Synthesis and evaluation of a spiro-isobenzofuranone class of histamine H3 receptor inverse agonists

Spiro-isobenzofuranones 1a and 1b were discovered as potent, selective, and brain-penetrable non-imidazole H3 receptor inverse agonists. Our corporate sample collection was screened to identify 2a as a lead. Recognizing the right-hand portion of 2a as an essential pharmacophore, an extensive screen of the left-hand piperidine portion was carried out to yield the potent spiro-derivatives 2t-x. Spiro-isobenzofuranone 2x, the most potent among the derivatives, was converted to the corresponding amide 1a, which possessed dramatically improved H3 activity (IC(50)=0.72 nM; more than 20-fold improvement over 2x). Further elaboration led to the identification of 1b, a 5-methoxy derivative with an IC(50) of 0.54 nM. Our studies demonstrated that derivatives 1a and 1b to be potent, selective, and brain-penetrable H3 inverse agonists.     

Development of a novel polycationic adsorbent for cryogel removal.

Cryogel, prevalent in the plasma of rheumatism patients, is a plasma fibronectin (pFN)-extra domain A containing FN (EDA(+)FN)-fibrinogen (Fbg) complex formed by adding heparin (HP) at a low temperature (4 degrees C). Although EDA(+)FN does not usually exist in normal plasma, its prevalence in rheumatic patients causes cryogelation in plasma. Removal of cryogel is thus a promising and novel approach to treating rheumatism. As HP-EDA(+)FN aggregate, which is induced by the main component of cryogel, is considered to be an anion, cationic materials capable of eliminating this anionic conjugate were innovated in this study. We found that an amino group density of 100-130 micromol/g (dry weight) of adsorbents prompted selective adsorption of the EDA(+)FN-HP complex. Elimination of EDA(+)FN as high as 80% accompanied by removal of the components of total FN (pFN) (10%) and Fbg (10%) in the model patient plasma was established.     

Novel plasma-separation dilayer gellan-gellan-sulfate adsorber for direct removal of extra domain A containing fibronectin from the blood of rheumatoid arthritis patients

Rheumatoid arthritis (RA) patients, in whom cryogelation occurs in the presence of heparin, exhibit abnormally high concentrations of extra domain A containing fibronectin [EDA(+)FN] in their plasma. The selective removal of EDA(+)FN from patient blood is therefore of potential therapeutic benefit. Gellan-sulfate is a candidate ligand for the removal of EDA(+)FN due to its high affinity for FN. In this study, we prepare a novel adsorber for the direct removal of EDA(+)FN from patient blood. The adsorber has both a plasma separation function and EDA(+)FN trapping zones, and is prepared by cross-linking gellan-sulfate with epichlorohydrine. The ratio of gellan-sulfate to gellan in the adsorber is 48%. The surface and internal structure of gellan beads were observed by a range of microscopic techniques, and the beads were found to have a dilayer structure, consisting of a porous outer layer and an underlying gellan-sulfate phase as the adsorber. The affinity constants of the gellan-sulfate beads for EDA(+)FN were almost the same in blood as in buffer because the porous gellan coating acts to separate plasma from the cellular fraction of the blood. The removal rate of plasma proteins and blood cells from mock RA blood was measured for coated and uncoated gellan-sulfate beads. Removal rates were 30-32% for EDA(+)FN, 6-10% for fibrinogen, 10-14% for antithrombin III, 8% for C3, 4-7% for C4, and 0% for albumin. The removal rates of uncoated beads were 11% for white blood cells, 0% for red blood cells and 33% for platelets, whereas removal rates of 0% for white blood cells, 0% for red blood cells and 20% for platelets were achieved for coated beads. The coating effectively inhibits the adsorption of white blood cells and platelets. Existing problems with direct adsorbers, including selectivity and plasma separation, have been solved by this material.     

Immobilized gellan sulfate surface for cell adhesion and multiplication: development of cell-hybrid biomaterials using self-produced fibronectin

A new concept for cell-hybrid biomaterial is proposed in which human unbilical vein endothelial cells (HUVEC) are adhered to an immobilized gellan sulfate (GS) surface. Extra domain A containing fibronectin (EDA(+)FN) released from HUVEC is necessary for cell adhesion and multiplication. The material design in this study is based on these self-released cell adhesion proteins. The interaction between GS and EDA(+)FN was evaluated using the affinity constant (KA); the value obtained was 1.03x10(8) (M(-1)). These results suggest that the adhesion of HUVEC to GS may be supported by the adhesion of EDA(+)FN to GS. We also found that this new material adheres to HUVEC, allowing the reintroduction of EDA(+)FN, which is self-produced by the cell. This material is relatively easy to produce, not requiring the usual coating of adhesion proteins in pretreatment.     

Chronic intracerebroventricular infusion of MCH causes obesity in mice. Melanin-concentrating hormone

Melanin-concentrating hormone (MCH) is a cyclic amino acid neuropeptide localized in the lateral hypothalamus. Although MCH is thought to be an important regulator of feeding behavior, the involvement of this peptide in body weight control has been unclear. To examine the role of MCH in the development of obesity, we assessed the effect of chronic intracerebroventricular infusion of MCH in C57BL/6J mice that were fed with regular or moderately high-fat (MHF) diets. Intracerebroventricular infusion of MCH (10 microg/day for 14 days) caused a slight but significant increase in body weight in mice maintained on the regular diet. In the MHF diet-fed mice, MCH more clearly increased the body weight accompanied by a sustained hyperphagia and significant increase in fat and liver weights. Plasma glucose, insulin, and leptin levels were also increased in the MCH-treated mice fed the MHF diet. These results suggest that chronic stimulation of the brain MCH system causes obesity in mice and imply that MCH may have a major role in energy homeostasis.     

Characterization of MCH-mediated obesity in mice

Melanin-concentrating hormone (MCH) is a cyclic orexigenic peptide expressed in the lateral hypothalamus. Recently, we demonstrated that chronic intracerebroventricular infusion of MCH induced obesity accompanied by sustained hyperphagia in mice. Here, we analyzed the mechanism of MCH-induced obesity by comparing animals fed ad libitum with pair-fed and control animals. Chronic infusion of MCH significantly increased food intake, body weight, white adipose tissue (WAT) mass, and liver mass in ad libitum-fed mice on a moderately high-fat diet. In addition, a significant increase in lipogenic activity was observed in the WAT of the ad libitum-fed group. Although body weight gain was marginal in the pair-fed group, MCH infusion clearly enhanced the lipogenic activity in liver and WAT. Plasma leptin levels were also increased in the pair-fed group. Furthermore, MCH infusion significantly reduced rectal temperatures in the pair-fed group. In support of these findings, mRNA expression of uncoupling protein-1, acyl-CoA oxidase, and carnitine palmitoyltransferase I, which are key molecules involved in thermogenesis and fatty acid oxidation, were reduced in the brown adipose tissue (BAT) of the pair-fed group, suggesting that MCH infusion might reduce BAT functions. We conclude that the activation of MCH neuronal pathways stimulated adiposity, in part resulting from increased lipogenesis in liver and WAT and reduced energy expenditure in BAT. These findings confirm that modulation of energy homeostasis by MCH may play a critical role in the development of obesity.     

Genomic organization and expression pattern of mouse neuroglycan C in the cerebellar development

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate proteoglycan with an epidermal growth factor module that is expressed predominantly in the brain. Cloning studies with mouse NGC cDNA revealed the expression of three distinct isoforms (NGC-I, -II, and -III) in the brain and revealed that the major isoform showed 94. 3% homology with the rat counterpart. The NGC gene comprised six exons, was approximately 17 kilobases in size, and was assigned to mouse chromosome band 9F1 by fluorescence in situ hybridization. Western blot analysis demonstrated that, although NGC in the immature cerebellum existed in a proteoglycan form, most NGC in the mature cerebellum did not bear chondroitin sulfate chain(s), indicating that NGC is a typical part-time proteoglycan. Immunohistochemical studies showed that only the Purkinje cells were immunopositive in the cerebellum. In the immature Purkinje cells, NGC, probably the proteoglycan form, was immunolocalized to the soma and thick dendrites on which the climbing fibers formed synapses, not to the thin branches on which the parallel fibers formed synapses. This finding suggests the involvement of NGC in the differential adhesion and synaptogenesis of the climbing and parallel fibers with the Purkinje cell dendrites.     

Identification of new geometric isomers of methyl linoleate hydroperoxide and their chromatographic behavior

New geometric isomers, methyl (9Z,11Z)-13-hydroperoxy-9,11-octadecadienoate and methyl (10Z,12Z)-9-hydroperoxy-10,12-octadecadienoate, were proved to be present in methyl linoleate hydroperoxide produced by autoxidation. They were identified from their UV, MS, and 1H-NMR spectra after conversion to the corresponding oxo derivatives: methyl (9Z,11Z)-13-oxo-9,11-octadecadienoate and methyl (10Z,12Z)-9-oxo-10,12-octadecadienoate. Their chromatographic behavior is described.