Better Peptides via Chemical Glycosylation: Somatostatin Analogues Having a Human Complex-Type N-Glycan with Improved Drug Properties.

Somatostatin (somatotropin release-inhibiting factor, SRIF) is a growth hormone inhibitory factor in the form of a 14- or 28-amino acid peptide. SRIF affects several physiological functions through its action on five distinct SRIF receptor subtypes (sst1-5). Native SRIF has only limited clinical applications due to its rapid degradation in plasma. To overcome this obstacle, we have developed glycosylated SRIF analogues that possess not only metabolic stability but also high affinity to all five receptor subtypes by attaching human complex-type oligosaccharides. Such glycosylated SRIF analogues with improved pharmacokinetic profiles could be potent and novel therapeutic drugs for SRIF-related diseases in which several SRIF receptor subtypes are closely involved, and also shed light on new indications. Our results show that chemical glycosylation can be a powerful tool for the development of peptide and protein analogues superior to the original molecules with enhanced drug properties.

Evaluation of acute toxicity of cancer-targeting albumin-based artificial metalloenzymes.

Recently, the development of abiotic metal-mediated drug delivery has been significant growth in the fields of anticancer approach and biomedical application. However, the intrinsic toxicity of abiotic metal catalysts makes in vivo use difficult. Our group developed a system of cancer-targeting albumin-based artificial metalloenyzmes (ArMs) capable of performing localized drug synthesis and selective tagging therapy in vivo for cancer therapy. The toxicity of the system at higher concentrations was investigated in vitro and in vivo in the study to demonstrate its safety for potential application in clinical trials. In cell-based experiments, the study revealed that the cytotoxicity of metal catalysts anchored within the binding cavity of the cancer-targeting ArMs could be significantly reduced compared to free-in-solution metal catalysts. Moreover, the in vivo data demonstrated that the cancer-targeting ArMs did not cause considerable damage in organs or change in the hematological parameters in a single-dose (160 mg/Kg) toxicity study in rats. Therefore, the system is safe, highlighting that it could be used in clinical trials for cancer treatment.

Simple Gd3+-Neu5NAc complexation results in NMR chemical shift asymmetries of structurally equivalent complex-type N-glycan branches.

In the present Communication, we propose a quite simple but previously overlooked approach for conveniently analyzing, assigning, and extracting sialic acid-containing N-glycan structures using high-resolution NMR spectroscopy without pre-installing metal chelators. Paramagnetic metals, such as Gd3+, appear to bind to the carboxyl groups of N-acetylneuraminic acid when introduced at room temperature, leading to the measurement of nonequivalent proton and carbon NMR spectral signals among otherwise "identical" glycan branched structures.

Delayed treatment with arundic acid reduces the MPTP-induced neurotoxicity in mice.

The authors investigated the protective effects of a novel astrocyte-modulating agent, arundic acid, in a 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine (MPTP) mouse model of Parkinson's disease. Male mice received four intraperitoneal (i.p.) injections of MPTP (20 mg/kg) at 2 h intervals. The content of dopamine and its metabolites in the striatum was reduced markedly 7 days after MPTP treatment. The delayed treatment with arundic acid (30 mg/kg, i.p.) administered 3, 4, 5 and 6 days after MPTP treatment did not affect the depletion of dopamine and its metabolites in the striatum. Our immunohistochemical study with anti-tyrosine hydroxylase antibody, anti-neuronal nuclei antibody, anti-glial fibrillary acidic protein antibody, anti-S 100beta antibody and anti-nestin antibody showed that the delayed treatment with arundic acid had a protective effect against MPTP-induced neuronal damage in the striatum and the substantia nigra of mice. Furthermore, this agent ameliorated the severe reductions in number of isolectin reactive microglia in the striatum and the substantia nigra 7 days after MPTP treatment. These results demonstrate that the inhibition of S 100beta synthesis in astrocytes may be the major component of the beneficial effect of arundic acid. Thus, our present findings provide that the therapeutic strategies targeted to astrocytic modulation with arundic acid offers a great potential for restoring the functional capacity of the surviving dopaminergic neurons in individuals affected with Parkinson's disease.

Arundic Acid ameliorates cerebral amyloidosis and gliosis in Alzheimer transgenic mice.

Like microglia, reactive astrocytes produce a myriad of neurotoxic substances in various brain pathologies, such as Alzheimer's disease (AD), trauma, and cerebral ischemia. Among the numerous products of reactive astrocytes, attention has recently been directed toward the possible detrimental role of S100B, because the protein has been shown to be highly expressed along with the progression of brain damage and to exert neurotoxic effects at high concentrations. The present study aimed to examine the possible role of astrocyte-derived S100B in the progression of cerebral amyloidosis and gliosis in transgenic mice overproducing mutant amyloid precursor protein (Tg APP(sw) mice, line 2576). For this purpose, arundic acid (Ono Pharmaceutical Co., Ltd., Mishima, Osaka, Japan), which is known to negatively regulate astrocyte synthesis of S100B, was orally administered to Tg APP(sw) mice for 6 months from 12 months of age, and the effects of the agent on the above parameters were examined. Here, we report that beta-amyloid deposits along with amyloid-beta peptide/S100B levels, as well as beta-amyloid plaque-associated reactive gliosis (astrocytosis and microgliosis), were significantly ameliorated in arundic acid-treated Tg APP(sw) mice relative to vehicle-treated Tg APP(sw) mice at 19 months of age. Based on the above results, arundic acid is considered to deserve further exploration as a promising therapeutic agent for AD.

[S100B: astrocyte specific protein].

The S100B is a Ca2+ binding proteins of EF-hand type and is produced primarily by astrocytes in the central nervous system. This protein has been implicated in the Ca2+-dependent regulation of a variety of intracellular functions such as protein phosphorylation, enzyme activities, cell proliferation and differentiation, dynamics of cytoskeleton constituents, structural organization of membranes, intracellular Ca2+ homeostasis, inflammation, and protection from oxidative cell damage. Recent studies suggest that released S100B exerts paracrine and autocrine effects on neurons and glia. On the other hand, elevations of S100B levels in blood or cerebrospinal fluid have been observed in patients with Alzheimer's disease, Down's syndrome, amyotrophic lateral sclerosis, multiple sclerosis, schizophrenia, depression, cerebral stroke and traumatic brain injury, and the levels have reached micromol/L-order at focal regions. It has been documented that the excessive S100B promotes the expression of inducible nitric oxide synthase or pro-inflammatory cytokines and exhibits detrimental effects on neurons. On studies using some animal models of the cerebral stroke or Alzheimer's disease, it is suggested that the excessive S100B produced by activated astrocytes precedes neurodegenerations. Authors discussed the relationship between neurological disorders and the S100B.

Relevance of astrocytic activation to reductions of astrocytic GABAA receptors.

Although astrocytes express gamma-aminobutyric acid subtype-A (GABAA) receptors in the mature brain, GABAA receptor expression in a cultivation state remains controversial. In this study, we investigated the alteration of astrocytic GABAA receptor expression in in vitro and in vivo studies to elucidate the relevance of astrocytic activation to reductions of astrocytic GABAA receptors. The GABA-evoked Cl- current (GABAA response) in cultured astrocytes was determined by recording in the whole-cell mode using a conventional patch-clamp technique under voltage-clamp conditions. The respective amplitudes of GABAA responses on days in vitro 1, 3-5, 7-10, and 12-15 were 1019+/-97, 512+/-76, 84+/-21, and 22+/-9 pA, respectively, suggesting that the GABAA response subsequently diminished with in vitro aging. In immunohistochemical and biochemical analyses, the expression of GABAA receptor beta-subunit decreased, whereas expressions of glial fibrillary acidic protein (GFAP) and S100B, hallmarks of astrocytic activation, increased dramatically in the cultured astrocytes with in vitro aging. With the use of [3H]SR95531, a GABAA-specific ligand, at 24 h after transient focal ischemia, binding was significantly reduced in the astrocytic fractions without affecting the synaptosomal fractions, and decreases in the mRNA expression level of GABAA receptor beta-subunits were concurrently observed. Interestingly, the loss of GABAA response in cultured astrocytes was mitigated by co-culturing with neurons or treatments with monoclonal S100B antibodies. These results indicate that astrocytic GABAA receptors are reduced with in vitro aging and cerebral ischemia, presumably through the overproduction of S100B in activated astrocytes.

Relationship between S100beta and GFAP expression in astrocytes during infarction and glial scar formation after mild transient ischemia.

The expression of astrocyte marker proteins (S100beta and GFAP) during infarction and glial scar formation after transient middle cerebral artery (MCA) occlusion was examined using double immunostaining. S100beta immunoreactivity markedly decreased in the core of the injured area when observed immediately after reperfusion and did not increase again. In the periphery, however, S100beta expression increased, showing that S100beta synthesis was up-regulated. S100beta+/iNOS+ astrocytes in the periphery were observed from day 1, when small infarct areas were detectable, up to day 5, when infarct expansion had almost ended. TUNEL+ cells in the periphery were present from days 1 to 5. S100beta+/TUNEL+ cells were observed centrally and around the periphery of the injured area, predicting that cell death contributes to the increase of S100beta concentration in the injured area. Our results suggest that (1) higher concentration of S100beta in the extracellular space due to S100beta leakage from damaged astrocytes leads to up-regulation of S100beta synthesis and induction of inducible nitric oxide synthase (iNOS) synthesis in astrocytes, contributing to infarct expansion that results in DNA damage or cell death via NO and ROS production, and (2) GFAP, but not S100beta, is a main contributor to glial scar formation. On day 1 postreperfusion, the microdiascopic images of the injured areas from the unstained thick sections or the areas detected by S100beta immunoreactivity were larger than those of the infarct areas detected by hematoxylin--eosin (HE)-staining. The difference between these sizes might be useful to predict infarct expansion.

Attenuation of a delayed increase in the extracellular glutamate level in the peri-infarct area following focal cerebral ischemia by a novel agent ONO-2506.

A novel agent, ONO-2506 [(R)-(-)-2-propyloctanoic acid, ONO Pharmaceutical Co. Ltd.] was previously shown to mitigate delayed infarct expansion through inhibition of the enhanced production of S-100beta, while inducing a prompt symptomatic improvement that attained a significant level as early as 24h after drug administration. To elucidate the mechanism underlying the prompt symptomatic improvement, the present study aimed to examine whether ONO-2506 modulates the level of extracellular glutamate ([Glu]e) in the rat subjected to transient middle cerebral artery occlusion (tMCAO). In this model, it had been shown that ONO-2506 reduces the infarct volume, improves the neurological deficits, and enhances the mRNA expression of glial glutamate transporters (GLT-1 and GLAST). The [Glu]e levels in the ischemic cortices were continuously measured using intracerebral microdialysis. The alterations in the [Glu]e levels in the sham-operated and tMCAO-operated groups with or without drug administration were compared. In the tMCAO groups, the [Glu]e level increased during tMCAO to a similar extent, returned to normal on reperfusion, and increased again around 5h. In the saline-treated group, however, the [Glu]e level further increased from 15 h on to reach about 280% of the normal level at 24h. This secondary increase in the [Glu]e level in the late phase of reperfusion was prevented by ONO-2506. The intracerebral infusion of glutamate transporter inhibitor, l-trans-pyrrolidine-2,4-dicarboxylic acid, at 24h after tMCAO induced an increase in the [Glu]e level, which was marked in both the sham-operated and ONO-2506-treated groups, but much less pronounced in the saline-treated group. The above results suggest that functional modulation of activated astrocytes by pharmacological agents like ONO-2506 may inhibit the secondary rise of [Glu]e level in the late phase of reperfusion, leading to amelioration of delayed infarct expansion and neurological deficits.