YAKUGAKU ZASSHI
 Abstracts 
Vol.130, No. 11 (2010)

--Foreword--

Signal Transduction in the Development of Vascular Disease and New Therapeutic Strategy

Motohiro NISHIDA

Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

--Review--

Mechanisms for Linking High Salt Intake to Vascular Tone: Role of Na⁺ Pump and Na⁺/Ca²⁺ Exchanger Coupling

Satomi KITA^* and Takahiro IWAMOTO

Department of Pharmacology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan

Excessive salt intake is a major risk factor for hypertension. However, the underlying molecular relationship between salt and hypertension is not fully understood. Recently discovered cardiotonic steroids, such as endogenous ouabain and other steroids, have been proposed as candidate intermediaries. Plasma cardiotonic steroids are significantly elevated in patients with essential hypertension and in salt-dependent hypertensive animals. Generally, it is believed that cardiotonic steroids inhibit Na⁺ pump activity and lead to an increase in the cytosolic Na⁺ concentration. Cellular Na⁺ accumulation raises the cytosolic Ca²⁺ concentration through the involvement of Na⁺/Ca²⁺ exchanger type 1 (NCX1). In isolated arteries from α2 Na⁺ pump knockout mice (α2^+/−), myogenic tone is increased, and NCX inhibitor normalizes the elevated myogenic tone in α2^+/− arteries. The NCX inhibitor lowers arterial blood pressure in salt-dependent hypertensive rats but not in other types of hypertensive rats or in normotensive rats. Furthermore, smooth muscle-specific NCX1 transgenic mice are hypersensitive to salt, whereas mice with smooth muscle-specific knockout of NCX1 (NCX1^SM−/−) have low salt sensitivity. These results suggest that functional coupling between the vascular α2 Na⁺ pump and NCX1 is a critical molecular mechanism for salt-induced blood pressure elevation.

Key words--Na⁺/Ca²⁺ exchanger; Na⁺ pump; excessive salt intake; hypertension; smooth muscle; myogenic tone

--Review--

Blood Flow Sensing Mechanism via Calcium Signaling in Vascular Endothelium

Kimiko YAMAMOTO^*,a and Joji ANDO^b

^aDepartment of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and ^bLaboratory of Biomedical Engineering, School of Medicine, Dokkyo Medical University, 880 Kita-kobayashi, Mibu-machi, Shimotsuga-gun, Tochigi 321-0293, Japan

The structure and function of blood vessels adapt to environmental changes, for example, physical development and exercise. This phenomenon is based on the ability of endothelial cells (ECs) to sense and respond to blood flow. ECs are in direct contact with blood flow and exposed to shear stress. A number of recent studies have revealed that ECs recognize changes in shear stress and transmit signals to the interior of the cell, which leads to cellular responses that involve changes in cell morphology, cell function, and gene expression. Cultured human pulmonary artery ECs (HPAECs) showed Ca²⁺ influx via an ATP-operated cation channel, P2X4, in response to shear stress. We have recently found that shear-induced activation of P2X4 requires endogenously released ATP and that shear stress induced HPAECs to release ATP, which was mediated by cell-surface ATP synthase located in caveolae. To gain insight into its significance, we generated a P2X4-deficient mouse. P2X4^−/− mice do not exhibit normal EC responses to flow, such as Ca²⁺ influx and subsequent production of NO, a potent vasodilator. Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2X4^−/− mice. Furthermore, P2X4^−/− mice have higher blood pressure than wild-type mice. Moreover, no adaptive vascular remodeling is observed in the P2X4^−/− mice. Thus, P2X4-mediated shear stress mechanotransduction plays an important role in the vascular homeostasis, including the control of blood pressure and vascular remodeling.

Key words--vascular endothelial cell; shear stress; mechanotransduction; Ca²⁺ signaling; nitric oxide

--Review--

Signaling Mechanism Involved in Regulation of Endothelial Cell-Cell Junctions

Shigetomo FUKUHARA^* and Naoki MOCHIZUKI

Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan

Endothelial cells lining blood vessels are in tight contact with each other, thereby maintaining vascular integrity. Compromising vascular integrity leads to an increase in vascular permeability, which is associated with chronic inflammation, edema, and tumor angiogenesis. Vascular endothelial (VE)-cadherin is an endothelium-specific cell-cell adhesion molecule involved in endothelial barrier functions. We previously reported that cyclic AMP-elevating agonists such as prostaglandins and adrenomedullin potentiate VE-cadherin-dependent cell adhesion by inducing activation of Rap1 small GTPase through Epac. We further investigated the mechanism whereby Rap1 potentiates VE-cadherin-dependent cell adhesion, and found that Rap1 induces the formation of circumferential actin bundles along the cell-cell junctions. Although it has been believed that α-/β-catenins anchor cadherin to the actin cytoskeleton to stabilize cadherin at cell-cell junctions (classical model), Nelson's and Weis' groups have recently suggested a new dynamic model in which α-/β-catenins do not stably connect actin to cadherin. However, our study clearly indicated that the circumferential actin bundles anchor VE-cadherin to the cell-cell junctions through α-/β-catenins. Thus Rap1 potentiates endothelial cell-cell junctions through the mechanism based on the static model.

Key words--endothelial permeability; vascular endothelial-cadherin; Rap1; actin; α-catenin

--Review--

Role of Angiontensin Receptors on Remodeling Perivascular Nerves

Narumi HOBARA,^*,a Mitsuhiro GODA,^b Naoya HASHIKAWA,^a Xin JIN,^b Yoshito ZAMAMI,^c Shingo TAKATORI,^b and Hiromu KAWASAKI^b

^aDepartment of Life Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan, ^bDepartment of Clinical Pharmaceutical Science, and ^cDepartment of Molecular Design of Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan

The aim of this study was to investigate age-related changes in the density of calcitonin gene-related peptide (CGRP)-containing nerve fibers in spontaneously hypertensive rats (SHR) and the effects of long-term inhibition of the renin-angiotensin system on these changes. An age-related decrease in the density of CGRP-like immunoreactive (LI)-containing nerve fibers but not neuropeptide Y (NPY)-LI-containing sympathetic nerve fibers was found in the mesenteric artery of SHR but not Wistar-Kyoto rats (WKY). The density of NPY-LI-containing nerve fibers was significantly greater in SHR than in WKY. SHR were treated for 7 weeks with angiotensin-converting enzyme inhibitor (0.005% temocapril), angiotensin II type-1 (AT1) receptor antagonist (0.025% losartan), or vasodilator (0.01% hydralazine) in their drinking water. Each drug treatment significantly lowered the systolic blood pressure measured using the tail-cuff method. Long-term treatment of SHR with temocapril and losartan significantly increased the density of CGRP-LI-containing nerve fibers in mesenteric arteries. Furthermore, to clarify the effect of the angiontensin II type-2 (AT2) receptor in the restoration of perivascular nerve innervation, we used the phenol-injured rat model, in which the perivascular nerves are markedly reduced by the topical application of phenol. Activation of AT2R significantly restored CGRP-LI innervation in phenol-injured rats. These results suggest that selective stimulation of AT2 receptors facilitates reinnervation of mesenteric perivascular CGRP-containing nerves.

Key words--hypertension; angiotensin II type 2 receptor; renin-angiotensin system; calcitonin gene-related peptide; nerve regeneration

--Review--

Regulation of Cardiovascular Functions by the Phosphorylation of TRPC Channels

Motohiro NISHIDA,^*,a Shota SAIKI,^a Naoyuki KITAJIMA,^a Michio NAKAYA,^a Yoji SATO,^b and Hitoshi KUROSE^a

^aDepartment of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, and ^bDivision of Cellular and Gene Therapy Products, National Institute of Health Sciences, 1-18-1 Kami-yoga, Setagaya-ku, Tokyo 158-8501, Japan

Calcium ions (Ca²⁺) play an essential role in homeostasis and the activity of cardiovascular cells. Ca²⁺ influx across the plasma membrane induced by neurohumoral factors or mechanical stress elicits physiologically relevant timing and spatial patterns of Ca²⁺ signaling, which leads to the activation of various cardiovascular functions, such as muscle contraction, gene expression, and hypertrophic growth of myocytes. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca²⁺ signaling pathway required for pathological hypertrophy. We have recently found that the inhibition of cGMP-selective phosphodiesterase 5 (PDE5) suppresses agonist- and mechanical stretch-induced hypertrophy through inhibition of Ca²⁺ influx in rat cardiomyocytes. The inhibition of PDE5 suppressed the increase in frequency of Ca²⁺ spikes induced by receptor stimulation or mechanical stretch. Activation of protein kinase G by PDE5 inhibition phosphorylated TRPC6 proteins at Thr⁶⁹ and prevented TRPC6-mediated Ca²⁺ influx. Substitution of Ala for Thr⁶⁹ in TRPC6 abolished the antihypertrophic effects of PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlies the prevention of cardiac hypertrophy by PDE5 inhibition. As TRPC6 proteins are also expressed in vascular smooth muscle cells and reportedly participate in vascular remodeling, TRPC6 blockade may be an effective therapeutic strategy for preventing pathologic cardiovascular remodeling.

Key words--transient receptor potential; calcium signaling; hypertrophy; remodeling; phosphodiesterase

--Foreword--

The Frontiers of White Biotechnology and the Development of Pharmaceutical Sciences

Seiji SHIBASAKI^*,a and Mitsuyoshi UEDA^b

^aDepartment of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530, Japan, and ^bDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan

--Review--

Novel Bioconversion Systems Using a Yeast Molecular Display System

Seiji SHIBASAKI

Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530, Japan

The budding yeast Saccharomyces cerevisiae has been used for the process of fermentation as well as for studies in biochemistry and molecular biology as a eukaryotic model cell or tool for the analysis of gene functions. Thus, yeast is essential in industries and researches. Yeast cells have a cell wall, which is one characteristic that helps distinguish yeast cells from other eukaryotic cells such as mammalian cells. We have developed a molecular display system using the protein of the yeast cell wall as an anchor for foreign proteins. Yeast cells have been designed for use in sensing and metal adsorption, and have been used in vaccines and for screening novel proteins. Currently, yeast is used not only as a tool for analyzing gene or protein function but also in molecular display technology. The phage display system, which is at the forefront of molecular display technologies, is a powerful tool for screening ligands bound to a target molecule and for analyzing protein-protein interactions; however, in some cases, eukaryotic proteins are not easily expressed by this system. On the other hand, yeast cells have the ability to express eukaryotic proteins and proliferate; thus, these cells display various proteins. Yeast cells are more appropriate for white biotechnology. In this review, displays of enzymes that are important in bioconversion, such as lipases and β-glucosidases, are going to be introduced.

Key words--yeast; molecular display; β-glucosidase; isoflavone; lipase

--Review--

Research on Search of the Carotenoid-producing Microorganisms in Marine Area and the Improvement of Production Ratio

Yoshikazu SAKAGAMI,^*,a Yasuji SUMIYA,^a,b and Sadawo KOMEMUSHI^a,c

^aFaculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan, ^bKanae Technos Co., Ltd., 93-27 tei, kunita-cho, Kanonji, Kagawa 768-0040, Japan, and ^cGraduate School of Engineering, Osaka City University, 3-3-138 Sugimoto Sumiyoshi-ku, Osaka 558-8585, Japan

Carotenoids are liposoluble pigments widely distributed in nature. More than 750 carotenoids are isolated from natural sources, but only a few kinds are used industrially. The production of carotenoid by microorganisms is to be expected, but few carotenoids originate from living things on land. And there is little knowledge about carotenoid-producing microorganisms in the oceans. The possibility still exists of discovering new carotenoid-producing microorganisms. Sunlight is very strong in subtropical regions. The surface of the sea and coral reefs in these regions is a severe environment for growth of microorganisms. While such conditions produce reactive oxygen species, the continuing strong irradiation can also lead to damaging and lethal photo-oxidative reactions. Many undiscovered microorganisms may possess protective mechanisms such as anti-oxidative activities for survival in this environment. This study focused on marine microorganisms inhabiting coral reefs in the Okinawa area, especially carotenoid-producing bacteria possessing anti-oxidative activities. Many carotenoid-producing microorganisms were collected from subtropical ocean areas (a total of 334 strains of pigmented microorganisms), and the chemical composition, some culture conditions and genetic characteristics of the carotenoids from these microorganisms were examined. Furthermore, similar research was performed using some creatures from the ocean surrounding Kochi Prefecture.

Key words--carotenoid-producing microorganism; Okinawa area; creature of ocean

--Review--

Microbe-inspired System Enzymology of Vitamin B₁₂ Metabolism

Tetsuo TORAYA

Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan

Vitamin B₁₂ is produced only by prokaryotes and utilized by animals as an essential micronutrient. Genetic complementation analysis of cell lines from patients indicated that at least eight gene products are involved in intracellular B₁₂ metabolism and utilization. We have investigated bacterial adenosylcobalamin-dependent enzymes and elucidated their structure-based fine mechanisms. They tend to undergo mechanism-based inactivation during catalysis, because they use highly reactive radicals for catalyzing chemically difficult reactions. We have discovered molecular chaperone-like reactivating factors for these enzymes that release a damaged cofactor forming apoenzyme. Methylcobalamin-dependent methionine synthase also undergoes inactivation, because it utilizes cob(I)alamin, a super nucleophile, for catalysis. Methionine synthase reductase is a reactivating partner for this enzyme. Recent studies suggested that activity-maintaining systems for B₁₂ enzymes are present in animal cells as well, and thus hints for designing therapeutic agents for B₁₂-related metabolic disorders might be obtained from the investigations of microbial B₁₂ metabolism.

Key words--vitamin B₁₂; B₁₂ metabolism; B₁₂ enzyme; inactivation; reactivating factor

--Review--

Novel L-Amino Acid Ligases Catalyzing Oligopeptide Synthesis

Kuniki KINO

Department of Applied Chemistry, School of Advanced Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan

L-Amino acid ligase (EC 6.3.2.28) is a microbial enzyme catalyzing formation of an alpha-peptide bond from unprotected L-amino acids in an ATP-dependent manner. The YwfE protein from Bacillus subtilis 168 was the first reported L-amino acid ligase, and it synthesizes various dipeptides. Thereafter, several L-amino acid ligases were newly obtained by in silico analysis using the ATP-grasp motif. But these L-amino acid ligases synthesize only dipeptide and no longer peptide. A novel L-amino acid ligase capable of catalyzing oligopeptide synthesis is required to increase the variety of peptides. We have previously found a new member of L-amino acid ligase, RizA, from B. subtilis NBRC3134, a microorganism that produces the peptide-antibiotic rhizocticin. We newly found that a gene at approximately 9 kbp upstream of rizA encoded a novel L-amino acid ligase RizB. Recombinant RizB synthesized homo-oligomers of branched-chain amino acids consisting of 2 to 5 amino acids, and also synthesized various heteropeptides. RizB is the first reported L-amino acid ligase that catalyzes oligopeptide synthesis. In addition, we obtained L-amino acid ligases showing oligopeptide synthesis activities by in silico analysis using BLAST, which is a set of similarity search programs. These L-amino acid ligases showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacteria adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones.

Key words--L-amino acid ligase; oligopeptide synthesis; in silico analysis; rhizocticin

--Review--

Drug Development from Natural Fermentation Products: Establishing a Manufacturing Process Which Maximizes the Potential of Microorganisms

Koji NAGAO,^*,a Satoshi UEDA,^a Munekazu KANDA,^b Nobutaka OOHATA,^a Michio YAMASHITA,^c and Motohiro HINO^a

^aAstellas Pharma Inc., Fermentation and Biotechnology Labs., ^bAstellas Toyama Inc., Fermentation Technology Research Center, 156 Nakagawara, Kiyosu, Aichi 452-0915, Japan, and ^cAstellas Foundation for Research on Metabolic Disorders, 3-11 Nihonbashi- Honcho 2-chome, Chuo-ku, Tokyo 103-8411, Japan

Natural fermentation products have long been studied as attractive targets for drug discovery due to their amazing diverse, complex chemical structures and biological activities. As such, a number of revolutionary drugs developed from natural fermentation products have contributed to global human health. To commercialize a drug derived from natural fermentation products, an effective chemical entity must be identified and thoroughly researched, and an effective manufacturing process to prepare a commercial supply must be developed. To construct such a manufacturing process for tacrolimus and micafungin, the following studies were conducted: first, we focused on controlling the production of the tacrolimus-related compound FR900525, a fermentation by-product of tacrolimus which was critical for quality assurance of the drug substance. FR900525 production was reduced by using a mutant strain which produced more pipecolic acid, the biosynthesis material of tacrolimus, than the original strain. Then, to optimize the fermentation process of FR901379, an intermediate of micafungin, a fed-batch culture was adopted to increase FR901379 productivity. Additionally, FULLZONE^TM impeller was installed into the scaled-up fermenter, reducing the agitation-induced damage to the mycelium. As a result, the mycelial form changed from filamentous to pellet-shaped, and the air uptake rate during fermentation was drastically improved. Finally, we conducted screening for FR901379 acylase-producing microorganisms, as FR901379 acylase is necessary to manufacture micafungin. We were able to easily discover FR901379 acylase-producing microorganisms in soil samples using our novel, convenient screening method, which involves comparing the difference in antibiotic activity between FR901379 and its deacylated product.

Key words--fermentation; natural product; manufacturing; process development

--Review--

Development of Revolutionary Enzymatic Reactions in Organic Solvents with Molecular Display

Mitsuyoshi UEDA

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan

We have seen increasing use of the term “White biotechnology”. White biotechnology involves the use of microbial cells and enzymes in the production of bulk and fine chemicals such as amino acids and polymers. This generally results in cleaner processes with minimum waste generation and energy use. Most of the organic syntheses using enzymes are carried out in nearly anhydrous organic solvents or solvent-free media. Ionic liquids have more recently emerged as another nonaqueous media, which, in view of their low vapor pressure, are viewed as “green solvents”. Organic solvents may alter the structure and activity of enzymes that usually function in an aqueous environment. One alternative is to immobilize the enzymes on solid supports to increase their function and stability in response to organic solvents or increased temperatures. Enzymes may be stabilized by chemical and physical processes. With chemical methods, enzymes are immobilized by strong covalent bonding, but changes in protein structure often result. In physical stabilization processes, the interactions between enzymes and solids usually are weaker, resulting in fewer changes in the enzyme's structure. Yeast cell surface engineering is an alternative approach that immobilizes enzymes on the yeast cell surface. Proteins are immobilized by using an outer shell cell-wall protein, the C-terminal half of alpha-agglutinin. Display of enzymes on the yeast cell surface has at least two advantages relative to other physical immobilization methods. First, the displayed enzymes can be readily produced in a standard fermentation. No further work is required to either purify or immobilize the enzymes. Second, enzyme displayed on the yeast cell surface can be modified directly by conventional genetic engineering, which enables error-prone PCR, DNA shuffling, and combinatorial mutagenesis to be used quickly and efficiently to create strains (whole-cell biocatalysts) with enhanced enzyme activity.

Key words--molecular display; cell surface engineering; white biotechnology; whole-cell biocatalysts; genetic immobilization; organic solvent

--Foreword--

Development of Cellular and Gene Therapy Products for Bioacitve Protein-based Therapy

Fuminori SAKURAI^*,a,b and Makiya NISHIKAWA^c

^aDepartment of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan, ^bLaboratory of Stem Cell Regulation, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaragi, Osaka 567-0085, Japan, and ^cDepartment of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimo-adachi-cho, Sakyo-ku, Kyoto 606-8501, Japan

--Review--

Development of Gene Delivery System into Skeletal Muscles by Bubble Liposomes and Ultrasound

Yoichi NEGISHI,^*,a Yoko ENDO-TAKAHASHI,^a Ryo SUZUKI,^b Kazuo MARUYAMA,^b and Yukihiko ARAMAKI^a

^aDepartment of Drug and Gene Delivery Systems, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan, and ^bDepartment of Biopharmaceutics, School of Pharmaceutical Sciences, Teikyo University, 1091-1 Suwarashi, Midori-ku, Sagamihara 252-5195, Japan

Skeletal muscle is a promising target tissue for the gene therapy of both muscle and non-muscle disorders. Gene transfer into muscle tissue can produce a variety of physiologically active proteins and may ultimately be applied to treatment of many diseases. A variety of methods have been studied to transfer genes into skeletal muscle, including viral and non-viral vectors. Recently, we have developed the polyethyleneglycol (PEG)-modified liposomes entrapping echo-contrast gas known as ultrasound (US) imaging gas. We have called the liposomes “Bubble liposomes” (BLs). We have further demonstrated that US-mediated eruption of BLs loaded with naked plasmid-DNA is a feasible and efficient technique for gene delivery. In this study, to assess the feasibility and the effectiveness of BLs for the gene therapy of disorders, we tried to deliver therapeutic genes (anti-inflammatory cytokine; IL-10 or anti-angiogenic factor; hK1-5) into skeletal muscles of arthritis or tumor model mice by the gene delivery system with BLs and US exposure. As a result, their disease symptom was efficiently improved by the systemic secretion of therapeutic proteins. Thus, this US-mediated BLs technique for muscle gene transfer may provide an effective noninvasive method for arthritis or cancer gene therapy in clinical use. In addition, it may be applicable for the gene therapy of other non-muscle and muscle disorders.

Key words--ultrasound; Bubble liposome; gene delivery system; gene therapy

--Review--

Development of Recombinant Adenovirus Carrying MicroRNA-regulated Gene Expression System

Fuminori SAKURAI^*,a,b and Hiroyuki MIZUGUCHI^a,b

^aDepartment of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan, and ^bLaboratory of Stem Cell Regulation, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaragi, Osaka 567-0085, Japan

Target tissue-specific delivery and transcription of foreign genes are desirable for safe and effective gene therapy. Two approaches for this purpose, “Targeted Delivery” and “Targeted Expression”, have been mainly reported. Among “Targeted Expression” approaches, microRNA (miRNA)-mediated “post-transcriptional de-targeting” has been recently demonstrated, and much attention has been focused on this approach. MiRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3'-untranslated region (UTR) of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. First, in order to reduce the hepatic transduction by Ad vectors, complementary sequences of liver-specific miRNA, miR-122a, were inserted into the 3'-UTR of the transgene expression cassette. Intratumor injection of this Ad vector resulted in approximately 100-fold lower hepatic expression than that of the conventional Ad vector, without reducing gene expression in the tumor. Second, complementary sequences for miRNAs selectively down-regulated in tumor cells were inserted into the E1 gene expression cassette in oncolytic Ads, which exhibit tumor cell-specific replication and antitumor effects. Recent studies demonstrated that expression of several miRNAs is exclusively reduced in tumor cells. Oncolytic Ads containing the miRNA complementary sequences showed reduced replication in the normal cells, without altering the antitumor effects. MiRNA-regulated gene expression system mediates “post-transcriptional de-targeting”, in which translation of transgene is suppressed in a tissue-specific manner; however, tissue-specific transgene expression can be achieved by taking tropism of gene delivery vehicles into consideration and reducing the transgene expression in untargeted organs via miRNA-regulated gene expression system.

Key words--miRNA; recombinant adenovirus; transcriptional targeting; post-transcriptional de-targeting

--Review--

Optimization of Gene Therapy Effect by Spaciotemporal Control of Expressed Proteins

Makiya NISHIKAWA,^* Yuki TAKAHASHI, and Yoshinobu TAKAKURA

Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan

Therapeutic effects of in vivo gene therapy, which aims to treat diseases by administering therapeutic genes to patients, are obtained via proteins expressed from the gene administered. Therefore, to optimize the therapeutic effects of such therapy, it is important to control not only the tissue distribution of gene vectors but also that of proteins expressed from the vector. Studies using protein pharmaceuticals have clearly demonstrated that the therapeutic effect depends largely on the spatiotemporal distribution of proteins, such as area under the curve and mean residence time. These results strongly suggest that precise control of the spatiotemporal distribution of proteins increases the efficacy of in vivo gene therapy. Based on these considerations, we tried to increase the therapeutic effect of plasmid DNA-based gene therapy by controlling the profile of proteins expressed form vectors. To increase the residence time of proteins, we developed plasmids with few CpG motifs and achieved sustained expression of proteins at therapeutic levels for as long as several months. Sustained expression of murine interferon γ was highly effective in inhibiting metastatic tumor growth and atopic dermatitis in mouse models. Thus, designing plasmid vectors is a promising approach not only to controlling the spatiotemporal distribution of proteins, but also to increasing the therapeutic potency of in vivo gene therapy.

Key words--gene therapy; plasmid DNA; interferon; hydrodynamic injection; sustained expression

--Review--

Gene Therapy with Vector-producing Multipotent Mesenchymal Stromal Cells

Takashi OKADA

Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi-cho, Kodaira, Tokyo 187-8502, Japan

Suicide gene therapy with retroviral vector-producing cells was feasible as an adjuvant to the surgical resection of recurrent glioblastoma, although any benefit appeared to be marginal. Further evaluation of the therapeutic strategy with the vector-producing cells must incorporate improved delivery of vectors and transgenes to the target cells. We have previously demonstrated the ability of vector-producing tumor cells engineered by the adenovirus-retrovirus hybrid vector to destroy satellite tumor cells, although therapeutic efficacy for aggressive tumor has to be further evaluated by the systemic delivery of the vector-producing cells. Mesenchymal stem cells (MSCs) should be an effective delivery vehicle to seek out tumor cells in vivo and transport cancer-killing gene or immune products with minimal rejection reaction by the host. We developed vector-producing tumor-tracking cells to improve suicide cancer gene therapy. Nucleofection was attempted to deliver retrovirus vector components into rodent MSCs. Athymic nude mice with subcutaneous 9L glioma were received vector-producing MSCs through the left ventricular cavity. Optical bioluminescence imaging in vivo revealed accumulation of the MSCs into the subcutaneous 9L tumors but not Rat-1 transplants. Consequently, the vector-producing MSCs significantly enhanced pro-drug killing of glioma cells compared to MSCs without ability to generate progeny virus. Our study demonstrated the effective MSCs-mediated tumor transduction with progeny vector production to improve suicide gene therapy. Although therapeutic benefit in the various orthotopic or metastatic tumor models has to be further validated, this transduction strategy would eradicate evasive tumors in situ.

Key words--mesenchymal stem cell; gene therapy; virus vector

--Review--

New Technologies for Immunotherapy against Cancer: Development of Cell Expansion Technology and Viruses as Immune Boosters

Yui HARADA and Yoshikazu YONEMITSU^*

Kyushu University Graduate School of Pharmaceutical Sciences, R&D Laboratory for Innovative Biotherapeutics, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Dendritic cells (DCs) play a crucial role in maintaining the immune system. Although DC-based cancer immunotherapy has been suggested as a potential treatment for various kinds of malignancies, clinical efficacies have been still unsatisfactory. To improve the clinical outcome of DC-based cancer immunotherapy, we are now focusing on 1) increase of numbers of therapeutic immune cells, i.e., DCs, and 2) the development of new methods for stimulating them. We have recently established a possible breakthrough, a simple cytokine-based culture method to realize a log-scale order of functional myeloid-type murine/human DCs. Moreover, we demonstrated that DCs activated by replication-deficient recombinant Sendai virus (rSeV) were highly effective than that seen in the use of current DC vaccine stimulated by conventional cytokines etc., for immunotherapy against malignancies. Therefore, our study strongly suggests that these improvements could overcome the current limitations of DC-based immunotherapy for malignancies.

Key words--dendritic cell; cell expansion; Sendai virus; cancer; immunotherapy

--Review--

Differentiation of Functional Cells from iPS Cells by Efficient Gene Transfer

Kenji KAWABATA,^*,a,b Katsuhisa TASHIRO,^a and Hiroyuki MIZUGUCHI^a,c

^aLaboratory of Stem Cell Regulation, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan, ^bDepartment of Biomedical Innovation and ^cDepartment of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan

Induced pluripotent stem (iPS) cells, which are generated from somatic cells by transducing four genes, are expected to have broad application to regenerative medicine. Although establishment of an efficient gene transfer system for iPS cells is considered to be essential for differentiating them into functional cells, the detailed transduction characteristics of iPS cells have not been examined. By using an adenovirus (Ad) vector containing the cytomegalovirus enhancer/beta-actin (CA) promoters, we have developed an efficient transduction system for mouse mesenchymal stem cells and embryonic stem (ES) cells. Also, we applied our transduction system to mouse iPS cells and investigated whether efficient differentiation could be achieved by Ad vector-mediated transduction of a functional gene. As in the case of ES cells, the Ad vector could efficiently transduce transgenes into mouse iPS cells. We found that the CA promoter had potent transduction ability in iPS cells. Moreover, exogenous expression of a PPARγ gene or a Runx2 gene into mouse iPS cells by an optimized Ad vector enhanced adipocyte or osteoblast differentiation, respectively. These results suggest that Ad vector-mediated transient transduction is sufficient to promote cellular differentiation and that our transduction methods would be useful for therapeutic applications based on iPS cells.

Key words--regenerative medicine; induced pluripotent stem (iPS) cell; adenovirus vector; mesenchymal stem cell; embryonic stem (ES) cell

--Review--

Synthetic Studies towards Kinamycin Antibiotics

Takuya KUMAMOTO

Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan

Kinamycin antibiotics, strongly active against gram-positive bacteria, were isolated from the culture broth of Streptomyces murayamaensis. The structures of kinamycins and prekinamycin, isolated from the same bacteria, were at first determined to be benzo[b]carbazole with cyanamide [N-C≡N]. Later re-examination of spectroscopic analysis concluded that those compounds should be benzo[b]fluorene with diazoalkane [C⁻-N⁺≡N]. However, the structure of the latter was re-revised to benzo[a]fluorene and renamed as isoprekinamycin. We have continued our effort for the synthesis of kinamycin antibiotics from the point of view of total synthesis and structural determination. In this review, our approach toward total synthesis of kinamycins was described.

Key words--kinamycin; antibiotics; prekinamycin; benzo[b]fluorene; diazoalkane; synthetic study

--Review--

Study for the Novel Factors Regulating Mitochondrial Dynamics

Midori SUENAGA

Department of Medical Pharmacology, Faculty of Pharmaceutical Science, Tokushima Bunri University, 180 Nishihama-Houji Yamashiro-cho, Tokushima 770-8514, Japan

Mitochondria are highly dynamic organelles and undergo continuous fission and fusion events in physiological situations. It was observed that mitochondrial morphology and number are changed in living cells during cellular differentiation, development, and under pathological conditions including muscle dystrophy, cardiomyopathy, and cancer. Defined sets of proteins are known to mediate mitochondrial fission and fusion and to constitute regulatory components controlling mitochondrial dynamics. In the present study, we first investigated mitochondrial dynamics during the cell cycle progression, and found that mitochondria exist as filamentous network structures throughout the cell cycle progression, changing their morphology, distribution, and abundance. In addition, we found that a mouse homolog of human DNA polymerase delta interacting protein 38, referred to as Mitogenin I, and mitochondrial single-stranded DNA-binding protein (mtSSB), identified as upregulated genes in the heart of mice with juvenile visceral steatosis, play a role in the regulation of mitochondrial morphology.

Key words--mitochondria; morphology; fission/fusion; dynamics; disease; cell cycle

--Regular Article--

A Systematic Review on the Short-term Efficacy and Safety of Nicorandil for Stable Angina Pectoris in Comparison with Those of β-Blockers, Nitrates and Calcium Antagonists

Yuki HANAI,^*,a,b Mitsuo MITA,^a Shigeru HISHINUMA,^a and Masaru SHOJI^a

^aDepartment of Pharmacodynamics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan, and ^bDepartment of Pharmacy, Toho University Omori Medical Center, 6-11-1 Omori-nishi, Ota-ku, Tokyo 143-8541, Japan

Nicorandil significantly reducted the incidence of major coronary events in patients with stable angina in a long-term trial, although there are few reports on its short-term efficacy in the treatment and prevention of angina symptoms. We performed a meta-analysis of the short-term efficacy of nicorandil compared with antianginal drugs for stable angina. We selected 20 reports (vs. β-blockers, n=6; vs. nitrates, n=6; vs. calcium antagonists, n=8) of prospective controlled trials from MEDLINE, the Cochrane Library, and Japana Centra Revuo Medicina. The trials were short in duration (median 5 weeks). We combined the results using odds ratios (OR) for discrete data and weighted mean differences (WMD) for continuous data. Compared with antianginal drugs, nicorandil did not show significant reduction of angina episodes per week (vs. β-blockers, −1.50 [95% confidence interval (CI): −4.09, 1.09]; vs. nitrates, 0.22 [95% CI: −1.22, 1.65]; vs. calcium antagonists, −0.23 [95% CI: −1.37, 0.90]). Furthermore, there were no significant differences in time to ischemia (total exercise duration, time to 1-mm ST depression, time to onset of pain). Although the total numbers of adverse events with each antianginal drug were similar, heart rate and blood pressure were significantly decreased by calcium antagonists but not changed by nicorandil (8.09 [95% CI: 3.20, 12.98] and 8.64 [95% CI: 3.28, 13.99], respectively). Thus this study suggests that short-term therapy with nicorandil is as effective as standard therapy and that nicorandil can also be used as a first-line agent in patients with stable angina.

Key words--nicorandil; angina pectoris; meta-analysis; β-blocker; calcium antagonist; nitrate

--Regular Article--

Development of Clinical Pharmacy Services to Improve Drug Adherence in Psychiatric Hospital Patients

Ai TAMAJI,^a,b Akira SAKAI,^b Motoko SATO,^b Yukiko IKEMA,^b Kiyohide MATSUZAKI,^b Hideaki TABUSE,^c and Yukihiro NODA^*,a

^aDivision of Clinical Science and Neuropsychopharmacology, Graduate School of Pharmaceutical Sciences, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan, ^bDepartment of Pharmacy, and ^cDepartment of Psychiatry, Holly Cross Hospital, 2431-160 Hisajiri, Izumi, Toki 509-5142, Japan

In the present study, we investigated whether the pharmacy services in our psychiatric hospital helped to improve the attitude of psychiatric patients to drugs. The subjects were 168 patients who received advice on medication at the hospital between August 2008 and December 2009. We found that anxiety about medication in 76% of these patients was relieved by the provision of clinical pharmacy services. This can be attributed to patients gaining an understanding of the importance of taking medication at a particular time, drug types, drug efficacy and drug-induced adverse events. Patient drug adherence scores using the 10-item version of the Drug Attitude Inventory (DAI-10) were significantly improved after pharmacy services were provided, indicating an improvement in drug adherence. There was a significant positive correlation between the DAI-10 score and understanding of the necessity for medication, but no correlation between the DAI-10 score and the amount of drug administered or number of doses taken per day. These results suggest that the clinical pharmacy services improve understanding of the importance of medication timing, drug type, drug efficacy and drug-induced adverse events, and also relieve medication anxiety, enhance understanding of the necessity of taking medication and improve patient attitude to a drug. We intend to further take comprehensive measures including educational, behavioral and emotional intervention.

Key words--psychiatry; clinical pharmacy service; schizophrenia; drug adherence

--Regular Article--

A Study on Incorporation of “Management and Guidance of Home-visiting by a Pharmacist” into a Care Plan -Observation Based on a Survey of Care Managers-

Yoko NANAUMI,^a Mitsuko ONDA,^*,b Hidehiko SAKURAI,^c Rie TANAKA,^b Kenichi TSUBOTA,^b Shunya MATOBA,^b Yusuke MUKAI,^b Yukio ARAKAWA,^b and Yukitoshi HAYASE^c

^aAdvance Pharma Research Office, 1026-11 Misasagi-cho, Nara 631-0803, Japan, ^bOsaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan, and ^cHokkaido Pharmaceutical University, 7-1 Katsuraoka-cho, Otaru, Hokkaido 047-0264, Japan

A survey of care managers (“CMs”) was conducted to identify top priority issues in promoting the management and guidance of home-visiting by a pharmacist (“management and guidance”) by focusing on the experience in incorporating the management and guidance into a care plan and its relevant factors. Major survey items included (1) number of years working as a CM, (2) basic occupation, (3) experience in incorporating the management and guidance into care plans, (4) understanding the management and guidance content, and (5) an awareness of the need for pharmacists' involvement in care plans. A x² test was conducted to determine if the experience in incorporating the management and guidance into care plans caused a difference in the distribution of the number of years as a CM, the basic occupation, understanding of the management and guidance content, and an awareness of the need for pharmacists' involvement in care plans. A regression analysis was conducted to determine the degree of association between the incorporation experience and each item. The numbers of years working as a CM, the basic occupation, understanding of the management and guidance content, and an awareness of the need for pharmacists' involvement in care plans, were found to be associated with the experience in incorporating the management and guidance into care plans. Understanding of the management and guidance content was most closely associated. To promote pharmacists' management and guidance for home-based care, CMs need to deepen their understanding of this service.

Key words--pharmacist; care manager; community pharmacy; long-term-care; home visiting

--Regular Article--

Specific Accumulation and Antitumor Effects of Hybrid Liposomes on the Growth of Lung Tumor Cells

Mamiko YUKIHARA, Yuji KOMIZU, Osamu TANOUE, Taku MATSUSHITA, Yoko MATSUMOTO, and Ryuichi UEOKA^*

Division of Applied Life Science, Graduate School of Engineering, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan

In general, chemotherapeutic effects were low for non-small cell lung cancer (NSCLC) in the lung tumor. We examined the accumulation and antitumor effects of hybrid liposomes (HL-23) composed of phospholipid (L-α-dimyristoylphosphatidylcholine: DMPC) and PEG surfactant [polyoxyethylene(23)dodecyl ether: C₁₂(EO)₂₃] on NSCLC cells in vitro. Accumulation of HL-23 including a fluorescence probe [1-Palmitoyl-2-[12(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-Glycero-3-Phosphocholine: NBDPC] was observed for NSCLC cells using a confocal laser microscope, but no accumulation of HL-23 in normal lung cells was observed. Furthermore, inhibitory effects of HL-23 on the growth of NSCLC cells were obtained on the basis of a WST-1 assay. It was also clarified that HL-23 induced apoptosis for NSCLC cells on the basis of Annexin-V binding and TUNEL assay. These results suggest that HL-23 could be applied in effective chemotherapies for NSCLC.

Key words--hybrid liposome; non-small cell lung cancer; apoptosis

--Regular Article--

Mechanism of Interaction between Risperidone and Tea Catechin (1)Complex Formation of Risperidone with Epigallocatechin Gallate

Hirohito IKEDA,^* Hanae MORIWAKI, Miho YUKAWA, Yukiko IWASE, and Hatsumi AKI

Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan

The mechanism of complexation between risperidone (RISP) and (−)-epigallocatechin gallate (EGCg) was clarified by ¹H-NMR and molecular modeling studies. RISP and EGCg formed an insoluble complex with a 1 : 1 stoichiometry in aqueous solution. In the ¹H-NMR spectra of RISP in DMSO-d₆, the chemical shifts of protons neighboring the N atom on the piperizine ring clearly moved downfield upon formation of the complex. In the molecular modeling study, the ¹H-chemical shifts for nine optimized structures of the complex were calculated to compare them with those of the experimental results. Only one conformer with the second minimum energy for the complex supported the downfield shifts of RISP protons. It was found from the structure of the complex that the two hydrogen bonds between hydroxyl groups of the galloyl ring in EGCg and N atoms in RISP, one of which was on the piperizine ring, were formed to stabilize the complex.

Key words--tea catechin; risperidone; epigallocatechin gallate; ¹H-NMR spectrometry; computational chemistry; molecular modeling

--Regular Article--

Risk Management of QT-prolonging Drugs by Community Pharmacists by Using a Mobile Electrocardiograph

Kohki SHINOZAKI

Hikuma Pharmacy, 5-17-8 Hikuma, Naka-ku, Hamamatsu, Shizuoka 430-0901, Japan

Prolongation of the QT interval is associated with a high risk of serious arrhythmia, i.e., torsades de pointes (TdP). However, in many cases, the QT-prolonging drug(s) is prescribed without performing a thorough check-up of the patient's condition, especially an electrocardiogram. In addition to patient interview, we used an electrocardiogram obtained with a mobile electrocardiograph (RMH-ECG) in a community pharmacy in order to improve the risk management for QT-prolonging drugs. A comparison of the results obtained using RMH-ECG (modified I) and 12-lead ECG (I) revealed that both corrected QT (QTc) values were almost identical, and the correlation coefficient was 0.96. In one month, 5 of 948 patients who visited our pharmacy and continuously took QT-prolonging drugs had additional risk factors for TdP (advanced age, female, and drug-drug interaction). We monitored the QT interval of one of these patients. She had received erythromycin for 19 months along with other drugs metabolized by a P450 (CYP3A4); benidipine and prednisolone (for over 2 years), and tacrolimus (for 13 weeks). Three RMH-ECG tests at every 2 weeks revealed that QTcs were normal (0.43-0.45 s); therefore, we dispensed drugs without any change in the prescription. Approximately 1 in 1200 individuals has a prolonged QT interval without any subjective symptoms, and the time window of drug-induced TdP is considered to be from several hours to months after taking these drugs. Therefore, we think that an ECG test should be performed in community pharmacies before dispensing QT-prolonging drugs and that the QT interval should be monitored.

Key words--mobile electrocardiograph; community pharmacy; QT-prolongation

--Regular Article--

HPLC Determination of Five Polyphenols in Rat Plasma after Intravenous Administrating Hawthorn Leaves Extract and Its Application to Pharmacokinetic Study

Si-Yuan WANG,^a Ji-Yan CHAI,^b Wen-Jie ZHANG,^a Xun LIU,^a Yang DU,^a Zhong-Zhe CHENG,^a Xi-Xiang YING,^*,a and Ting-Guo KANG^a

^aDepartment of Pharmaceutical Analysis, School of Pharmacy, Liaoning University of Traditional Chinese Medicine, 77 Shengming 1 Road, DD Port, Dalian 116600, China, and ^bTeaching Experiment Center, Liaoning University of Traditional Chinese Medicine, 79 Chongshang East Road, Shenyang 110032, China

A simple and specific HPLC-UV method was developed to simultaneously determine five active compounds including vitexin-4″-O-glucoside (VG), vitexin-2″-O-rhamnoside (VR), vitexin (VIT), rutin (RUT) and hyperoside (HP) in rat plasma after intravenous administrating the hawthorn leaves extract (HLE). With baicalin as internal standard (I.S.), sample pretreatment involved a one-step extraction with methanol of 0.2 ml plasma. The HPLC assay was carried out using a Phenomsil C18 analytical column with UV detection at 332 nm. The mobile phase consisted of methanol-acetonitrile-tetrahydrofuran-1% glacial acetic acid (6:1.5:18.5:74, v/v/v/v). The calibration curves were liner over the range of 2.030-500.5, 0.1513-75.64, 0.2507-12.54, 0.5128-25.64 and 0.4032-20.16 μg/ml for VG, VR, VIT, RUT and HP, respectively. The relative standard deviations (RSD) of the intra- and inter-day precisions for the analysis of the five analytes were between 1.0 and 8.9% with accuracies (relative error) below 8.2% for the analysis of the five analytes. The average extraction recoveries of five analytes were more than 82.67±4.74%. The HPLC method herein described was fully validated and successfully applied to the pharmacokinetic studies after intravenous administration of HLE solution to rats over three doses.

Key words--HPLC; hawthorn leaves extract (HLE); polyphenol; rat plasma; pharmacokinetics