A2B adenosine receptor inhibition by the dihydropyridine calcium channel blocker nifedipine involves colonic fluid secretion.

The adenosine A2B receptor is a critical protein in intestinal water secretion. In the present study, we screened compound libraries to identify inhibitors of the A2B receptor and evaluated their effect on adenosine-induced intestinal fluid secretion. The screening identified the dihydropyridine calcium antagonists nifedipine and nisoldipine. Their respective affinities for the A2B receptor (Ki value) were 886 and 1,399 nM. Nifedipine and nisoldipine, but not amlodipine or nitrendipine, inhibited both calcium mobilization and adenosine-induced cAMP accumulation in cell lines. Moreover, adenosine injection into the lumen significantly increased fluid volume in the colonic loop of wild-type mice but not A2B receptor-deficient mice. PSB-1115, a selective A2B receptor antagonist, and nifedipine prevented elevated adenosine-stimulated fluid secretion in mice. Our results may provide useful insights into the structure-activity relationship of dihydropyridines for A2B receptor. As colonic fluid secretion by adenosine seems to rely predominantly on the A2B receptor, nifedipine could be a therapeutic candidate for diarrhoea-related diseases.

Isolation of Adipose-Derived Stem/Stromal Cells from Cryopreserved Fat Tissue and Transplantation into Rats with Spinal Cord Injury.

Adipose tissue contains multipotent cells known as adipose-derived stem/stromal cells (ASCs), which have therapeutic potential for various diseases. Although the demand for adipose tissue for research use remains high, no adipose tissue bank exists. In this study, we attempted to isolate ASCs from cryopreserved adipose tissue with the aim of developing a banking system. ASCs were isolated from fresh and cryopreserved adipose tissue of rats and compared for proliferation (doubling time), differentiation capability (adipocytes), and cytokine (hepatocyte growth factor and vascular endothelial growth factor) secretion. Finally, ASCs (2.5 × 106) were intravenously infused into rats with spinal cord injury, after which hindlimb motor function was evaluated. Isolation and culture of ASCs from cryopreserved adipose tissue were possible, and their characteristics were not significantly different from those of fresh tissue. Transplantation of ASCs derived from cryopreserved tissue significantly promoted restoration of hindlimb movement function in injured model rats. These results indicate that cryopreservation of adipose tissue may be an option for clinical application.

Adenosine A2B Receptors: An Optional Target for the Management of Irritable Bowel Syndrome with Diarrhea?

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder, with the characteristic symptoms of chronic abdominal pain and altered bowel habits (diarrhea, constipation, or both). IBS is a highly prevalent condition, which negatively affects quality of life and is a significant burden on global healthcare costs. Although many pharmacological medicines have been proposed to treat IBS, including those targeting receptors, channels, and chemical mediators related to visceral hypersensitivity, successful pharmacotherapy for the disease has not been established. Visceral hypersensitivity plays an important role in IBS pathogenesis. Immune activation is observed in diarrhea-predominant patients with IBS and contributes to the development of visceral hypersensitivity. Adenosine is a chemical mediator that regulates many physiological processes, including inflammation and nociception. Among its receptors, the adenosine A2B receptor regulates intestinal secretion, motor function, and the immune response. We recently demonstrated that the adenosine A2B receptor is involved in visceral hypersensitivity in animal models of IBS. In this review, we discuss the possibility of the adenosine A2B receptor as a novel therapeutic target for IBS.

In situ depot formation of anti-HIV fusion-inhibitor peptide in recombinant protein polymer hydrogel.

Most peptide drugs have short half-lives, necessitating frequent injections that may induce skin sensitivity reactions; therefore, versatile prolonged-release delivery platforms are urgently needed. Here, we focused on an oxidatively and thermally responsive recombinant elastin-like polypeptide with periodic cysteine residues (cELP), which can rapidly and reversibly form a disulfide cross-linked network in which peptide can be physically incorporated. As a model for proof of concept, we used enfuvirtide, an antiretroviral fusion-inhibitor peptide approved for treatment of human immunodeficiency virus (HIV) infection. cELP was mixed with enfuvirtide and a small amount of hydrogen peroxide (to promote cross-linking), and the soluble mixture was injected subcutaneously. The oxidative cross-linking generates a network structure, causing the mixture to form a hydrogel in situ that serves as an enfuvirtide depot. We fabricated a series of enfuvirtide-containing hydrogels and examined their stability, enfuvirtide-releasing profile and anti-HIV potency in vitro. Among them, hydrophobic cELP hydrogel provided effective concentrations of enfuvirtide in blood of rats for up to 8 h, and the initial concentration peak was suppressed compared with that after injection of enfuvirtide alone. cELP hydrogels should be readily adaptable as platforms to provide effective depot systems for delivery of other anti-HIV peptides besides enfuvirtide. STATEMENT OF SIGNIFICANCE: In this paper, we present an anti-HIV peptide delivery system using oxidatively and thermally responsive polypeptides that contain multiple periodic cysteine residues as an injectable biomaterial capable of in situ self-gelation, and we demonstrate its utility as an injectable depot capable of sustained release of anti-HIV peptides. The novelty of this work stems from the platform employed to provide the depot encapsulating the peptide drugs (without chemical conjugation), which consists of rationally designed, genetically engineered polypeptides that enable the release rate of the peptide drugs to be precisely controlled.

Intravenous infusion of adipose-derived stem/stromal cells improves functional recovery of rats with spinal cord injury.

BACKGROUND AIMS: Adipose tissue has therapeutic potential for spinal cord injury (SCI) because it contains multipotent cells known as adipose-derived stem/stromal cells (ASCs). In this study, we attempted intravenous ASC transplantation in rats with SCI to examine the effect on functional recovery. METHODS: ASCs (2.5 × 106) were intravenously infused into SCI rats, after which hindlimb motor function was evaluated. Distribution of transplanted ASCs was investigated and growth factor/cytokine levels were determined. RESULTS: Intravenous transplantation of ASCs promoted the functional recovery in SCI rats and reduced the area of spinal cord cavitation. A distribution study revealed that ASCs gradually accumulated at the site of injury, but long-term survival of these cells was not achieved. Levels of growth factors increased only slightly in the spinal cord after ASC transplantation. Unexpectedly, cytokine-induced neutrophil chemoattractant (CINC)-1 showed a transient but substantial increase in the spinal cord tissue and blood of the ASC group. CINC-1 was secreted by ASCs in vitro, and the sponge implantation assay showed that CINC-1 and ASCs induced angiogenesis. CINC-1 promoted functional recovery in SCI rats, which was similar to the ASCs. Expression of glial cell line-derived neurotrophic factor was greater in the ASC group than in the CINC-1 group, although both promoted extracellular signal-regulated kinase (ERK)1/2 phosphorylation; Akt phosphorylation was enhanced in the spinal cord after ASC transplantation. CONCLUSIONS: Our findings indicated that intravenously transplanted ASCs gradually accumulated in the injured spinal cord, where cytokines such as CINC-1 activated ERK1/2 and Akt, leading to functional recovery.

Aminophylline suppresses stress-induced visceral hypersensitivity and defecation in irritable bowel syndrome.

Pharmacological therapy for irritable bowel syndrome (IBS) has not been established. In order to find candidate drugs for IBS with diarrhea (IBS-D), we screened a compound library of drugs clinically used for their ability to prevent stress-induced defecation and visceral hypersensitivity in rats. We selected the bronchodilator aminophylline from this library. Using a specific inhibitor for each subtype of adenosine receptors (ARs) and phosphodiesterases (PDEs), we found that both A2BARs and PDE4 are probably mediated the inhibitory effect of aminophylline on wrap restraint stress (WRS)-induced defecation. Aminophylline suppressed maternal separation- and acetic acid administration-induced visceral hypersensitivity to colorectal distension (CRD), which was mediated by both A2AARs and A2BARs. We propose that aminophylline is a candidate drug for IBS-D because of its efficacy in both of stress-induced defecation and visceral hypersensitivity, as we observed here, and because it is clinically safe.

Potential new chemotherapy strategy for human ovarian carcinoma with a novel KSP inhibitor.

Among synthetic kinesin spindle protein (KSP) inhibitor compounds, KPYB10602, a six-member lactam-fused carbazole derivative was the most potent in vitro against cell growth of human ovarian cancer, A2780. KPYB10602 caused dose-dependent suppression of tumor growth in vivo. Mitotic arrest due to KPYB10602 was confirmed in vitro, and was characterized by inhibition of securin degradation. Apoptosis after mitotic arrest was associated with an increase in the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Increase of reactive oxygen species (ROS) and caspase pathway were also involved. Furthermore, KPYB10602 caused little neurotoxicity in vivo. Therefore, KPYB10602 could be a promising candidate as an anti-tumor agent with reduced adverse events for treating human ovarian cancer.

Cell therapy with adipose tissue-derived stem/stromal cells for elastase-induced pulmonary emphysema in rats.

AIMS: The purpose of this study was to elucidate the mechanism underlying the effects of adipose tissue-derived stem/stromal cell (ASC) transplantation on porcine pancreatic elastase-induced emphysema. MATERIALS & METHODS: ASCs (2.5 × 10(6)) were transplanted into pancreatic elastase (250 U/kg)-treated rats, after which gas exchange and growth factor/cytokine levels in lung tissue were determined. RESULTS: ASC transplantation restored pulmonary function (arterial oxygen tension and alveolar-arterial oxygen tension difference) almost to that of normal animals. Enlargement of the alveolar airspaces was inhibited. HGF and CINC-1 levels were significantly higher in the ASC group even at 2 weeks after transplantation. Sponge implantation with CINC-1 induced neovascular formation with increased HGF. In vitro secretion of HGF and CINC-1 from ASCs was promoted in the presence of IL-1ß. CONCLUSION: Not only HGF, but also CINC-1, secreted from transplanted and viable ASCs presumably contributed to lung repair through angiogenesis.

The effect of Am-80, a synthetic retinoid, on spinal cord injury-induced motor dysfunction in rats.

The present study investigated the effect of 4[(5,6,7,8-tetrahydro-5,5,8,8,-tetramethyl-2-naphthalenyl)carbamoyl] benzoic acid (Am-80), a synthetic retinoid, on spinal cord injury (SCI) in rats. Treatment with Am-80 (orally and subcutaneously) significantly promoted recovery from SCI-induced motor dysfunction. On day 28 after injury, the lesion cavity was markedly reduced, while the expression of myelin basic protein (MBP; myelin), betaIIItubulin (neuron), and glial fibrillary acidic protein (GFAP; astrocyte) was increased, in comparison with SCI controls. Interestingly, expression of neurotrophin receptor, tyrosine kinase B (TrkB) was over 3-fold higher after Am-80 treatment than in SCI controls. A lot of TrkB-positive cells as well as brain-derived neurotrophic factor (BDNF)-positive ones were observed around the injured site. Am-80 (10 microM) combined with BDNF (100 ng/ml) promoted extensive neurite outgrowth and TrkB gene expression by cultured SH-SY5Y cells, as did all-trans retinoic acid (ATRA). Thymidine incorporation was dramatically suppressed, but there was little effect on cell viability. These findings suggest that Am-80 has the potential to be used for treating neurodegenerative disorders, including SCI. Its efficacy may be partly ascribed to promotion of cell viability and differentiation of neural stem cells through increased TrkB expression.

Therapeutic effect of lecithinized superoxide dismutase against colitis.

Ulcerative colitis (UC) involves intestinal mucosal damage induced by reactive oxygen species (ROS), in particular, superoxide anion. Superoxide dismutase (SOD) catalyzes dismutation of superoxide anion to hydrogen peroxide, which is subsequently detoxified by catalase. Lecithinized SOD (PC-SOD) is a new modified form of SOD that has overcome previous clinical limitations of SOD. In this study, we examined the action of PC-SOD using an animal model of UC, dextran sulfate sodium (DSS)-induced colitis. DSS-induced colitis was ameliorated by daily intravenous administration of PC-SOD. Unmodified SOD produced a similar effect but only at more than 30 times the concentration of PC-SOD. In vivo electron spin resonance analysis confirmed that the increase in the colonic level of ROS associated with development of colitis was suppressed by PC-SOD administration. The dose-response profile of PC-SOD was bell-shaped, but simultaneous administration of catalase restored the ameliorative effect at high doses of PC-SOD. Accumulation of hydrogen peroxide was observed with the administration of high doses of PC-SOD, an effect that was suppressed by the simultaneous administration of catalase. We also found that either a weekly intravenous administration or daily oral administration of PC-SOD conferred protection. These results suggest that PC-SOD achieves its ameliorative effect against colitis through decreasing the colonic level of ROS and that its ineffectiveness at higher doses is because of the accumulation of hydrogen peroxide. Furthermore, we consider that intermittent or oral administration of PC-SOD can be applied clinically to improve the quality of life of UC patients.

Mature adipocyte-derived cells, dedifferentiated fat cells (DFAT), promoted functional recovery from spinal cord injury-induced motor dysfunction in rats.

Transplantation of mature adipocyte-derived cells (dedifferentiated fat cells) led to marked functional recovery from spinal cord injury (SCI)-induced motor dysfunction in rats. When mature adipocytes were isolated from rat adipose tissue and grown in ceiling culture, transformation into fibroblast-like cells without lipid droplets occurred. These fibroblast-like cells, termed dedifferentiated fat cells (DFAT), could proliferate and could also differentiate back into adipocytes. DFAT expressed neural markers such as nestin, betaIII tubulin, and GFAP. Allografting of DFAT into SCI-induced rats led to significant recovery from hindlimb dysfunction. Grafted cells were detected at the injection site, and some of these cells expressed betaIII tubulin. DFAT expressed neurotrophic factors such as BDNF and GDNF prior to transplantation, and grafted cells were also positive for these factors. Therefore, these neurotrophic factors derived from grafted DFAT might have contributed to the promotion of functional recovery. These findings also suggest that mature adipocytes could become a new source for cell replacement therapy to treat central nervous system disorders.

Prolonging the in vivo residence time of prostaglandin E(1) with biodegradable nanoparticles.

PURPOSE: Prostaglandins have potent and diverse biologic activities, but their clinical application is severely restricted, mainly due to rapid inactivation in vivo. In order to modulate the pharmacokinetics of prostaglandin E(1) (PGE(1)), we prepared biodegradable nanoparticles as a drug carrier. METHODS: Nanoparticles encapsulating PGE(1) were prepared from a blend of poly(lactic acid) homopolymer and poly(ethylene glycol)-poly(lactide) block copolymer by the solvent diffusion method in the presence of iron. RESULTS: PGE(1) was efficiently and stably embedded in the nanoparticles through interaction with iron, despite being relatively hydrophilic and having unstable chemical properties. Depending on the isomers and molecular weight of poly(lactic acid) selected, PGE(1) was gradually released from the nanoparticles at various rates into diluted serum in vitro. Both stable retention of PGE(1) in the nanoparticles and coating of the nanoparticles with poly(ethylene glycol) led to an extremely extended blood residence time of PGE(1), as well as preferential accumulation in vascular lesions. CONCLUSIONS: These results suggest that the present strategy is useful to advance the clinical application of PGE(1) as a therapeutic agent for vascular disorders.

Efficient targeting to alveolar macrophages by intratracheal administration of mannosylated liposomes in rats.

The success of targeting systems to alveolar macrophages critically depends on internalization into these cells for pharmacological intervention. Direct respiratory delivery via inhalation of mannose modified liposomal carriers to alveolar macrophages is of great interest. To evaluate the targeting efficiency to alveolar macrophages by intratracheal administration of mannosylated liposomes (Man-liposomes), Man-liposomes with various ratio of mannosylated cholesterol derivatives, cholesten-5-yloxy-N-(4-((1-imino-2-D-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) as mannose receptor ligand were investigated with regard to their in vitro uptake in primary cultured alveolar macrophages and in vivo intratracheal administration in rats. The in vitro uptake of Man-liposomes took place in a concentration-dependent manner. The internalization of Man-liposomes with 7.5% (Man-7.5-liposomes) and 5.0% (Man-5.0-liposomes) Man-C4-Chol was considerably higher than that of Man-liposomes with 2.5% of Man-C4-Chol (Man-2.5-liposomes) and Bare-liposomes and significantly inhibited by an excess of mannan, suggesting mannose receptor-mediated endocytosis. After intratracheal administration of Man-7.5 and Man-5.0-liposomes in rats, a significantly high internalization and selective targeting to alveolar macrophages was observed. The enhanced cellular uptake in alveolar macrophages related to the mannose density of Man-liposomes was also confirmed both in vitro and in vivo confocal microscopy studies. These results demonstrate the efficient targeting to alveolar macrophages by the intratracheally administered Man-liposomes via mannose receptor-mediated endocytosis.

Plasma as a scaffold for regeneration of neural precursor cells after transplantation into rats with spinal cord injury.

The present study investigated whether plasma could be useful as a scaffold for cell transplantation in rats with spinal cord injury (SCI). Transplantation of cells with plasma promoted the recovery of SCI-induced motor dysfunction. Immunohistochemical analysis revealed that the grafted cells had differentiated into the neural lineage. When dissociated neural precursor cells were cultured with plasma, extensive neurite outgrowth was observed along with increased expression of p35 and NF68. Neural markers were also expressed by the cultured cells. Culture with plasma reduced thymidine incorporation, but promoted cell growth and increased the RNA contents. These findings suggest that the cells underwent differentiation into neurons in the presence of plasma. In conclusion, plasma could be a promising scaffold for cell transplantation therapy.

Lecithinized brain-derived neurotrophic factor promotes the differentiation of embryonic stem cells in vitro and in vivo.

The addition of lecithin molecules to brain-derived neurotrophic factor (BDNF) has been reported to markedly enhance its pharmacological effect in vivo. In the current study, we show that lecithinized BDNF (PC-BDNF) has a higher affinity than BDNF for neural precursor cells. Although BDNF only slightly increased the expression of the genes for Mash-1, p35, 68 kDa neurofilament, and TrkB receptor, PC-BDNF caused a significant increase in their expression. PC-BDNF also increased the level of neurofilament protein and dramatically increased TrkB mRNA gene expression, which was followed by a sustained activation of the p42/p44 extracellular-regulated kinases. Finally, transplantation of PC-BDNF-treated cells was more effective than BDNF-treated cells at improving impaired motor function caused by spinal cord injury. These findings showed that PC-BDNF has a better potential than BDNF for promoting neural differentiation, partly due to a higher cellular affinity. Furthermore, PC-BDNF-treated cells could be useful for transplantation therapy for central nervous system injuries.