDP+CPT-11(IP)療法において、CDDPは長期間にわたって抗腫瘍効果を示すことが示唆された。SN-38はday2からday5にかけて細胞生存率の回復を認め、CPT-11の1回日の投与により死滅したがん細胞が回復してきたところを再投与によりさらに攻撃するというレジメンの投与スケジュールに相関した。CDDP+VP-16(EP)療法において、各々の単剤のときと比較して抗腫瘍効果が増大することが示された。

DOI

Diclofenac-induced stimulation of SMCT1 (SLC5A8) in a heterologous expression system: A RPE specific phenomenon

Sudha Ananth, Lina Zhuang, Elangovan Gopal, Shiro Itagaki, Babu Ellappan, Sylvia B. Smith, Vadivel Ganapathy, Pamela Martin

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 394 ( 1 ) 75 - 80 2010.03 [Refereed]

SMCT1 is a Na(+)-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na(+)-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, beta-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance. (C) 2010 Elsevier Inc. All rights reserved.

DOI PubMed

Protective effects of quercetin-3-rhamnoglucoside (rutin) on ischemia-reperfusion injury in rat small intestine

Shirou Itagaki, Setsu Oikawa, Jiro Ogura, Masaki Kobayashi, Takeshi Hirano, Ken Iseki

FOOD CHEMISTRY ( ELSEVIER SCI LTD ) 118 ( 2 ) 426 - 429 2010.01 [Refereed]

Quercetin-3-rhamnoglucoside (rutin) has a wide spectrum of biochemical and pharmacological activities. Orally administered compounds are absorbed in the intestine. Although absorption of rutin from the jejunum of the rat was good, binding of rutin to the intestinal wall components may limit its absorption from the small intestine. The physiological importance of an orally administered compound depends on its interaction with target tissues. Since there is limited information on the importance of rutin in vivo, we focused on the protective effect of rutin on intestinal injury. The intestinal mucosa is extremely sensitive to reactive oxygen species. We used a rat mesenteric ischemia-reperfusion (I/R) injury model as a model of oxidative injury and investigated the antioxidant activities of rutin in vivo. We found that rutin, which has combined antioxidant activity from radical-scavenging, xanthine oxidase inhibition and chain-breaking effects, exhibits a protective effect on I/R injury in the rat small intestine. (C) 2009 Elsevier Ltd. All rights reserved.

DOI

In vitro Evaluation of Anticancer Effects of FOLFIRI and mFOLFOX 6 Chemotherapy Regimens and Their Effects on EGFR Expression

sei mei, sei mei, sei mei, sei mei, sei mei, sei mei, sei mei

Iryo Yakugaku (Japanese Journal of Pharmaceutical Health Care and Sciences) ( Japanese Society of Pharmaceutical Health Care and Sciences ) 36 ( 12 ) 855 - 862 2010 [Refereed]

Recently,the number of patients with colorectal cancer has been increasing.Many patients are not aware of any symptoms until the disease is advanced.It is important to select effective regimens for colorectal cancer therapy and combination therapy with a molecular-targeted agent has recently been used for this purpose.However,the effects of anticancer drugs on epidermal growth factor receptor (EGFR),a molecular-targeted protein,have not been clarified.We therefore evaluated the effects of folinic acid/5-fluorouracil/irinotecan (FOLFIRI) and folinic acid/5-fluorouracil/oxaliplatin (mFOLFOX citation=6)treatments,using the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) assay,as well as the effects of these treatments on EGFR expression in LoVo cells.
The results suggested that 5-fluorouracil and irinotecan have a stronger antiproliferative effect than oxaliplatin.The reduction in viability for LoVo cells treated with FOLFIRI was greater than that for Lovo cells treated with mFOLFOX 6 on day 22.Moreover,EGFR mRNA and protein levels were increased more by mFOLFOX 6 than by FOLFIRI.These results suggest that FOLFIRI therapy may be more effective than mFOLFOX 6 therapy against LoVo cells in consideration of EGFR expression.

DOI CiNii

Expression and Role of SNAT3 in the Placenta

C. Yoshioka, S. Yasuda, F. Kimura, M. Kobayashi, S. Itagaki, T. Hirano, K. Iseki

PLACENTA ( W B SAUNDERS CO LTD ) 30 ( 12 ) 1071 - 1077 2009.12 [Refereed]

Glutamine is the most versatile amino acid and its plasma concentration is the highest of all amino acid. Many transporters are therefore involved in glutamine uptake or efflux. Glutamine is actively released from the placenta into fetal circulation. In this study, we examined the alteration of transporters that transport glutamine into fetal circulation as gestation progresses. High expression levels of system A and y(+)L were found in the rat placenta in the late period of pregnancy and the expression levels of these transporters increased as gestation progressed (p < 0.05). On the other hand, the expression of SNAT3, the system N transporter, was detected in the early period of pregnancy and its expression level decreased as gestation progressed (p < 0.05). SNAT3 was also found to be expressed in isolated human primary cytotrophoblast cells and its expression level was decreased by their differentiation into syncytiotrophoblast cells (p < 0.05). Since this regulation is closely related to glutamine synthetase expression, SNAT3 may play a key role in providing glutamine corresponding to glutamine synthetase function in the early period of gestation. This is the first report on the expression of SNAT3 in the placenta in the early stage of pregnancy. (C) 2009 Elsevier Ltd. All rights reserved.

DOI

Liver X receptor regulates expression of MRP2 but not that of MDR1 and BCRP in the liver

Ikumi Chisaki, Masaki Kobayashi, Shirou Itagaki, Takeshi Hirano, Ken Iseki

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES ( ELSEVIER SCIENCE BV ) 1788 ( 11 ) 2396 - 2403 2009.11 [Refereed]

Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily. Multidrug resistance-associated protein 2 (MRP2), multidrug resistance 1 (MDR1) and breast cancer resistance protein (BCRP) play an important role in the efflux of a broad range of endogenous and xenobiotic compounds from hepatocytes. Since the effects of LXR activation on there transporters have been obscure, we investigated the effects of LXR agonists, TO901317 and 25-hydroxycholesterol, on MRP2, MDR1, BCRP expression in HepG2 cells and the rat liver. in an in vitro study, TO901317 increased ABCA1, an LXR target gene, and MRP2 mRNA and protein levels. On the other hand, TO901317 had little effect on MDR1 and BCRP mRNA levels. In an in vivo study, Abca1 and Mrp2 mRNA and protein levels were increased by TO901317. but TO901317 had no effect on Mdr1a and Bcrp mRNA levels in the rat liver. Moreover, TO901317-induced MRP2 mRNA expression was blocked by LXR alpha knockdown. In this study, we demonstrated that LXR activation induced expression of MRP2 but not that of MDR1 and BCRP in hepatocytes. The results suggest that agonists for LXR activate transcription of the MRP2 gene in order to promote excretion of endogenous and xenobiotic compounds from hepatocytes into bile. (C) 2009 Elsevier B.V. All rights reserved.

DOI

Liver X receptor regulates expression of MRP2 but not that of MDR1 and BCRP in the liver.

Chisaki I, Kobayashi M, Itagaki S, Hirano T, Iseki K

Biochimica et biophysica acta ( 11 ) 1788 ( 11 ) 2396 - 2403 2009.11 [Refereed]

Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily. Multidrug resistance-associated protein 2 (MRP2), multidrug resistance 1 (MDR1) and breast cancer resistance protein (BCRP) play an important role in the efflux of a broad range of endogenous and xenobiotic compounds from hepatocytes. Since the effects of LXR activation on there transporters have been obscure, we investigated the effects of LXR agonists, TO901317 and 25-hydroxycholesterol, on MRP2, MDR1, BCRP expression in HepG2 cells and the rat liver. in an in vitro study, TO901317 increased ABCA1, an LXR target gene, and MRP2 mRNA and protein levels. On the other hand, TO901317 had little effect on MDR1 and BCRP mRNA levels. In an in vivo study, Abca1 and Mrp2 mRNA and protein levels were increased by TO901317. but TO901317 had no effect on Mdr1a and Bcrp mRNA levels in the rat liver. Moreover, TO901317-induced MRP2 mRNA expression was blocked by LXRα knockdown. In this study, we demonstrated that LXR activation induced expression of MRP2 but not that of MDR1 and BCRP in hepatocytes. The results suggest that agonists for LXR activate transcription of the MRP2 gene in order to promote excretion of endogenous and xenobiotic compounds from hepatocytes into bile.

DOI CiNii

Liver X receptor regulates expression of MRP2 but not that of MDR1 and BCRP in the liver.

Chisaki I, Kobayashi M, Itagaki S, Hirano T, Iseki K

Biochimica et biophysica acta ( Elsevier B.V. ) 1788 ( 11 ) 2396 - 2403 2009.11 [Refereed]

Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily. Multidrug resistance-associated protein 2 (MRP2), multidrug resistance 1 (MDR1) and breast cancer resistance protein (BCRP) play an important role in the efflux of a broad range of endogenous and xenobiotic compounds from hepatocytes. Since the effects of LXR activation on there transporters have been obscure, we investigated the effects of LXR agonists, TO901317 and 25-hydroxycholesterol, on MRP2, MDR1, BCRP expression in HepG2 cells and the rat liver. in an in vitro study, TO901317 increased ABCA1, an LXR target gene, and MRP2 mRNA and protein levels. On the other hand, TO901317 had little effect on MDR1 and BCRP mRNA levels. In an in vivo study, Abca1 and Mrp2 mRNA and protein levels were increased by TO901317. but TO901317 had no effect on Mdr1a and Bcrp mRNA levels in the rat liver. Moreover, TO901317-induced MRP2 mRNA expression was blocked by LXRα knockdown. In this study, we demonstrated that LXR activation induced expression of MRP2 but not that of MDR1 and BCRP in hepatocytes. The results suggest that agonists for LXR activate transcription of the MRP2 gene in order to promote excretion of endogenous and xenobiotic compounds from hepatocytes into bile.

DOI PubMed CiNii

Intestinal Absorption and Secretion Mechanism of Carboxylate Drugs

Shirou Itagaki

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN ( PHARMACEUTICAL SOC JAPAN ) 129 ( 11 ) 1341 - 1349 2009.11 [Refereed] [Invited]

Oral drug delivery is generally the most desirable means of administration, mainly because of patient. acceptance, convenience in administration Intestinal absorption mechanisms of anionic drugs have been mainly explained by the passive diffusion of nonionized compounds. However, several studies have suggested the involvement of specific transporters in intestinal absorption of weak acids including monocarboxylates. (-)-N-(trans-4-Isopropylcyclohexanecarbonyl)-D-phenylalanine (nateglinide) is a oral hypoglycemic agent possessing a carboxyl group and a peptide-type bond in its structure. Although nateglinide quickly reaches the maximal serum concentration after oral administration, nateglinide itself is not transported by PepT1 or MCT1. We demonstrated that nateglinide transport occurs via a single system that is H+ dependent but is distinct from PepT1 or MCT1. In clinical, patients usually take many kinds of drugs at the same dine. Thus, drug-drug interactions involving transporters can often directly affect the therapeutic safety and efficacy of many drugs However, there have been few studies on food-drug interactions involving transporters. Dietary polyphenols have been widely assumed to be beneficial to human health. Polyphenols are commercially prepared and used as functional foods. We reported that ferulic acid, which is widely used as a functional food, affects the transport of clinical agents. The major dose-limiting toxicity after administration of trinotecan hydrochloride. 7-ethyl-10-(4-[1-piperidino]-1-piperidino)-carbonyloxycamptothecin (CPT-11) is severe diarrhea. We have found that a specific transport system mediates the uptake of active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) across the apical membrane in Caco-2 cells. Baicalin and sulfobromophthatlcin inhibit this transporter. Inhibition of this transporter would be a useful means for reducing late-onset diarrhea.

DOI

Transport by SLC5A8 With Subsequent Inhibition of Histone Deacetylase 1 (HDAC1) and HDAC3 Underlies the Antitumor Activity of 3-bromopyruvate

Muthusamy Thangaraju, Senthil K. Karunakaran, Shiro Itagaki, Elangovan Gopal, Selvakumar Elangovan, Puttur D. Prasad, Vadivel Ganapathy

CANCER ( JOHN WILEY & SONS INC ) 115 ( 20 ) 4655 - 4666 2009.10 [Refereed]

BACKGROUND: 3-Bromopyruvate is an alkylating agent with antitumor activity. It is currently believed that blockade of adenosine triphosphate production from glycolysis and mitochondria is the primary mechanism responsible for this antitumor effect. The current studies uncovered a new and novel mechanism for the antitumor activity of 3-bromopyruvate. METHODS: The transport of 3-bromopyruvate by sodium-coupled monocarboxylate transporter SMCT1 (SLC5A8), a tumor suppressor and a sodium (Na(+))-coupled, electrogenic transporter for short-chain monocarboxylates, was studied using a mammalian cell expression and the Xenopus laevis oocyte expression systems. The effect of 3-bromopyruvate on histone deacetylases (HDACs) was monitored using the lysate of the human breast cancer cell line MCF7 and human recombinant HDAC isoforms as the enzyme sources. Cell viability was monitored by fluorescence-activated cell-sorting analysis and colony-formation assay. The acetylation status of histone H4 was evaluated by Western blot analysis. RESULTS: 3-Bromopyruvate is a transportable substrate for SLC5A8, and that transport process is Na(+)-coupled and electrogenic. MCF7 cells did not express SLC5A8 and were not affected by 3-bromopyruvate. However, when transfected with SLC5A8 or treated with inhibitors of DNA methylation, these cells underwent apoptosis in the presence of 3-bromopyruvate. This cell death was associated with the inhibition of HDAC1/HDAC3. Studies with different isoforms of human recombinant HDACs identified HDAC1 and HDAC3 as the targets for 3-bromopyruvate. CONCLUSIONS: 3-Bromopyruvate was transported into cells actively through the tumor suppressor SLC5A8, and the process was energized by an electrochemical Na(+) gradient. Ectopic expression of the transporter in MCF7 cells led to apoptosis, and the mechanism involved the inhibition of HDAC1/HDAC3. Cancer 2009;115:4655-66. (C) 2009 American Cancer Society.

DOI PubMed

Response of the ABCG2 promoter in T47D cells and BeWo cells to sex hormone treatment

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

MOLECULAR BIOLOGY REPORTS ( SPRINGER ) 36 ( 7 ) 1889 - 1896 2009.09 [Refereed]

The aim of this study was to elucidate the effects of sex hormones on activity of the ABCG2 promoter in different cell lines. T47D cells and BeWo cells were used as models for ABCG2-expressing cell lines, and luciferase assays using ABCG2 promoter-luciferase constructs were performed. It was shown that progesterone increased the response of the ABCG2 promoter in T47D cells but not in BeWo cells. On the other hand, estradiol had no effect on response of the ABCG2 promoter in either cell line. However, response of the ABCG2 promoter was enhanced by overexpression of ER alpha in both T47D cells and BeWo cells. T47D cells had higher sensitivity to ER alpha than did BeWo cells. Furthermore, it was shown that the inductive effect of progesterone on the ABCG2 promoter was inhibited by addition of RU486 or mithramycin A. Therefore, it was thought that the ABCG2 promoter responded to stimulation of the progesterone receptor (PR)-Sp1 pathway in T47D cells. Furthermore, progesterone suppressed the response of the ABCG2 promoter by changing the expression levels of PR-A and PR-B in BeWo cells. These findings suggested that there are differences between cell lines in the regulation mechanism of ABCG2 expression by sex hormone treatment.

DOI

Food-Drug Interaction Involving the Ferulic Acid/H plus Transporter

Shirou Itagaki, Yoko Kobayashi, Yoshitaka Saito, Masaki Kobayashi, Takeshi Hirano, Ken Iseki

DRUG METABOLISM REVIEWS ( TAYLOR & FRANCIS INC ) 41 60 - 60 2009.08 [Refereed]

Contribution of organic anion transporting polypeptide OATP2B1 to amiodarone accumulation in lung epithelial cells

Satoru Seki, Masaki Kobayashi, Shirou Itagaki, Takeshi Hirano, Ken Iseki

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES ( ELSEVIER SCIENCE BV ) 1788 ( 5 ) 911 - 917 2009.05 [Refereed]

The accumulation mechanisms of amiodarone (AMD) involving transporters in lung alveolar epithelial type 11 cells were studied. The uptake of AMD was examined using human alveolar epithelia I-derived cell line A549 as a model. AMD was transported by the carrier-mediated system, and the apparent K-m and V-max values were 66.8 +/- 30.3 mu M and 49.7 +/- 9.7 nmol/mg protein/5 min, respectively. The uptake of AMD by A549 cells was Na+-independent and was inhibited by substrates of human organic anion transporting polypeptide (OATP). The inhibition profiles were similar to the inhibitory effects of several compounds on OATP2B1 -mediated E-3-S transport, and RT-PCR analysis showed mRNA expression of OATP2B1 and 1B3 in A549 cells. SiRNAs targeted to the OATP2B1 gene decreased the OATP2B1 mRNA expression level in A549 cells up to about 50% and reduced the uptake of AMD up to about 40%. These results indicate that AMD uptake mediated by carriers, including OATP2B1, might lead to accumulation of AMD in the lung and AMD-induced pulmonary toxicity (AIPT). (C) 2009 Elsevier B.V. All rights reserved.

DOI

In vitro and in vivo antioxidant properties of ferulic acid: A comparative study with other natural oxidation inhibitors

Shirou Itagaki, Toshirnitsu Kurokawa, Chie Nakata, Yoshitaka Saito, Setsu Oikawa, Masaki Kobayashi, Takeshi Hirano, Ken Iseki

FOOD CHEMISTRY ( ELSEVIER SCI LTD ) 114 ( 2 ) 466 - 471 2009.05 [Refereed]

Ferulic acid exhibits a wide range of therapeutic effects that are attributed to its potent antioxidant capacity. However, in vitro antioxidant properties of ferulic acid have not been elucidated in detail. Evidence that polyphenols, including ferulic acid, act as antioxidants in vivo is also limited. In order to elucidate in more detail the scientific background of antioxidant activities of ferulic acid, we carried out in vitro and in vivo experiments. We focused on superoxide anion scavenging activity, xanthine oxidase inhibition activity, and chain-breaking activity. The combined antioxidant activity from radical scavenging and xanthine oxidase inhibition of ferulic acid was much weaker than that of (-)-epigallocatechin gallate (EGCG) and ascorbic acid. On the other hand, EGCG, ascorbic acid and ferulic acid exhibited chain-breaking activity and prevented ischaemia-reperfusion-associated intestinal injury. Chain-breaking activity may play a contributory role in the protective effect of ferulic acid on oxidative injury in humans and in in vivo studies. (C) 2008 Elsevier Ltd. All rights reserved.

DOI CiNii

Contribution of organic anion transporting polypeptide OATP2B1 to amiodarone accumulation in lung epithelial cells.

Seki S, Kobayashi M, Itagaki S, Hirano T, Iseki K

Biochimica et biophysica acta ( 5 ) 1788 ( 5 ) 911 - 917 2009.05 [Refereed] [International journal]

The accumulation mechanisms of amiodarone (AMD) involving transporters in lung alveolar epithelial type II cells were studied. The uptake of AMD was examined using human alveolar epithelial-derived cell line A549 as a model. AMD was transported by the carrier-mediated system, and the apparent K(m) and V(max) values were 66.8+/-30.3 muM and 49.7+/-9.7 nmol/mg protein/5 min, respectively. The uptake of AMD by A549 cells was Na(+)-independent and was inhibited by substrates of human organic anion transporting polypeptide (OATP). The inhibition profiles were similar to the inhibitory effects of several compounds on OATP2B1-mediated E-3-S transport, and RT-PCR analysis showed mRNA expression of OATP2B1 and 1B3 in A549 cells. SiRNAs targeted to the OATP2B1 gene decreased the OATP2B1 mRNA expression level in A549 cells up to about 50% and reduced the uptake of AMD up to about 40%. These results indicate that AMD uptake mediated by carriers, including OATP2B1, might lead to accumulation of AMD in the lung and AMD-induced pulmonary toxicity (AIPT).

DOI PubMed

Search for Antioxidative Compounds Capable of Preventing Stomatitis Induced by Chemotherapy

TAKANO Rumi, HIRANO Takeshi, NAKATA Chie, KASASHI Kumiko, SUGAWARA Mituru, KOBAYASHI Masaki, ITAGAKI Shirou, ISEKI Ken

Iryo Yakugaku (Japanese Journal of Pharmaceutical Health Care and Sciences) ( Japanese Society of Pharmaceutical Health Care and Sciences ) 35 ( 4 ) 247 - 253 2009.04 [Refereed]

In the clinical setting,the frequency of adverse effects is directly linked to treatment results but during chemotherapy,they are unavoidable.One of them is stomatitis,a distressing toxic effect.In patients receiving myelosuppressive chemotherapy for solid tumors,the prevalence of stomatitis is 5-40% while its prevalence in patients receiving stem cell transplantation is 75-100%.Stomatitis-associated pain is the main source of cancer treatment-related pain and in some patients it is so severe that narcotic analgesia is required.
Oxidative stress may also be associated with chemotherapy-induced stomatitis and a mouthwash containing allopurinol,rebamipide and camostat for the removal of free radicals is widely used for the prevention and treatment of such stomatitis.However,such agents are not covered by the Japanese National Health Insurance scheme and there is little scientific evidence for their antioxidative properties and effectiveness in the prevention and treatment of chemotherapy-induced stomatitis.We therefore attempted to establish a more useful mouthwash.In the first part of this study,we investigated the antioxidant properties of various compounds.We then used H₂O₂-induced HO-1-N-1 cell injury as a model of stomatitis and found that epigallocatechin gallate had a strong preventive effect against it.Epigallocatechin gallate would therefore be a good component of a mouthwash for the prevention of stomatitis in patients undergoing cancer chemotherapy.

DOI CiNii

薬物動態因子の発現変動を反映した肝切除患者に対する薬物治療の最適化に関する研究.

板垣史郎, 井関健

医療の広場 49 18 - 20 2009.04 [Invited]

Sodium-coupled transport of the short chain fatty acid butyrate by SLC5A8 and its relevance to colon cancer - Discussion

Stephen A. McClave, Gail Cresci

JOURNAL OF GASTROINTESTINAL SURGERY ( SPRINGER ) 12 ( 10 ) 1781 - 1782 2008.10 [Refereed]

DOI PubMed

Placental folate transport during pregnancy

Satoru Yasuda, Satoko Hasui, Chiaki Yamamoto, Chihiro Yoshioka, Masaki Kobayashi, Shirou Itagaki, Takeshi Hirano, Ken Iseki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY ( TAYLOR & FRANCIS LTD ) 72 ( 9 ) 2277 - 2284 2008.09 [Refereed]

The aim of this study was to elucidate the mechanism of folate transport in the placenta over the course of pregnancy. We found that folate receptor of (FR alpha) and reduced folate carrier (RFC) localized on the apical side of human placental villi. Since folate binding to placental brush-border membrane vesicles (BBMVs) was strongly inhibited by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, it is possible that FR alpha, a glycosyl phosphatidylinositol linked glycoprotein, is a candidate for folate uptake from maternal blood to the placenta. Moreover, additional inhibitory effects of thiamine pyrophosphate (TPP) and hemin on folate uptake after PI-PLC treatment suggested that not only FRa but also RFC and heme carrier protein I (HCPI) are involved in the folate transport mechanism in the human placenta. It was also found that accumulation of folate after intravenous injection increased with the progress of gestation in the rat placenta and the fetus. Furthermore, increases in the expression levels of mRNA of rFR alpha, rRFC, and rHCP1 in the rat placenta during pregnancy were observed. These findings suggest that FR alpha, RFC, and HCP1 are important carriers of folate in the placenta during pregnancy. The results of this study suggest that increases in the expression levels of FRa, REC, and HCP1 in the placenta play an important role in the response to increased need for folate for the placenta and fetus during development with the progress of gestation.

DOI CiNii

Interaction of tryptophan derivatives with SLC6A14 (ATB(0,+)) reveals the potential of the transporter as a drug target for cancer chemotherapy

Senthil Karunakaran, Nagavedi S. Umapathy, Muthusarny Thangaraju, Takahiro Hatanaka, Shiro Itagaki, David H. Munn, Puttur D. Prasad, Vadivel Ganapathy

BIOCHEMICAL JOURNAL ( PORTLAND PRESS LTD ) 414 ( 3 ) 343 - 355 2008.09 [Refereed]

ATB(0,+) [SLC6A14 (Solute carrier family 6 member 14)] is an Na(+)/Cl(-)-coupled amino acid transporter whose expression is up-regulated in cancer. 1-Methyltryptophan is an inducer of immune surveillance against tumour Cells through its ability to inhibit indoleamine dioxygenase. fit the present Study, we investigated the role of ATB(0,+) in the uptake of 1-methyltryptophan as a potential mechanism for entry of this putative anticancer-drug into tumour cells. These Studies show that 1-methyltryptophan is a transportable substrate for ATB(0,+). The transport process is Na(+)/Cl(-)-dependent with an Na(+)/Cl(-)/1-methyltryptophan stoichiometry of 2:1:1. Evaluation of other derivatives of tryptophan has led to identification of (alpha-methyltryptophan as a blocker, not a transportable substrate, for ATB(0,+). ATB(0,+) can transport 18 of the 20 proteinogenic amino acids. alpha-Methyltryptophan blocks the transport function of ATB(0,+) with an IC(50) value of similar to 250 mu M under conditions simulating normal plasma concentrations of all these 18 amino acids. These results Suggest that a-methyltryptophan may induce amino acid deprivation in cells which depend on the transporter for their amino acid nutrition. Screening of several mammary epithelial cell lines shows that ATB(0,+) is expressed robustly in some cancer cell lines, but not in all; in contrast, non-malignant cell lines do not express the transporter. Treatment of ATB(0,+)-positive tumour cells with a-methyltryptophan leads to Suppression of their colony-forming ability, whereas ATB(0,+)-negative cell lines are not affected. The blockade of ATB(0,+) in these cells with alpha-methyltryptophan is associated with cell cycle arrest. These Studies reveal the potential of ATB(0,+) as a drug target for cancer chemotherapy.

DOI PubMed CiNii

Interaction of coenzyme Q10 with the intestinal drug transporter P-glycoprotein

Shirou Itagaki, Akiko Ochiai, Masaki Kobayashi, Mitsuru Sugawara, Takeshi Hiran, Ken Iseki

JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY ( AMER CHEMICAL SOC ) 56 ( 16 ) 6923 - 6927 2008.08 [Refereed]

In clinical trials, patients usually take many kinds of drugs at the same time. Thus, drug-drug interactions can often directly affect the therapeutic safety and efficacy of many drugs. Oral delivery is the most desirable means of drug administration. Changes in the activity of drug transporters may substantially influence the absorption of administered drugs from the intestine. However, there have been a few studies on food-drug interactions involving transporters. It is important to be aware of the potential of food-drug interactions and to act in order to prevent undesirable and harmful clinical consequences. Coenzyme Q10 (CoQ10) is very widely consumed by humans as a food supplement because of its recognition by the public as an important nutrient in supporting human health. Since intestinal efflux transporter P-glycoprotein (P-gp) is one of the major factors in drug-drug interactions, we focused on this transporter. We report here for the first time that CoQ10, which is widely used as a food supplement, affects the transport activity of P-gp.

DOI PubMed CiNii

Amiodarone increases the accumulation of DEA in a human alveolar epithelium-derived cell line

Satoru Seki, Shirou Itagaki, Masaki Kobayashi, Takeshi Hirano, Ken Iseki

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 31 ( 7 ) 1449 - 1452 2008.07 [Refereed]

Amiodarone (AMD)-induced pulmonary toxicity (AIPT) is the most life-threatening side-effect of AMD treatment. N-Monodesethylamiodarone (DEA), an active metabolite of AMD, also exhibits cytotoxicity and tends to accumulate in the lung more intensively than AMD. In this study, we characterized the mechanism of DEA accumulation using A549 cells as a model of the alveolar epithelium. Typical ATP-depletion compounds caused an approximately 30% increase in the accumulation of DEA in A549 cells, although these effects were less than those in Caco-2 cells. Triiodothyronine (T3), which exhibited an inhibitory effect on DEA efflux in Caco-2 cells, did not affect the accumulation of DEA in A549 cells. On the other hand, 100uM AMD caused an approximately 200% increase in DEA content in A549 cells, although AMD accumulation was not affected by 100 um DEA. Since the reducing effect of AMD on cellular ATP levels and that of FCCP were similar, the mechanism by which DEA accumulation is increased by AMD might be different from the ATP-dependent DEA efflux mechanism. The decrease in cell viability by DEA in the presence of AMD (ICS value of DEA for A549 cell viability: 25.4 2.4 ELM) was more pronounced than that by DEA alone (IC5o value: 11.5 3.0 um). This further DEA accumulation by AMD might be a factor responsible for the greater accumulation of DEA than that of AMD in the lung in long-term AMD-treated patients.

DOI CiNii

Alteration of Mrp2 and P-gp expression, including expression in remote organs, after intestinal ischemia-reperfusion

Jiro Ogura, Masaki Kobayashi, Shirou Itagaki, Takeshi Hirano, Ken Iseki

LIFE SCIENCES ( PERGAMON-ELSEVIER SCIENCE LTD ) 82 ( 25-26 ) 1242 - 1248 2008.06 [Refereed]

The present study was carried out in order to identify the changes in expression of multidrug resistance-associated protein (Mrp) 2 and P-glycoprotein (P-gp) in the intestine and remote organs after intestinal ischemia-reperfusion (I/R). Mrp2 expression in the jejunum and liver was decreased at 6 h after I/R. This decrease in Mrp2 expression was associated with an increase in the serum level of IL-6. These results suggest that the decreased Mrp2 expression after intestinal I/R was regulated by IL-6. The expression level of mdr1a in the ileum, which encodes P-gp, was decreased at 6 and 24 h after I/R, and the expression level of mdr1b, also encodes P-gp, was not altered at any time. P-gp protein expression in the ileum was decreased at 6 h after I/R. In the liver, mdr1a expression was decreased at 6 h after I/R, but mdr1b expression was increased at 6 h after I/R. Pgp protein was not altered at any time. In the kidney, mdr1a expression was decreased at 24 h after I/R, but mdr1b expression was not altered at any time. P-gp protein expression in the kidney was decreased at 24 h after I/R, as was mdr1a expression. These results suggest that P-gp expression after intestinal I/R differs in each organ. This is the first report to provide evidence that expression levels of transporters in remote organs are altered intestinal after I/R. (c) 2008 Elsevier Inc. All rights reserved.

DOI PubMed CiNii

Post-transcriptional regulation of breast cancer resistance protein after intestinal ischemia-reperfusion

Jiro Ogura, Masaki Kobayashi, Shirou Itagaki, Takeshi Hirano, Ken Iseki

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 31 ( 5 ) 1032 - 1035 2008.05 [Refereed]

Breast cancer resistance protein (BCRP), the product of the ABCG2 gene, is a recently identified ATP binding cassette half-transporter. BCRP is expressed in a variety of tumor cells and many normal human tissues. In the small intestine, BCRP can limit the influx and facilitate the efflux to prevent intracellular accumulation of BCRP substrates. Ischemia-reperfusion (I/R) induces the release of reactive oxygen species, and organs are severely damaged by I/R. It has been shown that the expression of transporters was altered in the organ after I/R. The present study was undertaken to clarify the expression of BCRP after intestinal I/R. We showed that the expression level of Bcrp was significantly decreased at I h after I/R. Bcrp mRNA level was not altered at I h after I/R. These results suggest that Bcrp expression was regulated by a post-transcriptional regulation mechanism after intestinal I/R. Bcrp mRNA level was increased at 24 h after I/R, and the expression level of Bcrp protein was of the same level or slightly increased compared with sham operated-rats. Bcrp was slightly located at the intestinal membrane at 24 h after intestinal I/R. These results suggested that Bcrp was not translocated to the intestinal membrane after intestinal I/R. There is little information on post-transcriptional regulation compared with information on transcriptional regulation. In this study, it was shown that Bcrp expression is regulated by post-transcriptional regulation after intestinal I/R. These results of this study may provide important information for further studies aimed at revealing the biological function of Bcrp.

DOI CiNii

Association between risk of myopathy and cholesterol-lowering effect: A comparison of all statins

Masaki Kobayashi, Ikumi Chisaki, Katsuya Narumi, Kazuhiro Hidaka, Toshiki Kagawa, Shirou Itagaki, Takeshi Hirano, Ken Iseki

LIFE SCIENCES ( PERGAMON-ELSEVIER SCIENCE LTD ) 82 ( 17-18 ) 969 - 975 2008.04 [Refereed]

In the present study, we examined the mechanisms underlying the cytotoxicity of pitavastatin, a new statin, and we compared the in vitro potencies of muscle cytotoxicity using a prototypic embryonal rhabdomyosarcoma cell line (RD cells), a typical side effect of statins and compared the cholesterol-lowering effects of statins using Hep G2 hepatoma cells. Pitavastatin reduced the number of viable cells and caused caspase-9 and -3/7 activation in a time- and concentration-dependent manner. The comparison of cytotoxities of statins showed that statins significantly reduced cell viability and markedly enhanced activity of caspase-3/7 in concentration-dependent manner. On the other hand, the effects of hydrophilic statins, pravastatin, rosuvastatin were very weak. The rank order of cytotoxicity was cerivastatin > sinavastatin acid> fluvastatin > atorvastatin > lovastatin acid > pitavastatin >> rosuvastatin, pravastatin. Statin-induced cytotoxicity is associated with these partition coefficients. On the other hand, the cholesterol-lowering effect of statins did not correlate with these partition coefficients and cytotoxicity. Thus, it is necessary to consider the association between risk of myopathy and cholesterol-lowering effect of a statin for precise use of statins. (c) 2008 Elsevier Inc. All rights reserved.

DOI PubMed

Sodium-coupled monocarboxylate transporters in normal tissues and in cancer

Vadivel Ganapathy, Muthusamy Thangaraju, Elangovan Gopal, Pamela M. Martin, Shiro Itagaki, Seiji Miyauchi, Puttur D. Prasad

AAPS JOURNAL ( SPRINGER ) 10 ( 1 ) 193 - 199 2008.03 [Refereed] [Invited]

SLC5A8 and SLC5A12 are sodium-coupled monocarboxylate transporters (SMCTs), the former being a high-affinity type and the latter a low-affinity type. Both transport a variety of monocarboxylates in a Na+-coupled manner. They are expressed in the gastrointestinal tract, kidney, thyroid, brain, and retina. SLC5A8 is localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to neurons and the retinal pigment epithelium. The physiologic functions of SLC5A8 include absorption of short-chain fatty acids in the colon and small intestine, reabsorption of lactate and pyruvate in the kidney, and cellular uptake of lactate and ketone bodies in neurons. It also transports the B-complex vitamin nicotinate. SLC5A12 is also localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to astrocytes and Muller cells. SLC5A8 also functions as a tumor suppressor; its expression is silenced in tumors of colon, thyroid, stomach, kidney, and brain. The tumor-suppressive function is related to its ability to mediate concentrative uptake of butyrate, propionate, and pyruvate, all of which are inhibitors of histone deacetylases. SLC5A8 can also transport a variety of pharmacologically relevant monocarboxylates, including salicylates, benzoate, and gamma-hydroxybutyrate. Non-steroidal anti-inflammatory drugs such as ibuprofen, ketoprofen, and fenoprofen, also interact with SLC5A8. These drugs are not transportable substrates for SLC5A8, but instead function as blockers of the transporter. Relatively less is known on the role of SLC5A12 in drug transport.

DOI PubMed

A new method for determination of both thalidomide enantiomers using HPLC systems

Kaname Sgmbongi, Masanori Tanaka, Kelsuke Sakurada, Masakl Kobayasfi, Shlrou Itagaki, Takeshi Hirano, Ken Iseki

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 31 ( 3 ) 497 - 500 2008.03 [Refereed]

Although thalidomide was withdrawn due to teratogenicity and neuropathy, there is now growing clinical interest in this compound because of its immunomodulatory and anti-angiogenic properties. In 1998, thalidomide was approved by the U.S. Food and Drug Administration for the treatment of erythema nodosum leprosum (ENL), an inflammatory complication of Hansen's disease, through a restricted-use program. Thalidomide was approved for the treatment of relapsed or refractory multiple myeloma (MM) as an orphan drug in Japan. Direct deproteinization method was shown to be useful for quantitation of enantioselective thalidomide blood level. Stabilized blood was deproteinized with methanol and 2 m trichloroacetic acid. The supernatant was injected onto reverse-phase column (CHIRALPAK AD-RH). The mobile phase consisted of 10% acetonitrile, 70% methanol and 20% 0.025 M citrate buffer (pH 3.0), and the flow rate was 0.5 ml/min. Wavelength of detection was 220 nm. (-)-(S)-thalidomide and (+)-(R)-thalidomide were separated at 13.5 min and 17.6 min, respectively. The accuracy of this method was almost the same as that of the measurement technique with extraction and concentration. In clinical practice, MM patients usually take many kinds of drugs at the same time. Actually, this patient takes a lot of drugs with thalidomide. However, we found no interference of these drugs and thalidomide on the chromatogram. This simple and reliable HPLC determination method for both enantiomers of thalidomide is thought to be very useful for thalidomide studies.

DOI CiNii

The inhibitory effects of fluoroquinolones on L-carnitine transport in placental cell line BeWo

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

INTERNATIONAL JOURNAL OF PHARMACEUTICS ( ELSEVIER SCIENCE BV ) 351 ( 1-2 ) 113 - 118 2008.03 [Refereed]

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 members of the OCTN family play an important role in L-carnitine transport in the placenta. Investigation of drug-drug or drug-nutrient interaction in the placenta is important for establishment of safety drug medication during pregnancy. The aim of this study was to determine the effects of fluoroquinolones, inhibitors of OCTN2, on L-carnitine transport in the placenta which is known to have a high expression level of OCTN2. We investigated the inhibitory effect of five fluoroquinolones, ciprofloxacin (CPFX), gatifloxacin (GFLX), ofloxacin (OFLX), levofloxacin (LVFX) and grepafloxacin (GPFX), on L-carnitine transport mediated by OCTN2 in placental cell line BeWo cells. We found that all of the fluoroquinolones inhibited L-carnitine transport, GPFX being the strongest inhibitor. We also found that the inhibitory effects of LVFX and GPFX depended on their existence ratio of zwitterionic forms as, we reported previously. Furthermore, we elucidated the LVFX transport mechanism in BeWo cells. LVFX was transported actively by transporters. However, we found that LVFX transport was Na+-independent and L-carnitine had no inhibitory effect on LVFX transport, suggesting that LVFX acts as inhibitor of OCTN2, not as a substrate for OCTN2. (C) 2007 Elsevier B.V. All rights reserved.

DOI PubMed

The mechanism of carrier-mediated transport of folates in BeWo cells: The involvement of heme carrier protein 1 in placental folate transport

Satoru Yasuda, Satoko Hasui, Masaki Kobayashi, Shirou Itagaki, Takeshi Hirano, Ken Iseki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY ( TAYLOR & FRANCIS LTD ) 72 ( 2 ) 329 - 334 2008.02 [Refereed]

The aim of this study was to elucidate the mechanism of folate transport in the placenta. A study of folate was carried out to determine which carriers transport folates in the human choriocarcinoma cell line BeWo, a model cell line for the placenta. We investigated the effects of buffer pH and various compounds on folate uptake. In the first part of the study, the expression levels of the mRNA of the folate receptor alpha (FR alpha), the reduced folate carrier (RFC), and heme carrier protein 1 (HCP1) were determined in BeWo cells by RT-PCR analysis. Folate uptake into BeWo cells was greater under an acidic buffer condition than under a neutral one. Structure analogs of folates inhibited folate uptake under all buffer pH conditions, but anion drugs (e.g., pravastatin) inhibited folate uptake only under an acidic buffer condition. Although thiamine pyrophosphate (TPP), a substrate of RFC, had no effect on folate uptake, hemin (a weak inhibitor of folate uptake via HCPI) decreased folate uptake to about 80% of the control level under an acidic buffer condition. Furthermore, kinetic analysis showed that hemin inhibited the low-affinity phase of folate uptake under an acidic buffer condition. We conclude that pH-dependent folate uptake in BeWo cells is mediated by at least two carriers. RFC is not involved in folate uptake, but FR alpha (high affinity phase) and HCP1 (low affinity phase) transport folate in BeWo cells.

DOI CiNii

Contribution of multidrug resistance-associated protein 2 to secretory intestinal transport of organic anions

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

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 31 ( 1 ) 146 - 148 2008.01 [Refereed]

Various mechanisms can influence the intestinal absorption and oral bioavailability of drugs. The barrier effects of efflux transporters may be one of the critical factors limiting the bioavailability of certain drugs. It has been reported that multidrug resistance-associated protein 2 (Mrp2) is expressed in the mucosal membrane of the epithelium of the small intestine and secretes various drugs into the jejunum lumen. However, it is possible that total intestinal secretion of Mrp2 substrates is accounted for the contribution of Mrp2 and other transporter(s) to the intestinal secretion of Mrp2 substrates. In this study, we found that phenolsulfonphthalein and pravastatin, both Mrp2 substrates, are transported by different transport systems in the intestine. These results suggest that contribution of transporters to the drug transport may be a critical factor affecting drug disposition and drug-drug interaction. In addition to evaluating the substrate specificity of a transporter, it is important to be aware of the contribution of a transporter to drug disposition.

DOI CiNii

Functional analysis of phenolsulfonphthalein transport system in Long-Evans Cinnamon rats.

Itagaki S, Chiba M, Kobayashi M, Sugawara M, Kobayashi M, Hirano T, Iseki K

Biochimica et biophysica acta ( 1 ) 1778 ( 1 ) 270 - 275 2008.01 [Refereed] [International journal]

It has been reported that the transport function for organic anions on the kidney is maintained in multidrug resistance-associated protein 2 (Mrp2)-deficient rats. Different from Mrp2-deficient rats, Long-Evans Cinnamon (LEC) rats have impaired urinary excretion of Mrp2-substrate, phenolsulfonphthalein (PSP). PSP is transported by the potential-sensitive urate transport system in rat brush-border membranes. We analyzed the function of PSP transport system in LEC rats. Unlike Long-Evans Agouti (LEA) rats, the initial uptake of PSP and urate into the renal brush-border membrane vesicles of LEC rats were not significantly enhanced in the presence of positive intravesicular potential, suggesting that the potential-sensitive urate transport system is impaired in LEC rats. LEC rats should be useful for elucidating the potential-sensitive urate transport system in rats at the molecular level.

DOI PubMed

Functional analysis of phenolsulfonphthalein transport system in Long-Evans Cinnamon rats

Shirou Itagaki, Makoto Chiba, Masaki Kobayashi, Mitsuru Sugawara, Michlya Kobayashi, Takeshi Hirano, Ken Iseki

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES ( ELSEVIER SCIENCE BV ) 1778 ( 1 ) 270 - 275 2008.01 [Refereed]

It has been reported that the transport function for organic anions on the kidney is maintained in multidrug resistance-associated protein 2 (Mrp2)-deficient rats. Different from Mrp2-deficient rats, Long-Evans Cinnamon (LEC) rats have impaired urinary excretion of Mrp2-substrate, phenolsulfonphthalein (PSP). PSP is transported by the potential-sensitive urate transport system in Tat brush-border membranes. We analyzed the function of PSP transport system in LEC rats. Unlike Long-Evans Agouti (LEA) rats, the initial uptake of PSP and urate into the renal brush-border membrane vesicles of LEC rats were not significantly enhanced in the presence of positive intravesicular potential, suggesting that the potential-sensitive urate transport system is impaired in LEC rats. LEC rats should be useful for elucidating the potential-sensitive urate transport system in rats at the molecular level. (c) 2007 Elsevier B.V. All rights reserved.

DOI

Functional characterization of human monocarboxylate transporter 9 (SLC16A9)

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

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN ( PHARMACEUTICAL SOC JAPAN ) 128 47 - 48 2008 [Refereed]

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

Effects of sex hormones on regulation of ABCG2 expression in the placental cell line BeWo.

S Yasuda, S Itagaki, T Hirano, K Iseki

JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES ( CANADIAN SOC PHARMACEUTICAL SCIENCES ) 9 ( 1 ) 133 - 139 2006.01 [Refereed]

PURPOSE: The aim of this study was to elucidate the effects of sex hormones that are secreted during gestation from the placenta on ABCG2 mRNA and protein expression levels by using the placental cell line BeWo. METHODS: We investigated the effects of estrogens ( estrone, 17-beta-estradiol and estriol) on the expression level of ABCG2 mRNA by RT-PCR. The expression level of ABCG2 protein was analyzed by Western blot analysis. We also investigated the localization of ABCG2 in BeWo cells by Western blot analysis of the plasma membrane fraction and by immunohistochemistry. RESULTS: It was found that all estrogens induce the expression of ABCG2 mRNA in a concentration-dependent manner. Furthermore, Western blot analysis showed that 17-beta-estradiol induces the expression of ABCG2 protein. Western blot analysis of the plasma membrane fraction and immunohistochemistry showed that ABCG2 localized on only the apical side of BeWo cells and that 17-beta-estradiol had no effect on the localization of ABCG2. In addition, progesterone suppressed the induction of ABCG2 expression by 17-beta-estradiol at 1-10 mu M. CONCLUSION: The expression of ABCG2 in the placenta is regulated by estrogen and progesterone during gestation.

PubMed

Cloning and functional identification of slc5a12 as a sodium-coupled low-affinity transporter for monocarboxylates (SMCT2)

Srinivas, SR, E Gopal, L Zhuang, S Itagaki, PM Martin, YJ Fei, Ganapathy, V, PD Prasad

BIOCHEMICAL JOURNAL ( PORTLAND PRESS LTD ) 392 655 - 664 2005.12 [Refereed]

We report in the present paper, on the isolation and functional characterization of slc5a12, the twelfth member of the SLC5 gene family, from mouse kidney. The slc5a12 cDNA codes for a protein of 619 amino acids. Heterologous expression of slc5a12 cDNA in mammalian cells induces Na+-dependent transport of lactate and nicotinate. Several other short-chain monocarboxylates compete with nicotinate for the cDNA-induced transport process. Expression of slc5a12 in Xenopus oocytes induces electrogenic and Na+-dependent transport of lactate, nicotinate, propionate and butyrate. The substrate specificity of slc5a12 is similar to that of slc5a8, an Na+-coupled transporter for monocarboxylates. However, the substrate affinities of s1c5a12 were much lower than those of slc5a8. slc5a12 mRNA is expressed in kidney, small intestine and skeletal muscle. In situ hybridization with sagittal sections of mouse kidney showed predominant expression of slc5a12 in the outer cortex. This is in contrast with slc5a8, which is expressed in the cortex as well as in the medulla. The physiological function of slc5a12 in the kidney is likely to mediate the reabsorption of lactate. In the intestinal tract, slc5a 12 is expressed in the proximal parts, whereas slc5a8 is expressed in the distal parts. The expression of slc5a12 in the proximal parts of the intestinal tract, where there is minimal bacterial colonization, suggests that the physiological function of slc5a12 is not to mediate the absorption of short-chain monocarboxylates derived from bacterial fermentation but rather to mediate the absorption of diet-derived short-chain monocarboxylates. Based on the functional and structural similarities between slc5a8 and slc5a12, we suggest that the two transporters be designated as SMCT1 (sodium-coupled monocarboxylate transporter 1) and SMCT2 respectively.

DOI PubMed

Transepithelial transport of telmisartan in Caco-2 monolayers

Y Goto, S Itagaki, S Umeda, M Kobayashi, T Hirano, K Iseki, K Tadano

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 28 ( 12 ) 2235 - 2239 2005.12 [Refereed]

Telmisartan is the most recently marketed angiotensin 11 type I receptor antagonist. Drug-drug interactions involving transporters can directly affect the therapeutic safety and efficacy of many important drugs. In clinical practice, telmisartan is coadministered with many kinds of drugs. However, little is known about the contribution of transporters to the intestinal transport of telmisartan. The aim of this study was to determine the transport mechanism of telmisartan across intestinal epithelial cells. In the presence of an inwardly directed proton gradient, the apical-to-basal transport of telmisartan was greater than basal-to-apical transport. Thus, we focused on the uptake mechanism of telmisartan across brush-border membranes. The uptake of telmisartan by Caco-2 cells was shown to be energy- and proton-dependent. Although some monocarboxylates inhibited the uptake of telmisartan, L-lactic acid, which is a typical substrate of the monocarboxylate transporter (MCT) 1-MCT4, did nor affect the uptake of telmisartan. Preloading of acetic acid enhanced the uptake of telmisartan, showing a trans-stimulation effect. These results suggest that the carrier-mediated transport system is involved in the uptake of telmisartan by Caco-2 cells and that the apical-localized transport system is similar to MCTs, but not MCT1-MCT4. It is possible that telmisartan reduce the absorption of coadministered drugs by sharing the MCTs. Since MCTs have an important role in the intestinal absorption of pharmacologically active compounds, it is important to be aware of the potential of telmisartan-drug interactions involving MCTs and to act in order to prevent undesirable and harmful consequences.

DOI CiNii

Expression level of ABCG2 in the placenta decreases from the mid stage to the end of gestation

S Yasuda, S Itagaki, T Hirano, K Iseki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY ( TAYLOR & FRANCIS LTD ) 69 ( 10 ) 1871 - 1876 2005.10 [Refereed]

The aim of this study was to elucidate the expression pattern of ABCG2 in the placenta from the mid stage to the end of gestation. rABCG2 expression was investigated in rats on the 14th gestation day (gd) and the 20th gd. Expression of the rABCG2 gene and expression of rABCG2 protein in the placenta were detected on gd 14 by RT-PCR and Western blot analysis respectively. The expression level of rABCG2 on gd 20 was less than that on gd 14. We investigated whether progesterone, secreted from the placenta, regulates the expression of ABCG2 in BeWo cells. Expression levels of the ABCG2 gene and protein in BeWo cells were decreased by progesterone treatment. We conclude that progesterone plays a role in reduction in the expression level of ABCG2 in the placenta with the advance of gestation from the mid stage to the end of gestation.

DOI PubMed CiNii

Phenolsulfonphthalein transport by potential-sensitive urate transport system

S Itagaki, S Shimamoto, M Sugawara, M Kobayashi, K Miyazaki, T Hirano, K Iseki

EUROPEAN JOURNAL OF PHARMACOLOGY ( ELSEVIER SCIENCE BV ) 518 ( 2-3 ) 83 - 89 2005.08 [Refereed]

The purpose of this study was to elucidate the transporter-mediated secretion systems for phenolsulfonphthalein in brush-border membranes. In human and rat renal brush-border membranes, a potential-sensitive transport system has been shown to be involved in the efflux of organic anions. The uptake of phenolsulfonphthalein into rat renal brush-border membrane vesicles was stimulated by an inside-positive membrane potential. This potential-sensitive uptake of phenolsulfonphthalein was inhibited by probenecid, pyrazinoate and urate. p-Aminohippurate had no effect on the potential-sensitive uptake of phenolsulfonphthalein. Moreover, urate competitively inhibited the uptake of phenolsulfonphthalein. On the other hand, the uptake of phenolsulfonphthalein was slightly increased in the presence of an outward Cl- gradient. These results suggest that phenolsulfonphthalein has high affinity for the potential-sensitive urate transport system but has low affinity for an anion exchanger. (c) 2005 Elsevier B.V. All rights reserved.

DOI PubMed

Nateglinide uptake by a ceftibuten transporter in the rat kidney brush-border membrane.

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

Biochimica et biophysica acta ( 1 ) 1715 ( 1 ) 19 - 24 2005.08 [Refereed] [International journal]

Nateglinide, a novel oral hypoglycemic agent, possesses a carbonyl group and a peptide-type bond in its structure. We previously reported that nateglinide transport occurs via a single system that may be identical to the ceftibuten/H(+) cotransport system by the rat small intestine. We speculated that the absorption system present on the intestinal epithelium may be similar to that found on the renal tubular epithelium. The aim of this study was to characterize the transporters on the apical side of the kidney that may contribute to the reabsorption of ceftibuten and nateglinide. The uptake of nateglinide by rat renal brush-border membranes is associated with an H(+)-coupled transport system. Ceftibuten competitively inhibited H(+)-dependent nateglinide uptake. In contrast, Gly-Sar, cephradine and cephalexin had no effect on nateglinide uptake. Nateglinide competitively inhibited H(+)-driven transporter-mediated ceftibuten uptake. We conclude that nateglinide transport occurs via a single system that is H(+)-dependent and may be identical to the ceftibuten/H(+) cotransport system.

DOI PubMed

Nateglinide uptake by a ceftibuten transporter in the rat kidney brush-border membrane

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

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES ( ELSEVIER SCIENCE BV ) 1715 ( 1 ) 19 - 24 2005.08 [Refereed]

Nateglinide, a novel oral hypoglycemic agent, possesses a carbonyl group and a peptide-type bond in its structure. We previously reported that nateglinide transport occurs via a single system that may be identical to the ceftibuten/H+ cotransport system by the rat small intestine. We speculated that the absorption system present on the intestinal epithelium may be similar to that found on the renal tubular epithelium. The aim of this study was to characterize the transporters on the apical side of the kidney that may contribute to the reabsorption of ceftibuten and nateglinide. The uptake of nateglinide by rat renal brush-border membranes is associated with an H+-coupled transport system. Ceftibuten competitively inhibited H+-dependent nateglinide uptake. In contrast, Gly-Sar, cephradine and cephalexin had no effect on nateglinide uptake. Nateglinide competitively inhibited H+-driven transporter-mediated ceftibuten uptake. We conclude that nateglinide transport occurs via a single system that is H+-dependent and may be identical to the ceftibuten/H+ cotransport system. (c) 2005 Elsevier B.V. All rights reserved.

DOI

Substrate specificity of the nateglinide/H+ cotransport system for phenolic acids

Y Saito, S Itagaki, Y Otsuka, Y Kobayashi, H Okumura, M Kobayashi, T Hirano, K Iseki

JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY ( AMER CHEMICAL SOC ) 53 ( 15 ) 6100 - 6104 2005.07 [Refereed]

In clinical, patients usually take many kinds of drugs at the same time. Thus, drug-drug interactions involving transporters can often directly affect the therapeutic safety and efficacy of many important drugs. However, there have been few studies on food-drug interactions involving transporters. Dietary polyphenols have been widely assumed to be beneficial for human health. Polyphenols are found ubiquitously, and they are commercially prepared and used as functional foods. We have reported that ferulic acid, which is one of the most well-known polyphenols and is used as a functional food, affected the transport of nateglinide, an antidiabetic drug, by Caco-2 cells. In this study, we investigated the effects of other polyphenols on the nateglinide/H+ transport system. We report here that caffeic acid and p-coumaric acid have a different inhibitory manner on the uptake of nateglinide. The results of this study are useful to identify the substrate specificity of the nateglinide/H+ cotransporter.

DOI PubMed CiNii

Transport mechanism for L-lactic acid in human myocytes using human prototypic embryonal rhabdomyosarcoma cell line (RD cells)

M Kobayashi, Fujita, I, S Itagaki, T Hirano, K Iseki

BIOLOGICAL & PHARMACEUTICAL BULLETIN ( PHARMACEUTICAL SOC JAPAN ) 28 ( 7 ) 1197 - 1201 2005.07 [Refereed]

Monocarboxylate transporter (MCT), which cotransport L-lactic acid and protons across cell membranes, are important for regulation of muscle pH. However, it has not been demonstrated in detail whether MCT isoform contribute to the transport Of L-lactic acid in skeletal muscle. The aim of this study was to characterize L-lactic acid transport using an human rhabdomyosarcoma (RD) cell line as a model of human skeletal muscle. mRNAs of MCT 1, 2 and 4 were found to be expressed in RD cells. The [C-14] L-lactic acid uptake was concentration-dependent with a K-m of 1.19 mm. This K-m value was comparable to its K-m values for MCTI or MCT2. MCT1 mRNA was found to be present markedly greater than that MCT2. Therefore, MCTI most probably acts on L-lactic acid uptake at RD cells. [C-14] L-Lactic acid efflux in RD cells was inhibited by alpha-cyano-4-hydroxycinnamate (CHC) but not by butyric acid, a substrate of MCT1. Accordingly, MCT2 or MCT4 is responsible for L-lactic acid efflux by RD cells. MCT4 mRNA was found to be present significantly greater than that MCT2. We conclude that MCTI is responsible for L-lactic acid uptake and L-lactic acid efflux is mediated by MCT4 in RD cells.

DOI PubMed CiNii

Uptake of irinotecan metabolite SN-38 by the human intestinal cell line Caco-2

T Itoh, S Itagaki, Y Sumi, T Hirano, Takemoto, I, K Iseki

CANCER CHEMOTHERAPY AND PHARMACOLOGY ( SPRINGER ) 55 ( 5 ) 420 - 424 2005.05 [Refereed]

Purpose: The aim of this study was to investigate the transport mechanisms of transporters that contribute to the intestinal uptake of 7-ethyl-10-hydroxycamptothecin (SN-38). Methods: Human intestinal epithelial Caco-2 cells were used to investigate the mechanistic basis of transepithelial uptake of SN-38. We investigated the characteristics of SN-38 uptake into Caco-2 cells. The effects of baicalin and sulfobromophthalein (BSP) on the uptake of SN-38 by Caco-2 cells were examined. Results: Uptake of SN-38 was significantly reduced at 4 degrees C. Baicalin inhibited the uptake of SN-38 in a concentration-dependent manner. BSP significantly reduced the uptake of SN-38. However, probenecid, pravastatin and grepafloxacin did not affect the uptake of SN-38. Conclusions: The results suggest that a specific transport system mediates the uptake of SN-38 across the apical membrane in Caco-2 cells.

DOI PubMed

Secretory transport of irinotecan metabolite SN-38 across isolated intestinal tissue

S Itagaki, Y Sumi, S Shimamoto, T Itoh, T Hirano, Takemoto, I, K Iseki

CANCER CHEMOTHERAPY AND PHARMACOLOGY ( SPRINGER ) 55 ( 5 ) 502 - 506 2005.05 [Refereed]

Purpose: The purpose of this study was to investigate the transport mechanisms of transporters that contribute to the intestinal efflux of 7-ethyl-10-hydroxycamptothecin (SN-38). Methods: The intestinal transport of SN-38 was studied in rat intestinal tissue mounted in Ussing chambers. Results: In the ileum, the level of transport from the serosal layer to the mucosal layer was significantly greater than that from the mucosal layer to the serosal layer, whereas a significant difference was not observed in the jejunum. This secretory transport required metabolic energy and was diminished by sulfobromophthalein. However, mitoxantrone, an inhibitor of breast cancer resistance protein (BCRP), did not affect the ileal secretion of SN-38. Conclusions: The results suggest that a specific transport system, which is distinct from BCRP, plays a major role in the secretion of SN-38 and that this secretory transport system predominantly exists in the ileum.

DOI PubMed

Food-drug interaction between ferulic acid and nateglinide involving the fluorescein/H+ cotransport system

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

JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY ( AMER CHEMICAL SOC ) 53 ( 7 ) 2499 - 2502 2005.04 [Refereed]

In clinical, patients usually take many kinds of drugs at the same time. Thus, drug-drug interactions involving transporters can often directly affect the therapeutic safety and efficacy of many drugs. However, there have been few studies on food-drug interactions involving transporters. Dietary polyphenols have been widely assumed to be beneficial to human health. Polyphenols are commercially prepared and used as functional foods. We report here for the first time that ferulic acid, which is widely used as a functional food, affects the transport of clinical agents. It is important to be aware of the potential of food-drug interactions and to act in order to prevent undesirable and harmful clinical consequences.

DOI PubMed CiNii

Intestinal uptake of nateglinide by an intestinal fluorescein transporter

S Itagaki, Y Otsuka, S Kubo, H Okumura, Y Saito, M Kobayashi, T Hirano, K Seki

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES ( ELSEVIER SCIENCE BV ) 1668 ( 2 ) 190 - 194 2005.03 [Refereed]

Nateglinide, a novel oral hypoglycemic agent, rapidly reaches its maximum serum concentration after oral administration, suggesting that it is rapidly absorbed in the intestine. However, nateglinide itself is not transported by MCT1 or PEPT1. The aim of this study was to characterize the transporters on the apical side of the small intestine that are responsible for the rapid absorption of nateglinide. It has been reported that the uptake of fluorescein by Caco-2 cells occurs via an H+-driven transporter and that the intestinal fluorescein transporter is probably not MCTI. We examined the contribution of the fluorescein transporter to the uptake of nateglinide by Caco-2 cells. Fluorescein competitively inhibited H+-dependent nateglinide uptake. All of fluorescein transporter inhibitors examined reduced the uptake of nateglinide. Furthermore, nateglinide inhibited fluorescein uptake. We conclude that the intestinal nateglinide/H+ cotransport system is identical to the intestinal fluorescein/H+ cotransport system. (C) 2004 Elsevier B.V All rights reserved.

DOI

Intestinal uptake of nateglinide by an intestinal fluorescein transporter.

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

Biochimica et biophysica acta ( 2 ) 1668 ( 2 ) 190 - 194 2005.03 [Refereed]

DOI

Intestinal uptake of nateglinide by an intestinal fluorescein transporter.

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

Biochimica et biophysica acta 1668 ( 2 ) 190 - 194 2005.03 [Refereed] [International journal]

Nateglinide, a novel oral hypoglycemic agent, rapidly reaches its maximum serum concentration after oral administration, suggesting that it is rapidly absorbed in the intestine. However, nateglinide itself is not transported by MCT1 or PEPT1. The aim of this study was to characterize the transporters on the apical side of the small intestine that are responsible for the rapid absorption of nateglinide. It has been reported that the uptake of fluorescein by Caco-2 cells occurs via an H+-driven transporter and that the intestinal fluorescein transporter is probably not MCT1. We examined the contribution of the fluorescein transporter to the uptake of nateglinide by Caco-2 cells. Fluorescein competitively inhibited H+-dependent nateglinide uptake. All of fluorescein transporter inhibitors examined reduced the uptake of nateglinide. Furthermore, nateglinide inhibited fluorescein uptake. We conclude that the intestinal nateglinide/H+ cotransport system is identical to the intestinal fluorescein/H+ cotransport system.

DOI PubMed

H+-dependent transport mechanism of nateglinide in the brush-border membrane of the rat intestine

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

JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS ( AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS ) 312 ( 1 ) 77 - 82 2005.01 [Refereed]

(-)-N-(trans-4-Isopropylcyclohexanecarbonyl)-D-phenylalanine (nateglinide) is a novel oral hypoglycemic agent possessing a carboxyl group and a peptide-type bond in its structure. Although nateglinide quickly reaches the maximal serum concentration after oral administration, nateglinide itself is not transported by PepT1 or MCT1. The aim of this study was to characterize the transporters on the apical side of the small intestine that are responsible for the rapid absorption of nateglinide. The uptake of nateglinide by rat intestinal brush-border membrane vesicles is associated with a proton-coupled transport system. Ceftibuten competitively inhibited H+-dependent nateglinide uptake. Glycylsarcosine (Gly-Sar), cephradine, and cephalexin did not significantly inhibit the uptake of nateglinide. The combination of Gly-Sar and nateglinide greatly reduced the uptake of ceftibuten. The effect of the combined treatment was significantly greater than that of Gly-Sar alone. Furthermore, nateglinide competitively inhibited H+-driven ceftibuten transporter-mediated ceftibuten uptake. Ceftibuten transport occurs via at least two H+-dependent transport systems: one is PepT1, and the other is the ceftibuten/H+ cotransport system. On the other hand, we demonstrated that nateglinide transport occurs via a single system that is H+ dependent but is distinct from PepT1 and may be identical to the ceftibuten/ H+ cotransport system.

DOI PubMed

Characterization of Secretory Intestinal Transport of Phenolsulfonphthalein

Shirou Itagaki, Makoto Chiba, Soji Shimamoto, Mitsuru Sugawara, Michiya Kobayashi, Katsumi Miyazaki, Takeshi Hirano, Ken Iseki

Drug Metabolism and Pharmacokinetics ( Japanese Society for the Study of Xenobiotics ) 20 ( 1 ) 72 - 78 2005 [Refereed]

DOI PubMed CiNii

Mechanism of L-lactic acid transport in L6 skeletal muscle cells.

Kobayashi M, Itagaki S, Hirano T, Iseki K

Drug metabolism and pharmacokinetics ( 5 ) 19 ( 5 ) 363 - 368 2004.10 [Refereed]

DOI PubMed CiNii

Pharmacokinetic modulation of irinotecan metabolites by sulphobromophthalein in rats

T Itoh, S Itagaki, K Sasaki, T Hirano, Takemoto, I, K Iseki

JOURNAL OF PHARMACY AND PHARMACOLOGY ( ROYAL PHARMACEUTICAL SOC GREAT BRITAIN ) 56 ( 6 ) 809 - 812 2004.06 [Refereed]

The purpose of this study was to modulate the pharmacokinetics of irinotecan metabolites, SN-38 and SN-38-glucuronide, by possibly reducing biliary excretion, which in turn could lower irinotecan toxicity. SN-38-glucuronide is associated with severe diarrhoea that occurs after irinotecan therapy as a result of enteric injury caused by SN-38. Sulphobromophthalein is used clinically as a drug for testing liver function and is considered to be a safe drug. We investigated the effect of sulphobro-mophthalein on the disposition of irinotecan metabolites after CPT-11 (7-ethyl-10-[10-4-(1-piperidino)-1-piperidino]-carbonyloxy-camptothecin) administration. Wistar rats were administered CPT-11 (500 mug/body) in saline as a bolus injection into the femoral vein through a catheter. The volume of drug solution injected into each animal was 1 mL. Rats were either administered CPT-11 alone or simultaneously with sulphobromophthalein (20 mg/body). After administration, blood and bile samples were taken at appropriate intervals and analysed by HPLC. Co-administration of sulphobromophthalein partially inhibited the biliary excretion of SN-38-glucuronide with a concomitant increase in its area under the plasma concentration-time curve (AUC) but did not significantly alter the biliary excretion and AUC of the active metabolite SN-38. These results suggested that cotreatment of CPT-11 with sulphobromophthalein might decrease intraluminal SN-38 concentrations without altering the pharmacokinetics of SN-38.

DOI PubMed

Comparison of urinary excretion of phenosulfonphthalein in an animal model for Wilson's disease (Long-Evans Cinnamon rats) with that in normal Wistar rats: Involvement of primary active organic anion transporter

Shirou Itagaki, Soji Shimamoto, Takeshi Hirano, Ken Iseki, Mitsuru Sugawara, Sachiho Nishimura, Michio Fujimoto, Michiya Kobayashi, Katsumi Miyazaki

Journal of Pharmacy and Pharmaceutical Sciences ( 2 ) 7 ( 2 ) 227 - 234 2004.05 [Refereed]

Purpose: The aim of this study was to determine the cause of the decline in phenolsulfonphthalein (PSP) excretion in Long-Evans Cinnamon (LEC) rats. Methods: The uptake of PSP into rat renal basolateral membrane vesicles (BLMV) was studied. Cyclosporin A (CYA) was used to modulate an ATP-dependent primary active transporter. PSP was intravenously injected into rats with or without CYA. The transcellular transport of PSP was examined by using primary cultured renal proximal tubule cells (PTC). Results: No significant difference was found between the uptake of PSP into renal BLMV of Wistar rats and that into renal BLMV of LEC rats. In the presence of CYA, the urinary excretion and the plasma concentrations of PSP in Wistar rats were decreased and increased, respectively. In primary cultured renal PTC from Wistar rats, the basal-to-apical transport of PSP was greater than that in the opposite direction and the basal-to-apical transport of PSP was substantially reduced by the addition of CYA. However, CYA did not affect the basal-to-apical transport of PSP in PTC from LEC rats. Conclusions: The results suggest that PSP is transported by primary active organic anion transporter and that the activity level of this transporter is reduced in LEC rats.

PubMed

Biliary excretion of irinotecan and its metabolites.

T Itoh, Takemoto, I, S Itagaki, K Sasaki, T Hirano, K Iseki

JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES ( CANADIAN SOC PHARMACEUTICAL SCIENCES ) 7 ( 1 ) 13 - 18 2004.01 [Refereed]

PURPOSE: The aim of this study was to investigate the excretion of irinotecan hydrochloride (CPT-11) and its metabolites into the gastrointestinal lumen via the biliary route after intravenous administration of lactone and carboxylate forms of CPT-11. METHODS: Biliary excretions of CPT-11 and its metabolites, SN-38 and SN-38-glucuronide, were investigated by an in vivo administration study using rats. The biliary excretion profiles for both the lactone and carboxylate forms of CPT-11 and its metabolites were determined. RESULTS: After the i.v. injection of the lactone form of CPT-11, the cumulative biliary excretion of SN-38-glucuronide was much greater than that of CPT-11 and SN-38, and biliary excretion of SN-38 was less than that of CPT-11. Further, CPT-11 and SN-38 were mainly excreted into bile as carboxylate forms. After the administration of the CPT-11 carboxylate form, biliary excretion of SN-38-glucuronide was significantly smaller than that after the administration of CPT-11 lactone form. On the other hand, biliary excretion of CPT-11 and SN-38 was greater after dosing with the CPT-11 carboxylate form than that after the CPT-11 lactone form. CONCLUSIONS: The results suggest that the rate of conversion of lactone to carboxylate forms of CPT-11 and its metabolites plays a major role in the biliary excretion of these compounds.

PubMed

Comparison of the Disposition Behavior of Organic anions in an Animal Model for Wilson's Disease (Long-Evans Cinnamon rats) with that in Normal Long-Evans Agouti rats

Shirou Itagaki, Mitsuru Sugawara, Michiya Kobayashi, Katsumi Miyazaki, Takeshi Hirano, Ken Iseki

Drug Metabolism and Pharmacokinetics ( Japanese Society for the Study of Xenobiotics ) 19 ( 2 ) 150 - 154 2004 [Refereed]

DOI PubMed CiNii

Major role of organic anion transporters in the uptake of phenolsulfonphthalein in the kidney

S Itagaki, M Sugawara, M Kobayashi, S Nishimura, M Fujimoto, K Miyazaki, K Iseki

EUROPEAN JOURNAL OF PHARMACOLOGY ( ELSEVIER SCIENCE BV ) 475 ( 1-3 ) 85 - 92 2003.08 [Refereed]

Phenolsulfonphthalein is used for testing renal function. However, its excretion mechanism has not been elucidated. The purpose of this study was therefore to elucidate the transporter-mediated excretion system for phenolsulfonphthalein. p-Aminohippuric acid, a substrate of rat organic anion transporter1 (rOat1), and cimetidine, a substrate of rOat3, reduced the urinary excretion of phenolsulfonphthalein. The uptake of phenolsulfonphthalein by kidney slices was found to consist of two components. The IC50 values of rOat1 substrates were higher than those of rOat3 substrates. In the presence of cimetidine, the Eadie-Hofstee plot gave a single straight line. The profile of the phenolsulfonphthalein uptake component in the presence of cimetidine was similar to that of the low-affinity component in the absence of cimetidine. We conclude that rOat1 and rOat3 are involved in the renal uptake of phenolsulfonphthalein and that phenolsulfonphthalein is a high-affinity substrate for rOat3 but is a relatively low-affinity substrate for rOat1. (C) 2003 Elsevier B.V. All rights reserved.

DOI PubMed

Mechanism of Active Secretion of Phenolsulfonphthalein in the Liver via Mrp2 (abcc2), an Organic Anion Transporter

Shirou Itagaki, Ken Iseki, Mitsuru Sugawara, Michiya Kobayashi, Katsumi Miyazaki

Drug Metabolism and Pharmacokinetics ( 4 ) 18 ( 4 ) 238 - 244 2003 [Refereed]

Phenolsulfonphthalein (PSP) has been selected as a model drug that is eliminated from both the kidney and liver in rats. Although the renal PSP transport system has been studied, few details of the biliary excretion of PSP have been reported. We investigated the biliary excretion system for PSP in rats. It has been reported that the biliary excretion of many organic anions from hepatocytes into bile is mediated by a primary active transporter, referred to as multidrug resistance-associated protein 2 (Mrp2/abcc2). The biliary excretion of PSP in SD rats was significantly decreased in the presence of Mrp2 inhibitors. The biliary excretion of PSP in Eisai hyperbilirubinemic rats (EHBR), hereditarily Mrp2-defective rats, was significantly lower than that in SD rats. Moreover, an efflux experiment using Caco-2 cells was carried out to confirm Mrp2-mediated PSP transport. Mrp2 inhibitors significantly decreased PSP efflux from Caco-2 cells. These results suggest that Mrp2 contributes to the biliary excretion of PSP in SD rats. © 2003, The Japanese Society for the Study of Xenobiotics. All rights reserved.

DOI CiNii

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