Discovery of N-sulfonyl-7-azaindoline derivatives as potent,orally available and selective M4 muscarinic acetylcholine receptor agonists

We designed and synthesized novel N-sulfonyl-7-azaindoline derivatives as selective M₄ muscarinic acetylcholine receptor agonists. Modification of the N-carbethoxy piperidine moiety of compound 2, an M₄ muscarinic acetylcholine receptor (mAChR)-preferring agonist, led to compound 1, a selective M₄ mAChR agonist. Compound 1 showed a highly selective M₄ mAChR agonistic activity with weak hERG inhibition in vitro. A pharmacokinetic study of compound 1 in vivo revealed good bioavailability and brain penetration in rats. Compound 1 reversed methamphetamine-induced locomotor hyperactivity in rats (1–10 mg/kg, po).     

乙酰胆碱协同剂对蚕豆保卫细胞原生质体内向钾电流的调节作用

用膜片钳全细胞记录方式研究了乙酰胆碱对蚕豆保卫细胞原生质体内向钾电流的作用与机理。发现乙酰胆碱的协同剂———烟碱和毒蕈碱分别促进内向钾电流 30 %～ 40 %。EGTA处理能有效减弱毒蕈碱对内向钾电流的促进效应 ,但不影响烟碱对内向电流的促进效应。这些结果提示 ,乙酰胆碱促进气孔开放的作用可能是通过受体直接或间接调节内向钾电流实现的     

Behavioral effects of inhibition of cannabinoid metabolism: The amidase inhibitor AM374 enhances the suppression of lever pressing produced by exogenously administered anandamide

Biochemical investigations have identified putative enzymatic pathways for the synthesis and metabolism of endogenous cannabinoids. Anandamide amidase is an enzyme that metabolizes anandamide into arachadonic acid and ethanolamine. Using in vitro methods, various inhibitors of amidase have been identified. The present studies were undertaken to determine if the amidase inhibitor AM 374 could enhance the effects of intraperitoneal (IP) injections of anandamide. Three studies were conducted to investigate the effects of various drug treatments on fixed ratio 5 operant lever pressing for food reinforcement. In the first study, the effects of different doses of anandamide were assessed, and it was demonstrated that 5.0 and 10.0 mg/kg anandamide IP significantly suppressed lever pressing, while 2.5 mg/kg produced very little effect. The second study tested the effects of intraventricular (ICV) injections of AM 374, and it was observed that doses up to 10.0, 20.0 and 40 microg AM 374 had no significant effect upon lever pressing. The third study investigated the combined effect of AM374 with a low dose of anandamide. Rats received two drug injections: one ICV and one IP. Four different drug treatments were assessed: 1) ICV vehicle + IP vehicle, 2) ICV vehicle + 2.5 mg/kg anandamide IP, 3) ICV 20.0 microg AM 374 + IP vehicle, and 4) ICV 20 microg AM 374 + 2.5 mg/kg anandamide IP. Combined administration of AM 374 plus anandamide led to a significant decrease in lever pressing compared to either AM374 or anandamide administered alone. Observations of the animals treated with the combination of AM374 plus anandamide indicated that the drug combination resulted in motor slowing, which is consistent with the notion that stimulation of cannabinoid receptors produced a motor deficit that interfered with lever pressing. Although AM374 produced no effect on its own, this amidase inhibitor did enhance the behavioral effect of a low dose of anandamide. These results are consistent with the notion that AM 374 inhibited the enzymatic breakdown of exogenously injected anandamide. This type of procedure can be used to assess a variety of different compounds for their ability to inhibit cannabinoid metabolism.     

Differential properties between TRK-820 and U-50,488H on the discriminative stimulus effects in rats

It has been demonstrated that the newly synthesized kappa-opioid receptor agonist TRK-820, which has a unique structure that is different from those of other prototypical kappa-opioid receptor agonists such as U-50,488H, exert some behavioral effects that differ from those induced by U-50,488H. Therefore, the present study was designed to examine the possible difference between the discriminative stimulus effects of TRK-820 and U-50,488H in rats. Substitution tests with several kappa-opioid receptor agonists were initiated in rats trained to discriminate between TRK-820 (40 microg/kg) or U-50,488H (3.0 mg/kg) and saline. In the cross-substitution tests, U-50,488H substituted for the discriminative stimulus effects of TRK-820, whereas TRK-820 did not substitute completely for those of U-50,488H, indicating that the discriminative stimulus effects of TRK-820 and U-50,488H were somewhat different. In the substitution tests, E-2078, but not R-84760, substituted for the discriminative stimulus effects of both TRK-820 and U-50,488H. KT-90, CI-977 and ICI-199441 each substituted for the discriminative stimulus effects of U-50,488H, but not to those of TRK-820. These results imply that these kappa-opioid receptor agonists possess U-50,488H-like discriminative stimulus effects. Furthermore, that U-50,488H and the other kappa-opioid receptor agonists substituted for the discriminative stimulus effects of U-50,488H, produced aversive effects in rats. These findings suggest the possibility that unlike those of TRK-820, the cue of the discriminative stimulus effects of U-50,488H may be, at least in part, associated with its aversive effects.     

Thrombin as a Regulator of Inflammation and Reparative Processes in Tissues

Activation of blood coagulation and thrombin formation accompany inflammation, wound healing, atherogenesis, and other processes induced by endothelial injury. Systems of hemostasis and inflammation play an important role in the pathogenesis of acute coronary syndromes. This paper reviews thrombin functions involved in its interaction with PAR family receptors, activation of platelets, endothelial cells, leukocytes, smooth muscle cells, and mast cells. Mechanisms of regulatory effects of thrombin on mast cells associated with nitric oxide release are discussed.     

Co-administration of APD668, a G protein-coupled receptor 119 agonist and linagliptin,a DPPIV inhibitor,prevents progression of steatohepatitis in mice fed on a high trans-fat diet

Non-Alcoholic SteatoHepatitis (NASH) is the more severe form of Non-Alcoholic Fatty Liver Disease (NAFLD) and is characterized by the presence of hepatic steatosis, oxidative stress, inflammation, hepatocyte injury with or without fibrosis. Recently, GPR119 receptor has emerged as a novel therapeutic target for the treatment of dyslipidemia and non-alcoholic steatohepatitis. In the present study, we investigated the effect of APD668, a GPR119 agonist alone or in combination with linagliptin, a DPPIV inhibitor on the progression of steatohepatitis in mice fed on a high trans-fat diet. In this study, monotherapy with either APD668 or linagliptin caused a reduction in the levels of ALT, AST, glucose, cholesterol and epididymal fat mass but the effect was more pronounced upon treatment with combination of both drugs.On the other hand, combined treatment of APD668 with linagliptin demonstrated a non-significant additive effect in reduction of hepatic triglyceride (?78%) and cholesterol (?56%) compared to monotherapy groups. Moreover, co-administration of APD668 and linagliptin resulted in enhanced levels of active GLP-1 with additional benefit of significant synergistic decrease in body weight gain (?19%) in mice. We speculated that the enhanced effect observed with the combination treatment could be due to either 1) direct activation of GPR119 receptors present in liver and intestine or 2) enhanced active GLP-1 levels or 3) decreased degradation of GLP-1 in-vivo through DPPIV inhibition. Therefore, these findings clearly suggest that GPR119 receptor agonists in combination with DPPIV inhibitors may represent a promising therapeutic strategy for the treatment of non-alcoholic steatohepatitis.     

Effect of dietary conjugated linoleic acid and selenized yeast on the concentration of fatty acids and minerals in rats

Abstract

The main objective of this study was to evaluate the influence of diets enriched in individual conjugated linoleic acid (CLA) isomers, their mixture, and/or selenized yeast (Se-yeast) on the concentration of CLA isomers, long-chain polyunsaturated fatty acids (PUFA) and Se in the heart, muscles and liver of rats. The investigation was performed on 73 female Wistar rats (8 weeks of age, 200 g initial BW). After one week sub-maintenance feeding, rats received diets supplemented with 1% individual CLA isomers or 1 or 2% of a CLA isomers mixture, without or with 1.2 mg Se/kg (as Se-yeast) for 29 days. Feeding diets with 2% CLA isomer mixture reduced feed intake and body weight gain of rats, while addition of trans10,cis12 CLA and Se-yeast resulted in the highest body weight gain. CLA supplementation generally elevated the concentration of CLA isomers in heart and muscles significantly, although cis9,trans11 CLA accumulated preferentially. Regardless of the presence of Se-yeast, the dietary enrichment with CLA isomers caused a reduction in the capacity of Δ9-desaturase. Addition of Se-yeast to diets with individual CLA isomers or a 1% mixture of CLA isomers elevated the accumulation of CLA isomers in the heart and muscles, whereas all treatments with supplemented CLA and Se-yeast increased the accumulation of Se in rats compared with animals fed the diet containing Se only. Furthermore, CLA isomer supplementation decreased the concentration of PUFA and total fatty acids in the heart and muscles compared with control rats. Moreover, addition of CLA isomers interfered in the conversion of linoleic and linolenic acids to higher metabolites due to competition of CLA isomers for the same enzymes (Δ6-, Δ5-, Δ4-desaturases and elongase).     

Comparative study of oestrogenic properties of eight phytoestrogens in MCF7 human breast cancer cells

Previous studies have compared the oestrogenic properties of phytoestrogens in a wide variety of disparate assays. Since not all phytoestrogens have been tested in each assay, this makes inter-study comparisons and ranking oestrogenic potency difficult. In this report, we have compared the oestrogen agonist and antagonist activity of eight phytoestrogens (genistein, daidzein, equol, miroestrol, deoxymiroestrol, 8-prenylnaringenin, coumestrol and resveratrol) in a range of assays all based within the same receptor and cellular context of the MCF7 human breast cancer cell line. The relative binding of each phytoestrogen to oestrogen receptor (ER) of MCF7 cytosol was calculated from the molar excess needed for 50% inhibition of 3H]oestradiol binding (IC50), and was in the order coumestrol (35x)/8-prenylnaringenin (45x)/deoxymiroestrol (50x)>miroestrol (260x)>genistein (1000x)>equol (4000x)>daidzein (not achieved: 40% inhibition at 10(4)-fold molar excess)>resveratrol (not achieved: 10% inhibition at 10(5)-fold molar excess). For cell-based assays, the rank order of potency (estimated in terms of the concentration needed to achieve a response equivalent to 50% of that found with 17beta-oestradiol (IC50)) remained very similar for all the assays whether measuring ligand ability to induce a stably transfected oestrogen-responsive ERE-CAT reporter gene, cell growth in terms of proliferation rate after 7 days or cell growth in terms of saturation density after 14 days. The IC50 values for these three assays in order were for 17beta-oestradiol (1 x 10(-11)M, 1 x 10(-11)M, 2 x 10(-11)M), and in rank order of potency for the phytoestrogens, deoxymiroestrol (1 x 10(-10)M, 3 x 10(-11)M, 2 x 10(-11)M)>miroestrol (3 x 10(-10)M, 2 x 10(-10)M, 8 x 10(-11)M)>8-prenylnaringenin (1 x 10(-9)M, 3 x 10(-10)M, 3 x 10(-10)M)>coumestrol (3 x 10(-8)M, 2 x 10(-8)M, 3 x 10(-8)M)>genistein (4 x 10(-8)M, 2 x 10(-8)M, 1 x 10(-8)M)/equol (1 x 10(-7)M, 3 x 10(-8)M, 2 x 10(-8)M)>daidzein (3 x 10(-7)M, 2 x 10(-7)M, 4 x 10(-8)M)>resveratrol (4 x 10(-6)M, not achieved, not achieved). Despite using the same receptor context of the MCF7 cells, this rank order differed from that determined from receptor binding. The most marked difference was for coumestrol and 8-prenylnaringenin which both displayed a relatively potent ability to displace [3H]oestradiol from cytosolic ER compared with their much lower activity in the cell-based assays. Albeit at varying concentrations, seven of the eight phytoestrogens (all except resveratrol) gave similar maximal responses to that given by 17beta-oestradiol in cell-based assays which makes them full oestrogen agonists. We found no evidence for any oestrogen antagonist action of any of these phytoestrogens at concentrations of up to 10(-6)M on either reporter gene induction or on stimulation of cell growth.