Antinociception and (R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid antagonism by gabapentin in the rat spinal cord in vivo

Gabapentin, a novel anticonvulsant and analgesic with an unknown mechanism of action, was tested on spinal dorsal horn neurone activity evoked by iontophoretically applied N-methyl-D-aspartic acid (NMDA) and (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and on nociceptive responses of single motor units (SMU) in anaesthetised rats. Gabapentin (10-215 mg/kg, i.v.) dose-dependently and selectively inhibited AMPA-evoked neuronal responses (ED50 106+/-24 mg/kg); no effect on NMDA-evoked activity was observed. In the same dose-range, gabapentin (10-215 mg/ kg, i.v.) dose-dependently reduced SMU responses to noxious electrical and mechanical stimulation. We conclude that gabapentin acts as an AMPA antagonist in the rat spinal cord, and that this mechanism is likely to substantially contribute to the antinociceptive effect of the drug.     

Inhibitory effects of OD 78 [3-(4-bromo-phenoxy)-4,5-dihydroxybenzoic acid-methyl ester] on the proliferation and migration of TNF-α-induced rat aortic smooth muscle cells

The proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in the formation and progression of intimal thickening in early-phase atherosclerosis and in restenosis after vascular injury. Tumor necrosis factor-α (TNF-α) is released from macrophages in atherosclerotic lesions and from neointimal vascular smooth muscle cells after balloon-injury. Obovatol, a major biphenolic component isolated from the Magnolia obovata leaf, is known to have anti-inflammatory and antitumor activities. The goal of this study was to examine the cardioprotective effects of the obovatol derivative OD 78 on the TNF-α-induced proliferation and migration of rat aortic smooth muscle cells (RASMCs). The antiproliferative effects of OD 78 on RASMCs were examined by cell counting and [(3)H]-thymidine incorporation assays. Treatment of cells with 1-4 μM OD 78 inhibited the proliferation and DNA synthesis of TNF-α-stimulated RASMCs in a concentration-dependent manner, without cytotoxicity. Treatment with OD 78 inhibited TNF-α-mediated p38 phosphorylation, but did not change the activation of extracellular signal-regulated kinase or c-Jun N-terminal kinase. Furthermore, treatment with OD 78 decreased TNF-α-induced levels of cyclin E, cyclin D1, CDK2, proliferating cell nuclear antigen, and phosphorylated retinoblastoma protein, but not the CDK4 expression level. Also, OD 78 inhibits the migration of TNF-α-induced RASMC in transwells. OD 78 treatment strongly decreased matrix metalloproteinase-9 (MMP-9) expression in a dose-dependent manner, but the MMP-2 expression was unchanged. These results show that OD 78 may be developed as a potential antiproliferative agent for the treatment of angioplasty restenosis and atherosclerosis.     

The effects of dl-p-chlorophenylalanine and dl-α-methyl-p-tyrosine on the brain catecholamine,serotonin and free amino acid contents in rat

The changes in the brain levels of catecholamines, serotonin and free amino acids have been considered as a factor in the development of the physical dependence on and tolerance to some drugs. In order to show the relationships among the brain levels of these substances, three groups of rats were given i.p. dl-MpT alone, dl-pCPA alone, or dl-MpT and dl-pCPA together, twice a day for three days. Another group was kept as control. The brain levels of catecholamines, serotonin and free amino acids were determined. Although dl-MpT and dl-pCPA alone caused a decrease in the levels of catecholamines and serotonin respectively, the administration of dl-MpT and dl-pCPA together did not. The changes in the levels of free amino acids which were found were related to the metabolism of catecholamines and serotonin.     

Characterization of the anticonvulsant and neuroprotectant BIIR 561 CL in vitro: effects on native and recombinant α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors

BIIR 561 CL is a novel blocker of AMPA receptors and voltage-dependent sodium channels. In this study we further describe the effects of BIIR 561 CL on AMPA receptor-mediated membrane currents in rodent neurons, as well as in cells expressing recombinant human GluR1/2 receptors in more detail. BIIR 561 CL suppressed responses to kainate in neuronal cultures from rat cortex with an IC50 of 9.8 microM. Similar effects were observed using acutely dissociated neurons from the CA1 region of rat hippocampus (IC50 = 9.5 microM). Inhibition of kainate responses by BIIR 561 CL was prevented by preapplication of GYKI 53655, suggesting that both non-competitive inhibitors bind to a common site of the receptor. The effect of 10 microM BIIR 561 CL on kainate-induced currents was dependent on extracellular pH, with more pronounced block (84.1%) under acidic conditions (pHextern=6.4), compared to only 30.1% at a pHextern of 8.4. Thus, it can be hypothesized that BIIR 561 CL inhibits AMPA receptors in ischaemic brain regions more effectively than in healthy tissue. BIIR 561 CL inhibited responses to 1 mM glutamate in cells expressing recombinant human GluR1/2 receptors with similar potency, as compared to kainate responses in rat neurons (IC50=17.3 microM). The reference compound NBQX had an IC50 of 25.2 nM. None of the two compounds affected the glutamate-induced receptor desensitization at any tested concentration. The block by BIIR 561 CL was not use-dependent and had fast on- and off-kinetics (tauon=6.8 s; tauoff=1.3 s in hGluR1/2 receptors with 30 microM BIIR 561 CL). Thus, BIIR 561 CL can be anticipated to have a promising profile for the treatment of neurological disorders like brain ischaemia and head trauma.     

从D-葡萄糖合成(3S,4S,5R)-4-O-苄基-3-表莽草酸甲酯

本文报道用D-葡萄糖为原料,经九步反应,对映选择地合成了标题化合物(10),从而为制备新抗生素二(口恶)霉素及类似物提供了对映选择合成的途径。     

Inhibitory effects of (±)-propranolol on excitation-contraction coupling in isolated soleus muscles of the rat

1. The effect of a β-adrenoceptor antagonist, propranolol, was investigated on excitation-contraction coupling in small, intact bundles of soleus muscle fibres from the rat.
2. (±)-Propranolol significantly inhibited twitch and tetanic tension with IC₅₀ values of 6.7 μM and 3.5 μM, respectively.
3. (+)-Propranolol (which has 100 times less β-blocking activity than the (±) form) was approximately one third as effective as the (±) form at inhibiting isometric tension.
4. (±)-Propranolol (20 μM) had no significant effect on the amplitude of caffeine contractures, suggesting that it did not directly inhibit Ca²⁺ release from the sarcoplasmic reticulum.
5. The resting membrane potential measured after 15 min perfusion with 20 μM (±)-propranolol was not significantly different from control. However, this concentration of (±)-propranolol significantly reduced both the peak amplitude and the maximum rate of rise of the action potential. Both effects were only partially reversible after extensive washing.
6. (±)-Propranolol perfusion caused a modest reduction in the amplitude of sub-maximal K⁺ contractures at concentrations (5  μM) that markedly depressed tetanic tension.
7. The results indicate that (±)-propranolol can decrease isometric tension independently of β-receptor occupation by (i) reducing the amplitude and rate of rise of the action potential and (ii) by directly inhibiting excitation-contraction coupling. The relatively low IC₅₀ for the ‘membrane-stabilizing'' action of propranolol on tetanic tension (3.5 μM), combined with the ability of the drug to accumulate gradually in biological membranes, may contribute to a peripheral component of the tremorolytic and fatigue-inducing actions of propranolol on skeletal muscle.

     

Effect of inhibition of γ-glutamyltranspeptidase by AT-125 (Acivicin) on glutathione and cysteine levels in rat brain and plasma

Summary AT-125 (Acivicin) is an inhibitor of -glutamyltranspeptidase (-GTP) which initiates glutathione catabolism to cysteine. We measured plasma and brain glutathione and cysteine in rats treated with AT-125. Six h after AT-125 treatment, plasma glutathione had increased 6-fold and plasma cysteine had fallen significantly. Brain cysteine fell after 24 h of AT-125 treatment, and brain glutathione had also decreased 18%. AT-125 pretreatment inhibited brain uptake of ³⁵S when it was given as ³⁵S-GSH but had no effect when it was given as ³⁵S-cysteine. These results suggest that plasma glutathione is catabolized by -GTP, and cysteine derived from it is taken up by the brain. N-acetylcysteine was administered to AT-125 treated rats in an attempt to supply cysteine to the brain in the face of -GTP inhibition. N-acetylcysteine supported brain glutathione levels, suggesting that it can serve as a source of cysteine under these conditions.Abbreviations -GTP -glutamyltranspeptidase - GSH reduced glutathione - TCA trichloroacetic acid     

Pharmacokinetics and Biologic Activity of the Novel Mast Cell Inhibitor, 4-(3′-Hydroxyphenyl)-amino-6,7-dimethoxyquinazoline in Mice

Purpose. The purpose of the present study was to examine the pharma-codynamic and pharmacokinetic features of the novel mast cell inhibitor 4-(3-Hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P180) in mice. Methods. A high performance liquid chromatography (HPLC)-based quantitative detection method was used to measure plasma WHI-P180 levels in mice. The plasma concentration-time data was fit to a single compartment pharmacokinetic model by using the WinNonlin program to calculate the pharmacokinetic parameters. A cutaneous anaphylaxis model was used to examine the pharmacodynamic effects of WHI-P180 on anaphylaxis-associated vascular hyperpermeability. Results. The elimination half-life of WHI-P180 in CD-1 mice (BALB/ c mice) following i.v., i.p., or p.o. administration was less than 10 min. Systemic clearance of WHI-P180 was 6742 mL/h/kg in CD-1 mice and 8188 mL/h/kg in BALB/c mice. Notably, WHI-P180, when administered in two consecutive nontoxic i.p. bolus doses of 25 mg/kg, inhibited IgE/antigen-induced vascular hyperpermeability in a well-characterized murine model of passive cutaneous anaphylaxis. Conclusions. WHI-P180 is an active inhibitor of IgE-mediated mast cell responses in vitro and in vivo. Further preclinical characterization of WHI-P180 may improve the efficacy of WHI-P180 in vivo and provide the basis for design of effective treatment and prevention programs for mast cell mediated allergic reactions.     

(2S,3R,4S)-4-羟基异亮氨酸的不对称合成

对甲氧基苯胺和乙醛酸乙酯依次经缩合、(S')-脯氨酸催化不对称Mannich反应和DBU不对称转化制得(2S,3R)-2-(4-甲氧基苯胺基)-3-甲基-4-氧代戊酸乙酯,再经CAN氧化脱保护、硼氢化钠还原和氧氧化锂水解得(2S,3R,4S)-4-羟基异亮氨酸,总收率约30%.     

Irgasan®DP 300 (5-chloro-2-(2,4-dichlorophenoxy)phenol) induces cytochrome P450s and inhibits haembiosynthesis in rat hepatocytes cultured on Matrigel

1. The effect of Irgasan DP 300 (5-chloro-2-(2,4-dichlorophenoxy)phenol) on cytochrome P450 (P450) induction and haem biosynthesis was studied in rat hepatocytes cultured on Matrigel. 2. Irgasan DP 300 significantly induced 7-benzyloxyresorufin O-debenzylase activity, followed by 7-pentoxyresorufin O-depentylase and 7-ethoxyresorufin O-deethylase activities. 4-Nitrophenol hydroxylase, testosterone 6β-hydroxylase and methoxyresorufin O-demethylase activities were also slightly increased. The maximum induction of these enzyme activities was obtained at the same concentration of 125 μM in the culture medium. 3. Immunochemical blots using anti-rat cytochrome P450 antibodies revealed that Irgasan DP 300 preferably induced CYP2B1/2 along with a slight increase in 3A. These results indicate that Irgasan DP 300 is a phenobarbital-type inducer. 4. In the absence of exogenous 5-aminolevulinic acid (ALA), slight increases in protoporphyrin IX (2·6-fold) and coproporphyrin III (1·3-fold) were observed in the Irgasan DP 300-treated cultures. In contrast, when 75 μM ALA was present, Irgasan DP 300 (250 μM) caused an extensive accumulation of uroporphyrin I (13-fold). 5. Irgasan DP 300 inhibited rat hepatic uroporphyrinogen III synthase in vitro. 6. These results indicate that Irgasan DP 300 produced accumulation of hydroxymethylbilane in rat hepatocytes by inhibiting uroporphyrinogen III synthase, and consequently an accumulation of uroporphyrin I.     

Pharmacokinetics and metabolism of N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-L-phenylalanine 1-methyl ester, monohydrate (advantame) in the rat, dog, and man

The pharmacokinetics and metabolism of advantame were evaluated in rats, dogs, and humans. The oral pharmacokinetic studies using (14)C-advantame showed that advantame undergoes rapid but incomplete absorption, with an oral bioavailability of total radioactivity in the range of 4-23%. Data indicated that absorption was mainly as ANS9801-acid (de-esterified advantame), which was formed in the gastrointestinal tract as a result of the hydrolysis of the methyl ester group of the parent compound. In the dog, plasma ANS9801-acid was present largely in the form of an unidentified conjugate. Advantame (chiefly in the form of metabolites) was mainly excreted in the feces in rats, dogs, and humans (>80% in each species), with urinary excretion representing a minor route. The predominant metabolite of (14)C-advantame detected in the feces and the urine of rats, dogs, and humans was ANS9801-acid, with lower amounts of 3-[3-hydroxy-4-methoxyphenyl]-1-propylamine (termed HU-1) or N-(3-(3-hydroxy-4-methoxy phenyl))propyl-L-aspartic acid (termed HF-1) present, as well as other minor metabolites and areas of indistinct radioactivity. ANS9801-acid, HU-1, and HF-1 were detected and identified in the urine of rats, humans, and dogs, while ANS9801-acid and HF-1 were identified in the feces of humans and dogs. In the feces of rats, in addition to ANS9801-acid, other additional metabolites were detected, including demethylated ANS9801-acid (designated as RF-1) and another unidentified metabolite (designated as RF-2). Overall, the data show generally similar pharmacokinetics of advantame and ANS9801-acid in animals and in humans and close similarity with neotame. Metabolites of advantame that occur in humans are also found in the 2 species utilized in the toxicology studies, and the metabolism studies support the interpretation of safety data from studies conducted in rats and dogs.     

(2S,3S,5S)-2,5-二氨基-3-羟基-1,6-二苯基己烷的合成

L-苯丙氨酸用氯苄进行MO-苄基化、氰化、格氏反应、还原制得（2S，3S，5S）-5-氨基-2-二苄胺基-1，6-二苯基-3-己醇，再经钯炭催化脱苄制得抗病毒药洛匹那韦中间体（2S，3S，5S）-2,5-二氨基-3-羟基-1，6-二苯基己烷，总收率约为63％。     

Effects of (R)-α-methylhistamine and scopolamine on spatial learning in the rat assessed using a water maze

The effects of (R)--methylhistamine ((R)--MeHA, a selective H₃-receptor agonist) and scopolamine (SCOP, a muscarinic antagonist) were investigated on spatial learning and memory in the rat (Hooded Lister) using a water maze (WM). (R)--MeHA treatment (6.3 and 10 mg/kg IP) had no apparent effect on spatial learning but did result in enhanced spatial recall at the higher dose, assessed by a transfer (probe) test after training. In contrast, SCOP (0.5 mg/kg IP) induced a learning and memory deficit measured both during and after training. In animals treated with (R)--MeHA and SCOP, (R)--MeHA partially (6.3 mg/kg) and completely (10 mg/kg) reversed the SCOP-induced deficit during the training phase, while in the post-training transfer test, (R)--MeHA (10 mg/kg) significantly reduced the SCOP-induced memory deficit. None of the treatments described resulted in impaired visual acuity as demonstrated by a raised platform test. These results are consistent with a role for histamine in cognitive processes and suggest a possible interaction between central histamine and cholinergic mechanisms associated with rodent spatial learning and memory.     

Comparison of the effects of haloperidol,remoxipride and raclopride on “pre”- and postsynaptic dopamine receptors in the rat brain

Summary The ability of the dopamine receptor antagonists haloperidol, raclopride and remoxipride to prevent the B-HT 920-induced decrease in striatal and limbic L-DOPA accumulation in gamma-butyrolactone (GBL)- and NSD 1015-treated rats (termed GBL-reversal) was used to define the effects of these compounds on presynaptic dopamine receptors. The doses of the dopamine antagonists producing antagonism of GBL-reversal were in each case roughly similar to the doses required to increase dopamine turnover in striatal and limbic areas. The potencies of haloperidol, raclopride and remoxipride in the GBL model were compared with their potencies in behavioural models for postsynaptic dopamine receptors. Haloperidol produced antagonism of GBL-reversal over a similar dose range to that required for antagonism of apomorphine-induced hyperactivity and stereotypy syndromes. Raclopride was effective in the order of potency: antagonism of apomorphine-induced hyperactivity > antagonism of GBL-reversal > antagonism of apomorphine-induced stereotypy. For remoxipride, the dose-response curve for antagonism of GBL-reversal was superimposable over that for antagonism of apomorphine-induced stereotypies, with an ED₅₀ value about 12 times higher than that for antagonism of apomorphine-induced hyperactivity. Thus, the relative potencies of dopamine receptor antagonists at pre- and postsynaptic dopamine receptors vary considerably from compound to compound. Send offprint requests to O. Magnusson     

(1S, 3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115), a potent γ-aminobutyric acid aminotransferase inactivator for the treatment of cocaine addiction

Vigabatrin, a GABA aminotransferase (GABA-AT) inactivator, is used to treat infantile spasms and refractory complex partial seizures and is in clinical trials to treat addiction. We evaluated a novel GABA-AT inactivator (1S, 3S)-3-amino-4-difluoromethylenyl-1-cyclopentanoic acid (CPP-115, compound 1) and observed that it does not exhibit other GABAergic or off-target activities and is rapidly and completely orally absorbed and eliminated. By use of in vivo microdialysis techniques in freely moving rats and microPET imaging techniques, 1 produced similar inhibition of cocaine-induced increases in extracellular dopamine and in synaptic dopamine in the nucleus accumbens at (1)/(300) to (1)/(600) the dose of vigabatrin. It also blocks expression of cocaine-induced conditioned place preference at a dose (1)/(300) that of vigabatrin. Electroretinographic (ERG) responses in rats treated with 1, at doses 20-40 times higher than those needed to treat addiction in rats, exhibited reductions in ERG responses, which were less than the reductions observed in rats treated with vigabatrin at the same dose needed to treat addiction in rats. In conclusion, 1 can be administered at significantly lower doses than vigabatrin, which suggests a potential new treatment for addiction with a significantly reduced risk of visual field defects.     

(2S, 3S)-3-氨基-2-羟基-4-苯丁酰胺的合成

目的 合成α-酮基酰胺肽类钙蛋白酶抑制剂的必需结构片段 (2S, 3S)-3-氨基-2-羟基-4-苯丁酰胺（1）。 方法 以(S)-2-氨基-3-苯丙醇为起始原料,经过苄基保护、Parikh-Doering 氧化、氰化水解、酰胺化和脱保护共5步反应合成目标化合物。结果与讨论 目标化合物和中间体的结构均经1H-NMR和13C-NMR谱确证,该合成路线方法简捷,适于大量制备。     

Comparison of the protective effect of (R)-α-methylhistamine and its prodrugs on gastric mucosal damage induced by ethanol in the rat

• 1.1. The gastroprotective activity of two azomethine prodrugs of (R)-α-methylhistamine was examined in lesions induced by absolute ethanol (1 ml/rat intragastrically for 1 h).
• 2.2. Pretreatment with (R)-α-methylhistamine as well as with the prodrugs (30 and 100 mg/kg intragastrically [IG]) significantly reduced macroscopically visible lesions caused by ethanol, with protection being almost complete at 100 mg/kg.
• 3.3. Histologically, in rats pretreated with the three compounds at a dose of 100 mg/kg, the evidence of damage was rare, with the appearance of gastric mucosa being similar in the different treatment groups.
• 4.4. Present results are suggestive of a local component in the protective activity of (R)-α-methylhistamine.

     

Effects of positive modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors in a benzodiazepine-induced deficit of spatial discrimination in mice

Imbalance between GABAergic and glutamatergic neurotransmission has been recently hypothesized to trigger memory decline related either to ageing or to Alzheimer's disease (AD). Thereby, benzodiazepine-induced anterograde amnesia has been construed as a model of hippocampal-related cognitive dysfunctions. Since spatial memory is altered both by ageing and by benzodiazepines such as alprazolam, we investigated the pharmacological sensitivity of alprazolam-induced deficit in a delayed spatial discrimination (SD) task, notably with positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. We showed that alprazolam (0.1?mg/kg intraperitoneally) induced memory impairments as compared with vehicle-treated mice. The oral administration of modulators of AMPA receptors (IDRA-21: 10?mg/kg; S18986: 3 and 10?mg/kg) reversed the alprazolam-induced deficits. This study is first to show evidence that reference treatments of AD, such as memantine (a NMDA receptor antagonist) at 3?mg/kg per os (po) and donepezil (an acetylcholinesterase inhibitor) at 1?mg/kg po, also reversed the alprazolam-induced amnesia. Given such results, the SD task emerges as a valuable novel task to screen pro-cognitive compounds. Thus, we highlight the efficacy of modulators of AMPA-type glutamate receptors to counteract alprazolam-induced spatial deficits. These results could be viewed alongside the imbalance between excitation and inhibition observed during normal and pathological ageing.     

In vitro characterisation of the novel positive allosteric modulators of the mGlu(5) receptor, LSN2463359 and LSN2814617, and their effects on sleep architecture and operant responding in the rat

The demonstrated functional interaction of metabotropic glutamate 5 (mGlu(5)) receptors with N-methyl-d-aspartate (NMDA) receptors has prompted speculation that their activation may offer a potential treatment for aspects of schizophrenia. Development of selective mGlu(5) agonists has been difficult, but several different positive allosteric modulator (PAM) molecules have now been identified. This study describes two novel mGlu(5) PAMs, LSN2463359 (N-(1-methylethyl)-5-(pyridin-4-ylethynyl)pyridine-2-carboxamide) and LSN2814617 [(7S)-3-tert-butyl-7-[3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-A]pyridine], which are useful tools for this field of research. Both compounds are potent and selective potentiators of human and rat mGlu(5) receptors in?vitro, displaying curve shift ratios of two to three fold in the concentration-response relationship to glutamate or the glutamate receptor agonist, DHPG, with no detectable intrinsic agonist properties. Both compounds displaced the mGlu(5) receptor antagonist radioligand, [(3)H]MPEP in?vitro and, following oral administration reached brain concentrations sufficient to occupy hippocampal mGlu(5) receptors as measured in?vivo by dose-dependent displacement from the hippocampus of intravenously administered MPEPy. In?vivo EEG studies demonstrated that these mGlu(5) PAMs have marked wake-promoting properties but little in the way of rebound hypersomnolence. In contrast, the previously described mGlu(5) PAMs CDPPB and ADX47273 showed relatively poor evidence of in?vivo target engagement in either receptor occupancy assays or EEG disturbance. Wake-promoting doses of LSN2463359 and LSN2814617 attenuated deficits in performance induced by the competitive NMDA receptor antagonist SDZ 220,581 in two tests of operant behaviour: the variable interval 30?s task and the DMTP task. These effects were lost if the dose of either compound extended into the range which disrupted performance in the baseline DMTP task. However, the improvements in response accuracy induced by the mGlu(5) potentiators in SDZ 220,581-treated rats were not delay-dependent and, therefore, perhaps more likely reflected optimization of general arousal than specific beneficial effects on discrete cognitive processes. The systematic profiling of LSN2463359 and LSN2814617 alongside other previously described molecules will help determine more precisely how mGlu(5) potentiator pharmacology might provide therapeutic benefit. This article is part of a Special Issue entitled 'Cognitive Enhancers'.