COA-Cl Evokes Protective Responses Against H2O2-and 6-OHDA-Induced Toxic Injury in PC12 Cells.

COA-Cl, a novel adenosine-like nucleic acid analog, has recently been shown to exert neuroprotective effects and to increase dopamine levels both in vivo and in vitro. Therefore, we hypothesized that COA-Cl could protect dopaminergic neurons against toxic insults. Thus, the present study aimed to investigate the protective effects of COA-Cl against hydrogen peroxide (H2O2)- and 6-hydroxydopamine (6-OHDA)-induced toxicity in PC12 cells and to elucidate the possible mechanisms. PC12 cells were incubated with COA-Cl (100 µM) with or without H2O2 or 6-OHDA (200 µM) for 24 h. Treatment with COA-Cl attenuated the decrease in cell viability, SOD activity and the Bcl-2/Bax ratio caused by H2O2. In addition, COA-Cl attenuated the increase in LDH release, ROS production, caspase-3 activity, and apoptosis induced by H2O2. Further, COA-Cl enhanced the protection of PC12 cells against the toxicity caused by 6-OHDA, as evidenced by an increase in cell viability and the Bcl-2/Bax ratio, and a decrease in LDH release. Our results are the first to demonstrate that COA-Cl potentially protects PC12 cells against toxicity induced by H2O2 and 6-OHDA, implying that COA-Cl could be a promising neuroprotective agent for the treatment of Parkinson's disease.

COA-Cl induces dopamine release and tyrosine hydroxylase phosphorylation: In vivo reverse microdialysis and in vitro analysis.

We found that local perfusion of COA-Cl (0.1, 0.4, or 1.0 mM) into the dorsal striatum of living mice produced a significant and dose-dependent increase in extracellular DA levels, with the highest dose of 1.0 mM COA-Cl producing an approximately 5-fold increase in DA. Consistent with in vivo findings, 0.1 and 0.2 mM COA-Cl significantly and dose-dependently enhanced DA release 3.0 to 5.0-fold in PC12 cells, an in vitro model of DA-responsive neurons. Interestingly, the increase in striatal DA levels by COA-Cl in vivo was similar in magnitude to that observed in PC12 cells. Treatment with 0.1 mM COA-Cl significantly increased both Ser31 and Ser40 phosphorylation of tyrosine hydroxylase (TH) in PC12 cells, and Ser40 phosphorylation in iCell neurons, without altering total TH protein levels. Further, we examined whether COA-Cl could stimulate neurite outgrowth in PC12 cells and iCell neurons and found that COA-Cl significantly induced neurite outgrowth in both cell lines. Our results provide the first evidence that COA-Cl can stimulate dose-dependent DA release and activation of TH phosphorylation, suggesting that COA-Cl may be a promising therapeutic candidate for the treatment of neurological dysfunction associated with low DA.

Data on COA-Cl administration to the APP/PS2 double-transgenic mouse model of Alzheimer׳s disease: Improved hippocampus-dependent learning and unchanged spontaneous physical activity.

We herein present behavioral data regarding whether COA-Cl, a novel adenosine-like nucleic acid analog that promotes angiogenesis and features neuroprotective roles, improves cognitive and behavioral deficits in a murine model for Alzheimer׳s disease (AD). COA-Cl induced significant spatial memory improvement in the amyloid precursor protein/presenilin 2 double-transgenic mouse model of AD (PS2Tg2576 mice). Correspondingly, non-spatial novel object cognition test performance also significantly improved in COA-Cl-treated PS2Tg2576 mice; however, these mice demonstrated no significant changes in physical activity or motor performance. COA-Cl did not change the spontaneous activities and cognitive ability in the wild-type mice.

Effect of COA-Cl on transforming growth factor-ß1-induced epithelial-mesenchymal transition in RLE/Abca3 cells.

Transforming growth factor (TGF)-ß1 has received much attention as a major inducer of epithelial-mesenchymal transition (EMT) in pathological conditions such as cancer and organ fibrosis. In this study, we examined the effect of a novel nucleic acid analog, COA-Cl, on TGF-ß1-induced EMT using RLE/Abca3, a cell line having alveolar type II cell-like phenotype. Changes in the cell morphology consistent with EMT were induced by TGF-ß1, whereas, this response was suppressed by co-treatment of the cells with COA-Cl. In addition, co-treatment with COA-Cl abolished TGF-ß1-induced downregulation of cytokeratin 19 and upregulation of α-smooth muscle actin transcripts. In order to delineate the mechanism underlying the inhibitory effect of COA-Cl on TGF-ß1-induced EMT in RLE/Abca3 cells, we examined the role of zinc finger E-box binding homeobox (ZEB) family transcription factors in this phenomenon. Our results demonstrated that the treatment of cells with COA-Cl suppressed the TGF-ß1 mediated increase in the mRNA levels of ZEB2. Overall, it was concluded that COA-Cl may have an inhibitory effect on TGF-ß1-induced EMT-like phenotypical changes in RLE/Abca3 cells via suppression of ZEB2 mRNA expression.

Synthesis and Evaluation of Novel Cyclopropane Nucleoside as Potential Tube Formation Agents.

Five novel nucleoside analogs with mono or bis-hydroxymethylated cyclopropane rings at the N9-position of the 2-chloroadenine moiety (2-chloro-carbocyclic oxetanocin A [COA-Cl] analog) were synthesized and evaluated using human umbilical vein endothelial cells. All the prepared compounds (2a-e) showed good to moderate activity with angiogenic potency. cis-2'-(Hydroxymethyl)cycloprop-1'-yl derivative (2b) at 100 µM had greater angiogenic activity than the other compounds did, with relative tube areas of 2.71±0.45 (mean±standard deviation (S.D.)), which was superior to the potency of COA-Cl (2.30±0.59).

A key role of PGC-1α transcriptional coactivator in production of VEGF by a novel angiogenic agent COA-Cl in cultured human fibroblasts.

We previously demonstrated a potent angiogenic effect of a newly developed adenosine-like agent namedCOA-Cl.COA-Cl exerted tube forming activity in human umbilical vein endothelial cells in the presence of normal human dermal fibroblasts (NHDF). We therefore explored whether and howCOA-Cl modulates gene expression and protein secretion ofVEGF, a master regulator of angiogenesis, inNHDFRT-PCRandELISArevealed thatCOA-Cl upregulatedVEGF mRNAexpression and protein secretion inNHDFHIF1α(hypoxia-inducible factor 1α), a transcription factor, andPGC-1α(peroxisome proliferator-activated receptor-γcoactivator-1α), a transcriptional coactivator, are known to positively regulate theVEGFgene. Immunoblot andRT-PCRanalyses revealed thatCOA-Cl markedly upregulated the expression ofPGC-1αprotein andmRNACOA-Cl had no effect on the expression ofHIF1αprotein andmRNAin both hypoxia and normoxia. SilencingPGC-1αgene, but notHIF1αgene, by small interferingRNAattenuated the ability ofCOA-Cl to promoteVEGFsecretion. When an N-terminal fragment ofPGC-1αwas cotransfected with its partner transcription factorERRα(estrogen-related receptor-α) inCOS-7 cells,COA-Cl upregulated the expression of the endogenousVEGF mRNA However,COA-Cl had no effect on the expression ofVEGF, whenHIF1αwas transfected.COA-Cl inducesVEGFgene expression and protein secretion in fibroblasts. The transcriptional coactivatorPGC-1α, in concert withERRα, plays a key role in theCOA-Cl-inducedVEGFproduction.COA-Cl-induced activation ofPGC-1α-ERRα-VEGFpathway has a potential as a novel means for therapeutic angiogenesis.

Synthesis and Evaluation of Novel Carbocyclic Oxetanocin A (COA-Cl) Derivatives as Potential Tube Formation Agents.

Six novel carbocyclic oxetanocin A analogs (2-chloro-C.OXT-A; COA-Cl) with various hydroxymethylated or spiro-conjugated cyclobutane rings at the N(9)-position of the 2-chloropurine moiety were synthesized and evaluated using human umbilical vein endothelial cells. All prepared compounds (2a-f) showed good to moderate activity with angiogenic potency. Among these compounds, 100 µM cis-trans-2',3'-bis(hydroxymethyl)cyclobutyl derivative (2b), trans-3'-hydroxymethylcyclobutyl analog (2d), and 3',3'-bis(hydroxymethyl)cyclobutyl derivative (2e) had greater angiogenic activity, with relative tube areas of 3.43±0.44, 3.32±0.53, and 3.59±0.83 (mean±standard deviation (S.D.)), respectively, which was comparable to COA-Cl (3.91±0.78). These data may be important for further development of this class of compounds as potential tube formation agents.

Involvement of S1P1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells.

COA-Cl (2Cl-C.OXT-A) is a recently developed adenosine-like nucleic acid analog that promotes angiogenesis via the mitogen-activated protein (MAP) kinases ERK1/2. Endothelial S1P1 receptor plays indispensable roles in developmental angiogenesis. In this study, we examined the functions of S1P1 in COA-Cl-induced angiogenic responses. Antagonists for S1P1, W146, and VPC23019, substantially but still partly inhibited the effects of COA-Cl with regard to ERK1/2 activation and tube formation in cultured human umbilical vein endothelial cells (HUVEC). Antagonists for adenosine A1 receptor and purinergic P2Y1 receptor were without effect. Genetic knockdown of S1P1 with siRNA, but not that of S1P3, attenuated COA-Cl-elicited ERK1/2 responses. The signaling properties of COA-Cl showed significant similarities to those of sphingosine 1-phosphate, an endogenous S1P1 ligand, in that both induced responses sensitive to pertussis toxin (Gα i/o inhibitor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), (calcium chelator), and PP2 (c-Src tyrosine kinase inhibitor). COA-Cl elevated intracellular Ca(2+) concentration and induced tyrosine phosphorylation of p130Cas, a substrate of c-Src, in HUVEC. COA-Cl displaced [(3)H]S1P in a radioligand-binding competition assay in chem-1 cells overexpressing S1P1. However, COA-Cl activated ERK1/2 in CHO-K1 cells that lack functional S1P1 receptor, suggesting the presence of additional yet-to-be-defined COA-Cl target in these cells. The results thus suggest the major contribution of S1P1 in the angiogenic effects of COA-Cl. However, other mechanism such as that seen in CHO-K1 cells may also be partly involved. Collectively, these findings may lead to refinement of the design of this nucleic acid analog and ultimately to development of small molecule-based therapeutic angiogenesis.

Intestinal absorption, organ distribution, and urinary excretion of the rare sugar D-psicose.

BACKGROUND: The purpose of this study was to evaluate intestinal absorption, organ distribution, and urinary elimination of the rare sugar D-psicose, a 3-carbon stereoisomer of D-fructose that is currently being investigated and which has been found to be strongly effective against hyperglycemia and hyperlipidemia. METHODS: This study was performed using radioactive D-psicose, which was synthesized enzymatically from radioactive D-allose. Concentrations in whole blood, urine, and organs were measured at different time points until 2 hours after both oral and intravenous administrations and 7 days after a single oral administration (100 mg/kg body weight) to Wistar rats. Autoradiography was also performed by injecting 100 mg/kg body weight of (14)C-labeled D-psicose or glucose intravenously to C3H mice. RESULTS: Following oral administration, D-psicose easily moved to blood. The maximum blood concentration (48.5±15.6 µg/g) was observed at 1 hour. Excretion to urine was 20% within 1 hour and 33% within 2 hours. Accumulation to organs was detected only in the liver. Following intravenous administration, blood concentration was decreased with the half-life=57 minutes, and the excretion to urine was up to almost 50% within 1 hour. Similarly to the results obtained with oral administration, accumulation to organs was detected only in the liver. Seven days after the single-dose oral administration, the remaining amounts in the whole body were less than 1%. Autoradiography of mice showed results similar to those in rats. High signals of (14)C-labeled D-psicose were observed in liver, kidney, and bladder. Interestingly, no accumulation of D-psicose was observed in the brain. CONCLUSION: D-psicose was absorbed well after oral administration and eliminated rapidly after both oral and intravenous administrations, with short duration of action. The study provides valuable pharmacokinetic data for further drug development of D-psicose. Because the findings were mainly based on animal study, it is necessary to implement human trials to study the metabolism pathway, which would give an important guide for human intake and food application of D-psicose.

Development of transdermal therapeutic formulation of CNS5161, a novel NMDA receptor antagonist, by utilizing pressure-sensitive adhesives II: improved transdermal absorption and evaluation of efficacy and safety.

The aim of this study was to prepare a transdermal therapeutic formulation of CNS5161, an NMDA receptor antagonist developed as a drug for neuropathic pain. Since a silicone pressure-sensitive adhesive (PSA) was found to be the best PSA for CNS5161 among six different PSAs examined in our previous study, the effects of the loading concentration of CNS5161 on release and rat skin permeability were investigated using silicone PSAs. The release of CNS5161 was elevated with an increase in the drug concentration from 1% to 14%. The transdermal flux at the steady state reached a plateau at 8% and over, while crystallization of CNS5161 was not observed for any formulation even at high drug concentrations. The drug concentration in rat skin at the steady state was also saturated at 8% and over, which correlated well with the transdermal flux at the steady state. Therefore, skin permeation clearance defined to the skin concentration at the steady state was almost constant at 0.21/h from 2% to 14% of CNS5161, which suggests that drug concentrations in the skin would be a driving force for transport of the drug to the receptor side. Since increasing the concentration of CNS5161 in the PSA patch was not able to elevate the transdermal flux, 12 formulations containing several permeation enhancers were examined to improve the transdermal transport of CNS5161. Among them, the formulation containing propylene glycol, diisopropyl adipate, and polyvinylpyrrolidone significantly increased the transdermal flux by approximately 1.8-fold by improving the diffusivity of CNS5161 in the skin, and also significantly enhanced the analgesic effect of CNS5161. This formulation caused only slight skin irritation, which indicated that it would be a promising transdermal therapeutic system for CNS5161.

Development of transdermal therapeutic formulation of CNS5161, a novel N-methyl-D-aspartate receptor antagonist, by utilizing pressure-sensitive adhesives I.

The aim of this study was to investigate the feasibility of percutaneous absorption of CNS5161, a novel N-methyl-D-aspartate (NMDA) receptor antagonist developed as a potential treatment for neuropathic pain and other neurological disorders. Six pressure-sensitive adhesives (PSA) with different physicochemical properties, namely, styrene-isoprene-styrene (1) (SIS(1)), styrene-isoprene-styrene (2) (SIS(2)), silicone, acrylate with a hydroxyl group (acrylate(OH)), acrylate without a functional group (acrylate(none)) and acrylate with a carboxyl group (acrylate(COOH)), were investigated for their release of CNS5161 and its subsequent skin permeability. Among the adhesives examined, silicone PSA provided the highest value of transdermal flux of CNS5161, which could be attributable to the highest release rate from it due to its very high thermodynamic activity. Although CNS5161 was also in the supersaturated state in SIS(1) and SIS(2) PSAs, the release and transdermal permeation from these adhesives were slower than those from silicone PSA. As for the acrylic PSAs, the highest release rate and permeability of CNS5161 were observed for acrylate(OH) PSA, followed by acrylate(none) and acrylate(COOH) PSAs, but none of them was better in terms of either the release or the permeability of CNS5161 than silicone PSA. These results clearly indicated that silicone PSA would be the most suitable for transdermal delivery of CNS5161 and silicone PSA containing 10% CNS5161 would be suitable for clinical use in humans.

A novel nucleic acid analogue shows strong angiogenic activity.

A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100muM was stronger than that of vascular endothelial growth factor (VEGF), a positive control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.

Stage- and region-specific cyclooxygenase expression and effects of a selective COX-1 inhibitor in the mouse amygdala kindling model.

In an attempt to elucidate the involvement of cyclooxygenase (COX) enzymes, particularly COX-1, in epileptogenesis, the localization of COX-1 and COX-2 expression in the mouse kindling model was analyzed by immunohistochemistry. COX-2 was predominantly observed in brain neurons and its concentration in the hippocampus increased with progressing seizures, as reported previously. COX-1 was predominant in microglia and its concentration was also enhanced in the hippocampus and areas around the third ventricle during the progression of seizures. These regions are thought to play an important role in the propagation of limbic seizures. Moreover, the administration of SC-560 (a selective COX-1 inhibitor) or indomethacin (a non-selective COX inhibitor) retarded the progress of seizures. Although the precise function of COX-positive cells in microglia and elsewhere is not clear, our results suggest that COX-1 as well as COX-2 may be involved in epileptogenesis, and that certain COX inhibitors can potentially prevent the occurrence of seizures.

Analysis of the inhibitory mechanism of D-allose on MOLT-4F leukemia cell proliferation.

D-Allose, the C-3 epimer of D-glucose, is one of the rare sugars found in nature. In the present study, we have elucidated for the first time that various leukemia cell lines have different susceptibility to anti-proliferative activity of D-allose, and that this difference is related to the difference in induction of thioredoxin interacting protein (TXNIP) expression. We examined 5 leukemia cell lines (MOLT-4F, IM-9, HL-60, BALL-1 and Daudi), and found that MOLT-4F (T-cell lymphoblastic leukemia) had the highest susceptibility to D-allose, and that Daudi (Burkitt's lymphoma) had the lowest. D-Allose significantly slowed the cell cycle progression without causing apoptosis of MOLT-4F cells. Intracellular TXNIP expression was specifically and markedly enhanced in MOLT-4F cells by D-allose treatment, and subsequent increase of p27(kip1), a cell cycle inhibitor, was observed. On the other hand, D-allose did not increase TXNIP and p27(kip1) levels at all in Daudi cells. These results indicate that D-allose suppresses MOLT-4F cell proliferation possibly by the inhibition of cell cycle progression via induction of TXNIP expression.

Anti-angiogenic activities of nucleosides and nucleotides and their structure-activity relationship.

The effects of nucleosides and nucleotides on the tube formation of human umbilical vein endothelial cells (HUVEC) were compared. Twenty eight compounds including endogenous species were examined and many of them were revealed to have inhibitory potency. The potency was strengthened significantly when the sugar moiety of ribonucleosides was modified. In case that chlorine was introduced to the 2-position of adenine, the potency also increased. The structure-activity relationship was considered together with the effects on the proliferation of HUVEC.

Stage and region dependent expression of a radial glial marker in commissural fibers in kindled mice.

Amygdala kindling is regarded as a model of temporal lobe epilepsy in humans because of many points of similarity. In amygdala kindling, bilateralization of epileptic seizures follows from the accumulation of stimulation and commissural fibers may play a role in this process. However, new progenies of cells following amygdala kindling have not been reported and the precise nature of how bilateralization occurs is not clear. In the present study, we aim to clarify the emergence of radial glia during the progress of amygdala kindling in mouse brain. For this purpose, immunohistochemical staining for 3CB2, which is a specific marker of radial glia, was employed. Immunoreactivity for 3CB2 was observed in the forceps minor, radiation of trunk and forceps major regions at Clonus 3 and more strongly at Clonus 5. In the forceps major, the cingulate gyrus showed immunopositive staining at Clonus 3, but the corpus callosum and alveus hippocampi showed staining only at Clonus 5. In the fimbria hippocampus, the anterior commissure posterior showed staining at Clonus 5. However, the anterior commissure anterior was not stained at the stage progressed to Clonus 5. These findings indicate stage and region dependent expression of progenitor cells in commissural fibers and suggest that these changes may accompany the formation of new circuits in seizure progression during amygdala kindling.

Unique high-affinity synthetic mu-opioid receptor agonists with central- and systemic-mediated analgesia.

Unique opioid mimetic substances containing identical N-terminal aromatic residues separated by an unbranched alkyl chain containing two to eight methylene groups were developed. Regardless of the length of interposing alkyl chain, the bis-Tyr and bis-Phe compounds were inactive; however, replacement by a single Dmt (2',6'-dimethyl-L-tyrosine) residue enhanced activity by orders of magnitude. Moreover, the bis-Dmt compounds were another 10-fold more potent with an optimum intra-aromatic ring distance of about four to six methylene units. 1,4-Bis(Dmt-NH)butane (7) had high mu-opioid receptor affinity (K(i) = 0.041 nM) and functional mu-opioid agonist bioactivity (IC(50) = 5.3 nM) with in vivo central (intracerebroventricular) and systemic (subcutaneous) analgesia in mice (1.5- to 2.5-fold greater than and 10-12% relative to morphine, respectively); these activities were reversed by naloxone to the same degree. It appears that the bis-Dmt compounds indiscriminately act as both message and address domains.

Suppression of hyperemia and DNA oxidation by indomethacin in cerebral ischemia.

We investigated antioxidative activity and the effect of indomethacin, an agent that inhibits cyclooxygenase, on extracellular glutamate and cerebral blood flow in cerebral ischemia in gerbils. Pre-ischemic administration of indomethacin (5 mg/kg, i.p.) significantly rescued hippocampal CA1 neurons (9+/-6 cells/mm in the ischemia, 87+/-43 cells/mm in the indomethacin group, P<0.001). DNA fragmentation induced by ischemia was also examined using the terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) method and indomethacin reduced TUNEL positive cells (140+/-21 in the ischemia, 99+/-31 in the indomethacin group, P<0.01). In addition, indomethacin attenuated the increase in hippocampal blood flow during reperfusion, but not increased extracellular glutamate by ischemia. Eight-hydroxydeoxyguanosine (8-OH-dG), a highly sensitive marker of DNA oxidation, was measured 90 min following ischemia using high-pressure liquid chromatography. Indomethacin significantly decreased the level of ischemia-induced 8-OH-dG in the hippocampus (P<0.05). These results suggest that indomethacin may protect neurons by attenuating oxidative stress and reperfusion injury in ischemic insult.