Identification of a Natural Product Antagonist against the Botulinum Neurotoxin Light Chain Protease

Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. BoNTs are the most lethal known poisons affecting humans and has been recognized as a potential bioterrorist threat. Current treatments for botulinum poisoning are predominately prophylactic in nature relying on passive immunization with antitoxins. Inhibition of the BoNT light chain metalloprotease (LC) has emerged as a new therapeutic strategy for the treatment of botulism that may provide an effective post-exposure remedy. A high-throughput screening effort against the light chain of BoNT serotype A (LC/A) was conducted with the John Hopkins Clinical Compound Library comprised of over 1,500 existing drugs. Lomofungin, a natural product first isolated in the late 1960's, was identified as an inhibitor of LC/A, displaying classical noncompetitive inhibition kinetics with a K(i) of 6.7 ± 0.7 μM. Inhibitor combination studies reveal that lomofungin binding is nonmutually exclusive (synergistic). The inhibition profile of lomofungin has been delineated by the use of both an active site inhibitor, 2,4-dichlorocinnamic hydroxamate, and a noncompetitive inhibitor d-chicoric acid; the mechanistic implications of these observations are discussed. Lastly, cellular efficacy was investigated using a rat primary cell model which demonstrated that lomofungin can protect against SNAP-25 cleavage, the intracellular protein target of LC/A.     

Mibefradil,a Pharmacologically Distinct Calcium Antagonist

Mibefradil is the prototype of a new class of calcium antagonists that selectively block T-type voltage-gated plasma membrane calcium channels in vascular smooth muscle. The drug is structurally and pharmacologically different from traditional calcium antagonists. It does not have negative inotropism at therapeutic concentrations, and is not associated with reflex activation of neurohormonal and sympathetic systems. In clinical studies of hypertension, mibefradil 50 and 100 mg/day reduced trough sitting diastolic and systolic blood pressures in a dose-related manner. Dosages exceeding 100 mg/day generally did not result in significantly greater efficacy, but were associated with a higher frequency of adverse events. No first-dose hypotensive phenomenon was observed. Mibefradil has antiischemic properties resulting from dilation of coronary and peripheral vascular smooth muscle, and a slight reduction in heart rate. In clinical studies of chronic stable angina pectoris, dose-related increases in exercise duration, time to onset of angina, and time to 1-mm ST-segment depression during exercise tolerance tests occurred. Mibefradil reduced the number and duration of ischemic events recorded by 48-hour ambulatory electrocardiograph (ECG) monitoring, as well as number of anginal episodes and nitroglycerin consumption. Favorable hemodynamic and clinical profiles are reported, including high trough:peak ratios (> 80%), high oral bioavailability, and long elimination half-life (17–25 hrs) permitting once/day dosing. Dizziness, headache, leg edema, and lightheadedness are frequently reported, but overall the agent is well tolerated. First-degree atrioventricular block and sinus bradycardia are the most frequent ECG changes caused by the drug. In vitro studies indicate mibefradil inhibits cytochrome P450 1A2, 2D6, and 3A4, resulting in elevated plasma concentrations of drugs metabolized by those isoenzymes. Therefore, it is contraindicated in patients receiving terfenadine, astemizole, cisapride, lovastatin, or simvastatin.     

血小板活化因子受体拮抗剂——芳氨酮类化合物的合成及其抗炎活性

以取代亚苄基丙酮和二级胺盐酸盐经Mannich反应合成了一系列具有抗炎活性的芳氨酮类化合物，其结构经¹H NMR、MS、元素分析或HRMS确证。同时测定了所合成化合物对血小板活化因子(PAF)引起的大鼠胸腔多形核白细胞β-葡糖苷酸酶释放的抑制作用，初步药理实验结果表明，大部分芳氨酮类(MA)化合物对PAF引起的大鼠胸腔多形核白细胞β-葡糖苷酸酶的释放有明显的抑制作用，其中化合物MA12、MA13、MA18、MA21和MA33的抑制活性较强。提示化合物可通过抑制β-葡糖苷酸酶释放发挥抗炎作用，可能是潜在的PAF受体拮抗剂。     

BM－13505抗栓作用及机理研究

ＢＭ１３５０５是新合成的血栓烷受体拮抗剂，ＢＭ１３５０５０．４５和０．２３ｍｇ／ｋｇｉｖ延长大鼠颈动脉血管阻塞时间（ｐ＜０．００１和ｐ＜０．０１）．ＢＭ１３５０５２ｍｇ／ｋｇｉｖ有效地防止ＡＡ４ｍｇ／ｋｇ所致的大鼠脑血栓形成（ｐ＜０．０１）。ＢＭ１３５０５１０ｍｇ／ｋｇｉｐ可防止ＡＡ诱导的小鼠肺部栓塞（ｐ＜０．０１）。该药可延长小鼠尾出血时间。ＢＭ１３５０５显著抑制ＡＡ诱导的兔血小板聚集，半数抑制浓度ＩＣ＿（５０）为０．１７Ｖｍｏｌ／Ｌ，对ＡＤＰ和胶原诱导的血小板聚集无影响。ＢＭ１３５０５降低兔血小板及小鼠血浆中ＴＸＢ＿２水平，对血小板ｃＡＭＰ水平无影响。在不远的将来，ＢＭ１３５０５可能会发展成一种理想的抗血小板药及抗血栓药。     

BM—13505抗栓作用及机理研究

BM 13505是新合成的血栓烷受体拮抗剂，BM 13505 0.45和0.23mg/kg iv延长大鼠颈动脉血管阻塞时间（P＜0.001和P＜0.01）。BM 13505 2mg/kg iv有效地防止AA 4mg/kg所致的大鼠脑血栓形成（P＜0.01）。BM 13505 10mg/kg ip可防止AA诱导的小鼠肺部栓塞（P＜0.01）。该药可延长小鼠尾出血时间。BM 13505显著抑制AA诱导的兔血小板聚集，半数抑制浓度IC50为0.17μmol/L，对ADP和胶原诱导的血小板聚集无影响。BM13505降低兔血小板及小鼠血浆中TXB2水平，对血小板cAMP水平无影响，在不远的将来，BM13505可能会发展成一种理想的抗血小板药及抗血栓药。     

Intranasal Delivery of RS-93522, a Dihydropyridine-Type Calcium-Channel Antagonist

Pharmaceutical Research -     

Discovery of NVP-LDE225, a Potent and Selective Smoothened Antagonist

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Selectivity of Bevantolol Hydrochloride,a β-Adrenoceptor Antagonist,in Asthmatic Patients

Bevantolol hydrochloride, a new β-adrenoceptor antagonist, has been shown to be cardioselective in animals. To evaluate its selectivity in humans, a double-blind, crossover study was conducted in 8 asthmatics. Following a single oral dose of placebo, bevantolol 75 or 150 mg or propranolol hydrochloride 40 mg, forced expiratory volume in 1 second (FEV₁), heart rate, blood pressure and skeletal muscle tremor were measured before and after 4 increasing intravenous doses of terbutaline sulfate to establish terbutaline dose-reponse curves. The FEV₁ decreased after all active treatments. During terbutaline infusions there was an increase in FEV₁ after both bevantolol doses and placebo. The terbutaline dose-response curve after bevantolol shifted to the right, however. After propranolol, there was no increase in FEV₁ during terbutaline stimulation. Heart rate and skeletal muscle tremor showed a similar pattern during the experiment. In dosages that have previously been shown to produce at least the same degree of blockade of exercise-induced tachycardia, bevantolol has less influence on terbutaline-induced bronchodilation, heart rate increase and skeletal muscle tremor than did propranolol. Thus bevantolol has relative β₁-adrenoceptor antagonist selectivity. Drawing upon the results of a previous study in the same patients, we believe bevantolol, atenolol and metoprolol have similar β₁-selectivity.     

Cathinone,a Natural Amphetamine

Abstract: Cathinone is an alkaloid that has been discovered some fifteen years ago in the leaves of the khat bush. This plant grows in East Africa and in southern Arabia, and the inhabitants of these regions frequently chew khat because of its stimulating properties. Cathinone, which is S(–)-α-aminopropiophenone, was soon found to have a pharmacological profile closely resembling that of amphetamine; indeed, in a wide variety of in vitro and in vivo experiments it was demonstrated that cathinone shares the action of amphetamine on CNS as well as its sympathomimetic effects; thus, for example, drug-conditioned animals will not distinguish between cathinone and amphetamine. These various observations were confirmed by a clinical experiment showing that cathinone also in humans produces amphetamine-like objective and subjective effects. Finally, it was demonstrated that cathinone operates through the same mechanism as amphetamine, i.e. it acts by releasing catecholamines from presynaptic storage sites. Thus, much experimental evidence indicates that cathinone is the main psychoactive constituent of the khat leaf and that, in fact, this alkaloid is a natural amphetamine.     

The Disposition of Thioperamide,a Histamine H3-Receptor Antagonist,in Rats

Abstract— An HPLC method using an ovomucoid-conjugated column has been developed for measurement of thioperamide, a histamine H₃ antagonist, with a minimum quantitation limit of 0·05 μg mL^?1 The assay was used to study the disposition of thioperamide in rats. After bolus intravenous administration of thioperamide (10 mg kg^?1), the plasma concentration decreased monoexponentially with a half-life of 26·9 min. The apparent total body clearance of thioperamide from rat plasma was 74·6 mL min^?1 kg^?1. Although thioperamide was quickly transferred to various tissues, its concentrations in peripheral tissues were higher than that in the brain. However, the brain regional tissue/plasma ratios of thioperamide increased continuously after its injection.     

GYKI-46 903, a Non-Competitive Antagonist for 5-HT3 Receptors

Abstract: The effects of GYKI-46 903 ((+)endo-4-propionyloxy-6-(4-fluorophenyl)-1-azabicyclo [3.3.1]non-6-ene HCI), on 5-HT₃ receptors have been studied and compared with ondansetron in peripheral organs in vitro and in vivo, and in a receptor binding assay in membranes prepared from rat cerebral cortex. GYKI-46 903 was found to be a non-competitive antagonist at 5-HT₃ receptors present in non-stimulated longitudinal muscle strip of guinea-pig ileum (pD₂’against serotonin=5.54), and also in 5-methoxytryptamine-pretreated electrically stimulated ileal preparations (pD₂’against serotonin=5.26). On the contrary, ondansetron was found to be a competitive antagonist for 5-HT₃ receptors; the pA₂ value against serotonin was 7.40 in non-stimulated ileum, and it was 7.08 in electrically stimulated ileal preparation pretreated with 5-methoxytryptamine. In displacement studies, the pIC₅₀ values of GYKI-46 903 and ondansetron against [³H]granisetron binding to rat cerebral cortex membranes were 6.91 and 8.58 respectively. GYKI-46 903, when administered by intravenous infusion, antagonized the decrease in heart rate evoked by serotonin (Bezold-Jarisch reflex) in anaesthetized rats, and the maximal reversal was less than 50%. This was in striking contrast with ondansetron, which, after intravenous injection, completely antagonized the serotonin-induced bradycardia with an ID₅₀ value of 3.28 ug/kg. These data classify GYKI-46 903 as a non-competitive antagonist for 5-HT₃ receptors.     

内皮素及其受体拮抗剂与肿瘤

目的分析内皮素系统与肿瘤发生发展的关系,为内皮素受体成为抗肿瘤药物新的作用靶点提供依据,同时为抗肿瘤药物研究提供新思路。方法检索2000—2010年国内外医学期刊中内皮素系统与肿瘤发生发展及内皮素受体拮抗剂与肿瘤治疗的资料,综述内皮素及其受体拮抗剂与肿瘤的关系。结果内皮素受体是抗肿瘤药物新的作用靶点。结论内皮素受体拮抗剂将成为肿瘤治疗中的新型药物。     

Flupirtine,a Nonopioid Centrally Acting Analgesic,Acts as an NMDA Antagonist*

• 1.Flupirtine (Katadolon) is a member of a class of triaminopyridines and is used as a nonopioid analgesic agent with muscle relaxant properties.
• 2.In situ experiments have revealed that flupirtine protects against ischemic-induced insults to the retina and brain.
• 3.Data derived from in vitro and in vivo studies suggest that flupirtine functions as a weak N-methyl-d-aspartate (NMDA) antagonist with little evidence that it acts on AMPA–kainate type glutamate receptors.
• 4.No evidence could be found from binding studies to suggest that flupirtine has an affinity for any of the characterized binding sites associated with the NMDA receptor.
• 5.Studies on cultured cortical neurons show that the NMDA-induced influx of ⁴⁵Ca²⁺ is more readily decreased by flupirtine when a reducing agent (dithiothreitol) is present. However, when N′-ethylmaleimide, which is thought to alkylate the NMDA receptor redox site, is present, no obvious effect on the NMDA-induced influx of ⁴⁵Ca²⁺ is produced by flupirtine.
• 6.Flupirtine is also known to counteract the production of reactive oxygen species caused by ascorbate/iron as well as to prevent apoptosis in cells lacking NMDA receptors induced by oxidative stress.
• 7.To explain all the experimental data, it is suggested that flupirtine affects the redox state/pH/electrons in the cell. The specific way by which flupirtine antagonizes the NMDA receptor might be by an action on the known redox site of the receptor.

     

Discovery of AMG 853, a CRTH2 and DP Dual Antagonist

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CB1R部分拮抗剂alisol G的大鼠药动学研究及血脑屏障透过率初评

建立了UPLC-MS/MS方法测定大鼠血浆及脑脊液中大麻素受体1(CB1R)部分拮抗药泽泻醇G(alisol G)的浓度,评价其血脑屏障透过性。以利莫那班为内标,采用液液萃取前处理方法,色谱柱为ACQUITY BEH C18柱,流动相为甲醇∶0.1%乙酸溶液(90∶10),质谱采用正离子扫描的电喷雾离子源,多反应监测(MRM)模式,对m/z 455.16→m/z383.04(泽泻醇G)、m/z 464.88→m/z364.75(内标)进行定量分析。泽泻醇G血浆样品在10~4000ng/ml、脑脊液样品在4~400 ng/ml范围内线性良好;批内、批间RSD≤10.22%,样品回收率稳定(78.5%~86.5%)、贮存和处理条件下稳定性良好。本方法灵敏、可靠,能准确测定大鼠血浆及脑脊液中的泽泻醇G浓度。大鼠灌胃后血浆中cmax为312.64ng/ml,脑脊液中检测不到原型化合物,表明泽泻醇G不易透过血脑屏障。本研究结果为CB1R部分拮抗药泽泻醇G的后续研究开发提供了重要依据。     

Discovery of INCB3284, a Potent,Selective, and Orally Bioavailable hCCR2 Antagonist

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Absorption and Disposition of a Selective Aldosterone Receptor Antagonist,Eplerenone, in the Dog

Purpose. The present study was conducted to characterize the pharmacokinetics of eplerenone (EP), a selective aldosterone receptor antagonist, and its open lactone ring form in the dog. Methods. Pharmacokinetic studies of EP were conducted in dogs following i.v., oral, and rectal dosing (15 mg/kg) and following intragastric, intraduodenal, intrajejunal, and intracolonic dosing (7.5 mg/kg). Results. After oral administration, the systemic availability of EP was 79.2%. Systemic availabilities following administration via other routes were similar to that following oral administration. The half-life and plasma clearance of EP were 2.21 hr and 0.329 l/kg/hr, respectively. Plasma concentrations of the open lactone ring form were lower than EP concentrations regardless of the route of administration. The C-14 AUC in red blood cells was approximately 64% and 68% of the plasma AUC for i.v. and oral doses. Percentages of the dose excreted as total radioactivity in urine and feces were 54.2% and 40.6%, respectively, after i.v. administration, and 40.7% and 52.3%, respectively, after oral administration. The percentages of the dose excreted in urine and feces as EP were 13.7% and 2.5%, respectively, after i.v. administration, and 2.1% and 4.6% after oral administration, respectively. Approximately 11% and 15% of the doses were excreted as the open form following i.v. and oral doses. Conclusions. EP was rapidly and efficiently absorbed throughout the gastrointestinal tract, resulting in a good systemic availability. The drug did not preferentially accumulate in red blood cells. EP was extensively metabolized; however, first-pass metabolism after oral and rectal administration was minimal. EP and its metabolites appear to be highly excreted in the bile.     

Disposition and Metabolism of Setipiprant,a Selective Oral CRTH2 Antagonist,in Humans

Background

Setipiprant, a tetrahydropyridoindole derivative, is a CRTH2 (chemoattractant receptor-homologous molecule expressed on T-helper [Th]-2 cells) antagonist that has the potential to be effective in the treatment of patients with diseases with an allergic etiology, such as allergic rhinitis and asthma.

Objectives

This study investigated the disposition, metabolism, and elimination of setipiprant.

Study design

In this open-label study, a single oral dose of 1,000 mg ¹⁴C-labeled setipiprant was administered.

Participants

Six healthy male subjects were enrolled in this study.

Results

The radioactive dose was almost completely recovered in feces (88.2 %) and to a smaller extent in urine (11.7 %). The main recovery route for unchanged setipiprant was feces (50 % of the radioactive dose). The recovered amount of unchanged setipiprant in urine accounted for 3.7 %. The two main metabolites were M7 and M9 with the intact tetrahydropyridoindole core of setipiprant. M7 and M9 are supposedly two distinct dihydroxy-dihydronaphthalene isomers assumed to be formed by intermediate epoxidation of the naphthyl ring followed by a hydrolytic epoxide ring-opening. M7 and M9 accounted for 20.0 and 15.3 % of the administered radioactive dose. Both metabolites were mainly excreted via feces and to a lesser extent via urine. M7 was the only metabolite quantifiable in plasma, but at concentrations consistently below 10 % of those of the parent drug.

Conclusion

Setipiprant is mainly excreted in feces in the form of the parent drug and in smaller amounts as its metabolites M7 and M9.     

Characterization of the Anticonflict Effect of MK-801, a Non-Competitive NMDA Antagonist

Abstract: Brain serotonergic, noradrenergic and GABAergic mechanisms are all involved in the regulation of conflict behaviour, and the GABA_A/benzodiazepine receptor complex may play the most central role in this context. Since facilitation of GABAergic inhibitory transmission produces anticonflict effects, it has been suggested that antagonism of excitatory inputs may serve the same cause, and, indeed, blockade of excitatory neurotransmission mediated via N-methyl-D-aspartate (NMDA), receptors, produces anticonflict effects. In the present study, using a modified Vogel's rat conflict model, we have investigated whether the anticonflict effect of the non-competitive NMDA antagonist MK-801 can be linked to NMDA receptor blockade, and if stimulation of these receptors instead produces proconflict effects. The tentative involvement of noradrenergic, serotonergic or GABAergic effects in the MK-801-induced anticonflict effect was also studied. MK-801 produced a dose-dependent and specific anticonflict effect (maximal effect after 0.05 mg/kg, intraperitoneally,–90 min.). This anticonflict action was completely counteracted by NMDA in a dose (0.125 μg, intracerebroventricularly) not affecting behaviour per se. The highest dose tested of NMDA alone (0.5 μg) tended to produce a proconflict effect, but this action may be unspecific due to concomitant drug-induced motor-inhibition. Neither bicuculline and picrotoxin, antagonists at the GABA_A/benzodiazepine receptor complex, nor the adrenoceptor antagonists propranolol and prazosin significantly altered the MK-801-induced anticonflict effect, whereas L-5-HTP (50 mg/kg, intraperitoneally, after inhibition of peripheral decarboxylation with benzerazide) completely abolished the anticonflict effect of MK-801. The results indicate that the anticonflict effect of MK-801 is primarily mediated by antagonism of NMDA receptors, and that brain 5-HT systems may also be involved in this effect.