5-HT6 Receptor (Ki = 2.04 nM)

为了确定任何其他脱靶活性，Novascreen在其100多个靶位点的商业选择性小组中评估了化合物SUVN-502（5al），这些靶位点包括受体（64）、酶（5）、肽（18）、生长因子（5），离子通道（6）、类固醇、免疫因子、第二信使和前列腺素（支持信息）。1μM的化合物SUVN-502（5al）对所有受试受体的抑制率均小于50%，多巴胺D3、肾上腺素能α2A和2C除外。在一项独立实验中，5al在多巴胺D3和2570 nM肾上腺素能α2A受体上的Ki值为616 nM。在α2C受体上，功能亲和力（Kb）值为619 nM。在阿司咪唑结合试验中测试时，主要候选物5al没有显示出对hERG钾通道的显著结合，在人类1期临床试验中也没有发现QTc间期的变化
在Caco-2肠上皮细胞系中评估了SUVN-502（5al）的渗透性，该细胞系显示出高渗透性（Papp=19.6×10-6cm/s）。外排比[Papp（B-A）/Papp（A-B）]小于2，表明与外排转运蛋白如P-gp的相互作用最小。使用平衡透析法在大鼠、狗和人血浆中测定了5al的蛋白结合程度。发现5al在大鼠、狗和人中的血浆蛋白结合率分别为99.0%、98.6%和98.0%[1]。

在观察到单独治疗后乙酰胆碱水平升高后，评估了治疗剂量（3mg/kg口服）和亚治疗剂量（1mg/kg口服）的Masupidine/SUVN-502（5al）与多奈哌齐（1mg/kg皮下注射）和美金刚胺（1mg/kg静脉注射）联合使用对雄性Wistar大鼠腹侧海马乙酰胆碱调节的影响（图5）。本研究设计的前提是，这种组合可能会提高治疗效果，并提供多种治疗益处，例如减少治疗剂的剂量，这可能会避免与更高剂量给药相关的副作用。如果假设是正确的，与现有批准的治疗方案相比，这些方案可能会提供一种新的机制，提高药物疗效和耐受性。

多奈哌齐（1 mg/kg皮下注射）和美金刚（1 mg/kg静脉注射）联合治疗导致海马乙酰胆碱水平升高，最高达到基础水平的1203±106%。化合物Masupidine/SUVN-502（5al）与多奈哌齐和美金刚联合口服1和3mg/kg后，平均最大增幅分别为1383±194%和2136±288%。Masupidine/SUVN-502（5al）（口服1和3 mg/kg）、多奈哌齐和美金刚联合用药产生的乙酰胆碱增加明显高于多奈哌齐和美金刚联合使用产生的增加。联合治疗组乙酰胆碱的增加没有任何胆碱能副作用。在另一项涉及药代动力学评估的研究中，单独或以三种组合给药时，多奈哌齐、美金刚或Masupidine/SUVN-502（5al）的血浆暴露量没有显著差异，从而排除了药代动力学相互作用介导效应的可能性（存档数据）。上述临床前研究的结果为5-HT6R拮抗剂Masupidine/SUVN-502（5al）与多奈哌齐和美金刚联合治疗认知障碍的潜在治疗效用提供了支持。

在第一阶段人体临床试验研究中，Masupidine/SUVN-502（5al）在健康男性和女性受试者中单次递增剂量和健康老年男性受试者14天的多次递增剂量中显示出良好的安全性和药代动力学特征。性别和食物对Masupidine/SUVN-502（5al）的药代动力学没有显著影响。它在人类中具有良好的耐受性，具有足够的血浆暴露效果和适合每天一次治疗的良好半衰期。正在进行一项2期概念验证研究（ClinicalTrials.gov标识符：NCT02580305），以评估5al在接受稳定剂量多奈哌齐和美金刚胺的中度AD患者中的安全性和有效性。据我们所知，这是第一项临床试验，其中使用三重组合[5-H-6R拮抗剂（5al）+多奈哌齐+美金刚]来评估5-HT6R拮抗剂的疗效[1]。

放射配体结合分析法测定5-羟色胺6受体Ki：[1]
之前已经描述了所使用的程序。简而言之，使用的受体源和放射性配体分别是HEK-293细胞和[3H]赖氨酸二乙胺（LSD）中表达的人重组受体。最终配体浓度为1.5 nM，非特异性决定簇为甲磺酸甲氧基肝素[0.1µM]。甲磺酸甲氧基肝素用作阳性对照。反应在含有10 mM MgCl2、0.5 mM EDTA的50 mM Tris-HCl（pH 7.4）中在37°C下进行60分钟。通过在玻璃纤维过滤器上快速真空过滤终止反应。测定过滤器上捕获的放射性，并将其与对照值进行比较，以确定受试化合物与克隆的5-羟色胺5-HT6结合位点的任何相互作用，并报告为Ki值。进行了这些研究，并使用如上所述的标准放射性配体结合技术对数据进行了分析。在这些测试的测定条件下，甲磺酸甲氧苄啶（参考化合物）的Ki值为0.5±0.04 nM。

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5-HT6受体Kb的测定：[1]
之前已经描述了所使用的程序。表达重组人5-HT6受体和pCRE-Luc报告系统的稳定CHO细胞系用于基于细胞的检测。该测定提供了一种非放射性的方法来确定化合物与GPCR的结合。在该特定测定中，测量由受体的激活或抑制调节的细胞内环AMP的水平。重组细胞在cAMP反应元件的控制下携带萤光素酶报告基因。使用含有10%胎牛血清（FBS）的Hams F12培养基，将上述细胞以5 x 104个细胞/孔的密度铺在96孔透明底部白色板上，在37℃和5%CO2下孵育S4过夜，然后血清饥饿18-20小时。向细胞中加入越来越高浓度的测试化合物以及OptiMEM中的10µM血清素。在37℃的CO2培养箱中继续孵育4小时。4小时后，使用裂解缓冲液裂解细胞，向每个孔中加入萤光素酶测定缓冲液，并使用发光计数器记录每秒计数。根据获得的每秒计数（CPS），以10µM 5-HT为100%结合，以载体为0%结合，计算每个孔的结合百分比。将百分比界限值与化合物浓度作图，并使用Graph pad Prism 4软件的非线性迭代曲线拟合计算机程序对数据进行分析。Kb和IC50值是使用该测定中使用的激动剂浓度及其在同一软件中的EC50值计算的。在这些测试的测定条件下，甲磺酸甲氧苄啶（参考化合物）的Kb值为0.7±0.05 nM。

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5-HT1A结合实验程序：[1]
之前已经描述了所使用的程序。从重组人5-HT1A细胞系和放射性配体8-羟基-DPAT[丙基2,3--ring-1,2,3-3H]制备的膜是商业上购买的。缓冲液制备中使用的所有其他试剂都是商业上购买的。最终配体浓度为1.75nM；非特异性决定因素是5-HT[10µM]和5-HT1A膜蛋白（16µg/孔）。血清素被用作阳性对照。反应在含有0.5 mM EDTA、10 mM MgSO4和0.1%抗坏血酸缓冲液的50 mM Tris pH 7.4中在25℃下进行120分钟。通过快速过滤停止反应，然后使用预涂有0.33%聚乙烯亚胺的96孔收获板对结合混合物进行六次洗涤。将板干燥，使用MicroBeta-TriLux通过闪烁计数测定过滤器上收集的结合放射性。在S5存在非标记化合物的情况下，放射性配体结合以总结合的百分比表示，并与化合物的对数浓度作图。Ki值使用Graph pad Prism 4软件的非线性迭代曲线拟合计算机程序确定（表1）。在这些测试的测定条件下，血清素（参考化合物）的Ki值为0.2±0.03 nM。

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5-HT2A结合实验程序：[1]
使用的程序如前所述，由重组人5-HT2A细胞系制备膜，放射性配体盐酸氯胺酮[Ethylene-3H]-（R-41468）购自Perkin-Elmer。缓冲液制备中使用的所有其他试剂均购自Sigma。最终配体浓度为1.75nM；非特异性决定簇为1-NP[10µM]和5-HT2A膜蛋白（5µg/孔）。1-NP用作阳性对照。反应在含有0.5 mM EDTA缓冲液的67 mM Tris pH 7.6中在25℃下进行60分钟。通过快速过滤停止反应，然后使用预涂有0.33%聚乙烯亚胺的96孔收获板对结合混合物进行六次洗涤。将板干燥，使用MicroBeta-TriLux通过闪烁计数测定过滤器上收集的结合放射性。未标记化合物存在下的放射性配体结合以总结合的百分比表示，并与化合物的对数浓度作图。Ki值使用Graph pad Prism 4软件的非线性迭代曲线拟合计算机程序确定（表1）。在这些测试的测定条件下，1-（1-萘基）哌嗪盐酸盐（参考化合物）的Ki值为15.4±0.7 nM。

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5-HT2C结合实验程序：[1]
S6使用的程序已在前面描述过。简而言之，重组人5-HT2C细胞系的膜制剂，放射性配体Mesulergine，[NMethyl3H]购自Perkin-Elmer。缓冲液制备中使用的所有其他试剂均购自Sigma。最终配体浓度为1.25nM；非特异性决定因素是棉子丝氨酸[10µM]、5-HT2C膜蛋白（30µg/孔）和Ysi聚赖氨酸SPA珠，1.0 mg/孔。米安丝氨酸用作阳性对照。反应在含有10.8 mM MgCl 2、0.54 mM EDTA、10.8µM Pargyline、0.108%抗坏血酸、pH 7.4的54 mM Tris（pH 7.4）缓冲液中在25°C下进行180分钟。该板在MicroBeta TriLux中读取。未标记化合物存在下的放射性配体结合以总结合的百分比表示，并与化合物的对数浓度作图。Ki值使用Graph pad Prism 4软件的非线性迭代曲线拟合计算机程序确定（表1）。在这些测试的测定条件下，米安色林（参考化合物）的Ki值为2.8±0.3 nM。

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5-HT4B结合实验程序：[1]
7简而言之，从重组人5-HT4B细胞系制备的膜是商业上购买的，放射性配体GR113808，[N-甲基3H]是商业上购得的。缓冲液制备中使用的所有其他试剂均购自Sigma。最终配体浓度为0.5nM；非特异性决定簇为GR113808[10µM]和5-HT4B膜蛋白（5µg/孔）。GR113808用作阳性对照。反应在25 mM Tris-HCl（pH 7.4）缓冲液中在25°C下进行120分钟。通过快速过滤停止反应，然后使用预涂有0.33%聚乙烯亚胺的96孔收获板对结合混合物进行六次洗涤。S7干燥该板，使用MicroBeta-TriLux通过闪烁计数测定过滤器上收集的结合放射性。未标记化合物存在下的放射性配体结合以总结合的百分比表示，并与化合物的对数浓度作图。Ki值使用Graph pad Prism 4软件的非线性迭代曲线拟合计算机程序确定（表1）。在这些测试的测定条件下，GR113808（参考化合物）的Ki值为16.2±0.8 nM。

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大鼠5-HT7受体IC50值的测定：[1]
之前已经描述了所使用的程序。8表达重组大鼠5-HT7受体和pCRE-Luc报告系统的稳定CHO细胞系用于基于细胞的测定。该测定提供了一种非放射性的方法来确定化合物与GPCR的结合。在该特定测定中，测量由受体的激活或抑制调节的细胞内环AMP的水平。重组细胞在cAMP反应元件的控制下携带萤光素酶报告基因。使用含有10%胎牛血清（FBS）的Hams F12培养基，将上述细胞以5 x 104个细胞/孔的密度铺在96孔透明底部白色板上，在37℃和5%CO2下孵育过夜，然后血清饥饿18-20小时。向细胞中加入越来越高浓度的测试化合物以及OptiMEM中的10µM血清素。在37℃的CO2培养箱中继续孵育4小时。4小时后，使用裂解缓冲液裂解细胞，向每个孔中加入萤光素酶测定缓冲液，并使用发光计数器记录每秒计数。根据获得的CPS，以10µM血清素为100%结合，载体为0%结合，计算每个孔的结合百分比。将百分比界限值与化合物浓度作图，并使用Graph pad Prism 4软件的非线性迭代曲线拟合计算机程序对数据进行分析。IC50 S8值是使用该测定中使用的激动剂的浓度及其在同一软件中的EC50值计算的（表1）。在这些测试的测定条件下，甲磺酸甲氧苄啶（参考化合物）的Kb值为0.6±0.04 nM（IC50=117±6.6 nM）。

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蛋白质结合方案：[1]
使用快速平衡透析法测定血浆中Masupidine/SUVN-502（5al）的未结合部分。透析液室装有750µL 100 mM磷酸盐缓冲液（pH 7.4），一式三份。基质室装有500µL血浆，血浆中掺有终浓度为1µM的Masupidine/SUVN-502（5al）。0小时时，从两个腔室中取出50µL样品。密封S19板，在37°C下以100 rpm孵育6小时。6小时后，从两个腔室中取出50µL样品。将等体积的缓冲液或血浆分别添加到血浆/微粒体和缓冲液样品中，以创建相同的样品基质进行分析。用150µL含乙腈的内标沉淀样品。所有样品在4°C下以10000 rpm离心10分钟。将上清液转移到小瓶中，并通过LC-MS/MS进行分析。

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CYP抑制：[1]
如别处所述，使用人肝微粒体中CYP酶的标记探针底物反应来确定受试化合物的抑制谱。17最终的孵育混合物由磷酸盐缓冲液（100 mM，pH 7.4）、每种酶特异性的标记探针底物和人肝微粒体组成。通过加入NADPH（1 mM）至最终体积为200µL来引发反应。在为每种CYP亚型指定孵育时间后，用240µL含乙腈的内标终止120µL的孵育混合物。样品在4°C下以2500xg离心10分钟。

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CYP诱导：[1]
将来自三个供体的人冷冻肝细胞解冻，在通用冷冻恢复培养基中回收，并在100 g下离心10分钟。通过台盼蓝染料排除评估存活率。肝细胞被铺在胶原涂层的96孔板上，每孔约有65000个细胞，并在37°C、5%二氧化碳和95%空气的加湿环境中培养。将肝细胞培养1 S21天，然后用试验化合物和阳性对照诱导剂（1A2:50μM奥美拉唑；2B6:1000μM苯巴比妥；3A4:20μM利福平）处理。研究中使用了来自3个不同供体的人肝细胞。治疗又进行了3天。在3天的处理期后，将培养基换成含有特定P450底物的HIM，并将细胞孵育30分钟。孵育期结束时，将培养基收至96孔板中，并在-80°C下储存直至分析。使用四唑啉盐WST-1通过WST细胞活力测定法测定肝细胞的细胞活力。将肝细胞与含HIM的WST-1试剂以1:10的稀释度孵育2小时，然后使用紫外平板读数器在450nm处定量吸光度。根据制造商提供的分离说明，使用RNeasy 96试剂盒从肝细胞中分离总RNA。使用高容量cDNA逆转录试剂盒用100ng分离的RNA进行逆转录。使用7500快速实时PCR测量基因表达。使用Taqman通用混合物和特定引物组对RT反应进行定量。将靶基因的相对数量与通过ΔΔCT方法测定的内源性控制管家基因表达（GAPDH）的相对数量进行比较。

渗透性研究：[1]
Caco-2细胞中的跨上皮转运在瑞士RCC Gen biotec GmbH（RCCGBT）按照其标准操作程序进行。细胞在添加了10%胎牛血清、1%非必需氨基酸和1%庆大霉素的DMEM培养基中维持。使用Caco-2培养基将细胞以约300000个细胞/cm2的密度接种在12孔聚酯（PET）透孔培养皿中。细胞在37°C/5%CO2的加湿培养箱中培养。培养基每2-3天更新一次。在培养约10天后，每三天测定一次TEER，直至其达到平稳状态。试验中使用了TEER大于280Ωcm 2的Transwell腔。对于经上皮转运研究，在试验开始前测量TEER S18，取出培养基，用预热（37℃）的Hank's缓冲液洗涤细胞两次，以去除微量培养基。通过向顶腔或基底外侧腔中加入溶解的试验化合物，使其终浓度达到10µM，从而开始试验。在不同时间点（20、40、60、80、100和120分钟）采集100µl的样品。通过添加100µL Hank's缓冲液来替换去除的体积，并在评估结果时考虑浓度的降低。使用LCMS/MS分析对样品进行分析。[1]

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放射配体结合分析法测定肾上腺素能α2A受体Ki：[1]
简言之；使用的受体源和放射配体分别是HT29细胞和[3H]MK-912。最终配体浓度为0.75 nM，非特异性决定簇为L（-）-去甲肾上腺素-[100µM]。盐酸羟甲唑啉用作阳性对照。在含有1 mM EDTA、140 mM NaCl和100µM Gpp（NH）p（鸟苷-5'-酰亚胺二磷酸盐）的33 mM Tris-HCl（pH 7.5）中，于25°C下反应60分钟。通过在玻璃纤维过滤器上快速真空过滤终止反应。测定过滤器上捕获的放射性，并将其与对照值进行比较，以确定受试化合物与人肾上腺素能α2A结合位点的任何相互作用，并报告为Ki值。进行了这些研究，并使用如上所述的标准放射性配体结合技术对数据进行了分析。在这些测试的测定条件下，盐酸羟甲唑啉（参考化合物）的Ki值为1.3±0.06 nM。

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肾上腺素能α2C受体Kb值的测定：[1]
表达重组人肾上腺素能α2C受体和pCRE-Luc报告系统的稳定CHO细胞系用于基于细胞的检测。该测定提供了一种非放射性的方法来确定化合物与GPCR的结合。在该特定测定中，测量由受体的激活或抑制调节的细胞内环AMP的水平。重组细胞在cAMP反应元件的控制下携带萤光素酶报告基因。使用含有10%胎牛血清（FBS）的Hams F12培养基，将上述细胞以5 x 104个细胞/孔的密度铺在96孔透明底部白色板上，在37℃和5%CO2下孵育过夜，然后血清饥饿18-20小时。向细胞中加入越来越高浓度的试验化合物以及MEM Opti中的1µM肾上腺素和1µM福司克林。在37℃的CO2培养箱中继续孵育4小时。4小时后，使用裂解缓冲液裂解细胞，向每个孔中加入萤光素酶测定缓冲液，并使用发光计数器记录每秒计数。根据获得的CPS，以10µM肾上腺素为100%结合，以载体为0%结合，计算每个孔的结合百分比。将百分比界限值与化合物浓度作图，并使用Graph pad Prism 4软件的非线性迭代曲线拟合计算机程序对数据进行分析。Kb和IC50值是使用该测定中使用的激动剂浓度及其在同一软件中的EC50值计算的。在这些测试的测定条件下，螺酯碱（参考化合物）的Kb值为3.6±0.3 nM。

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放射性配体结合分析法测定多巴胺D3受体Ki：[1]
简言之；使用的受体源和放射性配体分别是CHO细胞和[3H]7-OH DPAT中表达的大鼠重组受体。最终配体浓度为0.8 nM，非特异性决定簇为多巴胺–[1µM]。（±）-7-OH-DPAT-HBr作为阳性对照。反应在含有120 mM NaCl的50 mM Tris-HCl（pH 7.4）中在25°C下进行60分钟。通过在玻璃纤维过滤器上快速真空过滤终止反应。测定过滤器上捕获的放射性，并将其与对照值进行比较，以确定测试化合物与克隆的多巴胺D3结合位点的任何相互作用，并报告为Ki值。进行了这些研究，并使用如上所述的标准放射性配体结合技术对数据进行了分析。在这些测试的测定条件下，（±）-7-OH-DPAT HBr（参考化合物）的Ki值为0.4±0.02 nM。

In Vivo Brain Microdialysis [1]
Male Wistar rats (240–300 g body weight) were stereotaxically implanted with a microdialysis guide cannula in ventral hippocampus (AP: −5.2 mm, ML: +5.0 mm, DV: −3.8 mm) under isoflurane anesthesia. Coordinates were taken according to the atlas for rat brain with reference points taken from bregma and vertical from the skull. The rats were allowed to recover individually for 4 days in a round-bottom Plexiglas bowl with free access to feed and water. After surgical recovery of 4–5 days, male Wistar rats were connected to a dual quartz lined two-channel liquid swivel on a counter balance lever arm, which allowed unrestricted movements of the animal. Sixteen hours before the study, a pre-equilibrated microdialysis probe (4 mm dialysis membrane) was inserted into the ventral hippocampus through the guide cannula. On the day of study, a probe was perfused with artificial cerebrospinal fluid (aCSF; NaCl 147 mM, KCl 3 mM, MgCl2 1 mM, CaCl2·2H2O 1.3 mM, NaH2PO4·2H2O 0.2 mM and Na2HPO4·7H2O 1 mM, pH 7.2) at a flow rate of 1.5 μL/min, and a stabilization period of 2 h was maintained. Five basal samples were collected at 20 min intervals prior to the treatment of compound Masupirdine/SUVN-502 (5al) (1 or 3 mg/kg p.o.) or vehicle. For studies involving combination, donepezil (1 mg/kg s.c.) and memantine (1 mg/kg s.c.) were administered 30 min after administration of Masupirdine/SUVN-502 (5al) . Dialysates were collected for an additional period of 6 h post-treatment (4 h post-treatment for studies involving combination) of Masupirdine/SUVN-502 (5al) , and dialysates were stored below −50 °C until quantification of acetylcholine by LC-MS/MS. Concentrations of acetylcholine were converted as percent change from mean basal acetylcholine concentrations with 100% defined as the average of five predose values. The percent changes in acetylcholine levels were compared with vehicle. In studies involving combination, percent changes in acetylcholine levels after combination of Masupirdine/SUVN-502 (5al) , donepezil, and memantine were compared with the donepezil and memantine combination using two-way analysis of variance (time and treatment), followed by Bonferroni’s post-test. Statistical significance was considered at a p value less than 0.05. Incorrect probe placement was considered as a criterion to reject the data from animal.

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Pharmacokinetic Study in Rats: [1]
Male Wistar rats (225 ± 25 g) were used as experimental animals. Three animals were housed in each cage. Two days prior to dosing day, male Wistar rats (225 - 250 g) were anesthetized with isoflurane for surgical placement of jugular vein catheter. Animals were fasted overnight before oral dosing (p.o.) and food pellets were allowed 2 h post dosing, whereas during intravenous dosing food and water were provided ad libitum. Three rats each were dosed with test compound orally (10 mg/kg) and intravenously (10 mg/kg). 10 mL/kg for oral and 2 mL/kg for intravenous was used as dosing volume and water as a vehicle for preparation of dose formulation. At each time point blood was collected through jugular vein and immediately replenished with an equivalent volume of normal saline into freely moving rats. Collected blood was transferred into a labeled vial containing 10 µL of heparin as anticoagulant. Blood samples were collected at following time points: pre dose, 0.08 (only i.v.), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post dose (n=3). Blood was centrifuged at 4000 rpm for 10 min. Plasma was prepared and stored at - 70 °C until analysis. The concentrations of the compounds were quantified in plasma by qualified LC-MS/MS method using suitable extraction technique. The compounds were quantified in the calibration range around 2-2000 ng/mL in plasma. Study samples were analyzed using calibration samples in the batch and quality control samples spread across the batch. Pharmacokinetic parameters Cmax, AUC0-t, t1/2 and bioavailability were calculated by non compartmental model using standard non-compartmental model by using WinNonLin 5.0.1 version Software package. We have evaluated the pharmacokinetic profiling of a reported reference 5-HT6 antagonist (SAM-531) in rats under similar experimental conditions to that of lead compounds (Table 2). The reported values are in agreement with the published report.

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Rodent Brain Penetration Study: [1]
Male Wistar rats (225 ± 25 g) were used as experimental animals. Three animals were housed in each cage. Animals were given water and food ad libitum throughout the experiment and maintained on a 12 h light/dark cycle. Male Wistar rats were fasted overnight and compound was administered per orally (p.o.; dosing volume 2.5 mL/kg) at 10 mg/kg (n = 3). Blood was collected by cardiac puncture at pre dose 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24 and 32 h after dosing (n=3/ time point), immediately brain was isolated and carcasses were discarded. The brain was homogenized with 4 volumes of ice cooled water. Plasma and brain homogenates (20%) were stored frozen at -20 °C until analysis. The plasma and brain levels of compound was determined by validated LC-MS/MS method using solid phase extraction technique, quantified in the calibration range of 2-2000 ng/mL in plasma and brain homogenates. Pharmacokinetic evaluation was performed using the validated software WinNonlin version 4.1. Extent of brain-plasma ratio was calculated (Cb/Cp) at Tmax.

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Protocol for Object Recognition Test: [1]
For object recognition test, Male Wistar rats 10-12 weeks old were used. Arena was 50 x 50 x 50 cm. open field was made up of acrylic. On day 1, rats were habituated to individual test arenas for 20 min in the absence of any objects. On day 2 (24 h after the habituation), rats were subjected to familiarization phase (T1). The rats were placed individually in the open field containing two identical objects (a1 and a2) for 3 min. The recognition trial (T2) trial was carried out after 24 h after the T1 trial. Rats were allowed to explore the open field in presence of one familiar object (a3) and one novel object (b) for 3 min. The exploratory behavior towards the objects during the familiarization and recognition phases were recorded. We have evaluated a reported reference 5-HT6 antagonist (SAM-531) in object recognition task model under similar experimental conditions, except the route of administration. SAM-531 showed efficacy at doses of 1 and 3 mg/kg as shown below (Figure 1).

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Pharmacokinetic Study in Dogs: [1]
Masupirdine/SUVN-502 (5al) was administered to fasted Beagle dogs (n = 3/dose/ group) at 10 mg/kg, orally on Day 8 and non-fasted dogs were treated with intravenous route at a dose of at 3.0 mg/kg on Day 1. For oral administration, compound was weighed directly into a gelatin capsule. Water for injection was used as a vehicle for intravenous administration at a dosing volume of 1 mL/kg. On Day 1 and Day 8 of the study, blood samples were obtained for plasma drug concentrations following oral (capsule) and intravenous (bolus) administration. Samples were collected at 15 min, 30 min, 45 min and 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48 and 72 h after the oral dose. Whereas, after the intravenous dose, blood samples were collected at 5 min, 10 min, 20 min, 30 min and 1, 2, 4, 6, 8, 12, 24, 48 and 72 h. On each occasion, approximately 3 ml of blood was drawn from the jugular vein and collected into lithium heparin blood collection tubes. Blood samples were centrifuged at 4000 rpm for 10 min at 4 °C. Plasma was transferred to plastic (polypropylene) tubes and placed on dry ice. The samples were stored at -80 ± 10 °C until analysis.

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Protocol for Fear Conditioning Model: [1]
Experiment was carried out over a period of two days. On day 1, rats were placed in the operant behavior chamber and allowed to acclimatize for 2 min. Rats received a conditioned stimulus (CS) (tone for 10 sec) followed by an unavoidable foot shock (unconditioned stimulus (US): electric shock of 0.5 - 0.7 mA for 3 sec). Following a 1 min interval between each administration, tone and shock were repeated to deliver a total of three CS-US pairings. Rats were administered Masupirdine/SUVN-502 (5al) (1 h). Scopolamine (0.3 mg/kg, s.c.) was administered 120 S22 min after training. On day 2, rats were placed in the operant behavior chamber and total freezing time scored for a period of 5 min.

The pharmacokinetic study of SUVN-502 (5al)was carried out in nonrodent species, Beagle dogs (at 10 mg/kg p.o. and 3 mg/kg i.v. dose). It was well absorbed into systemic circulation with high oral exposures (AUC0-t 1356 ± 1067 ng.h/mL), favorable terminal half-life 3.5 h, and high intravenous clearance (46.4 ± 10.9 mL/min/kg). The volume of distribution was 7.5 L/kg, indicating that 5al was widely distributed in tissues. Absolute oral bioavailability was found to be 35%. The CYP3A4 inhibition for compound 5al was assessed with testosterone, another probe substrate. It inhibited the testosterone hydroxylation with an IC50 value of 2.64 μM. Compound 5al was also evaluated for the inhibitory potential against other CYP isoforms, CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, and 2E1, in pooled human liver microsomes using specific CYP isoform marker activities. It had moderately inhibited mephenytoin hydroxylation (2C19) with IC50 values of 5.7 μM and did not inhibit the other CYP450 enzymes significantly (IC50 > 10 μM). Also the CYP induction potential of 5al was evaluated in human plateable hepatocytes using both mRNA expression and enzyme activities as end points. The results indicated that 5al did not induce CYP1A2, CYP2B6, and CYP3A4 at tested concentrations of 0.01, 0.1, and 1.0 μM. Compound 5al was evaluated in additional animal models of cognition and neurochemistry. It reversed the scopolamine induced emotional memory deficits in a contextual fear conditioning task at 1, 3, and 10 mg/kg p.o. dose (Figure 3).[1]

[1]. Discovery and Development of 1-[(2-Bromophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole Dimesylate Monohydrate (SUVN-502): A Novel, Potent, Selective and Orally Active Serotonin 6 (5-HT6) Receptor Antagonist for Potential Treatment of Alzheimer's Disease. J Med Chem.2017 Mar 9;60(5):1843-1859.

Optimization of a novel series of 3-(piperazinylmethyl) indole derivatives as 5-hydroxytryptamine-6 receptor (5-HT6R) antagonists resulted in identification of 1-[(2-bromophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole dimesylate monohydrate (Masupirdine/SUVN-502 (5al)) as a clinical candidate for potential treatment of cognitive disorders. It has high affinity at human 5-HT6R (Ki = 2.04 nM) and selectivity over 100 target sites which include receptors, enzymes, peptides, growth factors, ion channels, steroids, immunological factors, second messengers, and prostaglandins. It has high selectivity over 5-HT2A receptor. It is orally bioavailable and brain penetrant with robust preclinical efficacy. The combination of 5al, donepezil, and memantine (triple combination) produces synergistic effects in extracellular levels of acetylcholine in the ventral hippocampus. Preclinical efficacy in triple combination and high selectivity over 5-HT2A receptors are the differentiating features which culminated in selection of 5al for further development. The Phase-1 evaluation of safety and pharmacokinetics has been completed, allowing for the initiation of a Phase-2 proof of concept study. [1] In summary, the focused SAR around the 3-(piperazinylmethyl) indole scaffold led to the finding that the central indole core is optimal for affinity at 5-HT6R. Replacing it with 4-azaindole, 7-azaindole, or indazole resulted in moderately potent compounds. Smaller halo and alkoxy substitutions were tolerated, preferably at the C5 position of indole. 3-(Piperazinylmethyl) indole is optimal for affinity. Further chain elongation, i.e. 3-(piperazinylethyl)indole, is not tolerated. N-Aryl sulfonamide is critical for affinity; replacing it with benzyl and benzoyl gave less potent compounds. Halo and lower alkoxy are preferred substitutions on the N-aryl sulfonamide ring. The in vitro affinity, selectivity, physicochemical properties, in vivo efficacy, and safety evaluations resulted in the identification of Masupirdine/SUVN-502 (5al), as a developmental candidate. 5al is a crystalline dimesylate monohydrate salt with very high water solubility and permeability. It weakly inhibited recombinant cytochrome P450 3A4 while it does not inhibit CYP 2D6. Compound 5al is an orally bioavailable and brain penetrant 5-HT6R antagonist with robust efficacy in cognition models such as ORT and the contextual fear conditioning model. The triple combination of 5al, donepezil, and memantine produced additive augmentation in extracellular levels of acetylcholine in the ventral hippocampus without any cholinergic side effects, thus providing a neurochemical basis for the pro-cognitive activity observed in various behavioral models. The preclinical efficacy in triple combination coupled with selectivity over the 5-HT2A receptor is the differentiating feature of this compound. Compound 5al is nonmutagenic and nonclastogenic. The clinical portions of the single and multiple ascending dose studies assessing safety and pharmacokinetics have been completed, allowing for the initiation of the Phase-2 proof of concept study. The complete biological characterization of 5al covering the detailed rationale for the use of a triple combination in a Phase-2 POC trial will be reported in a forthcoming biology publication.[1]

C22H28BRN3O6S2

574.508222579956

669.048

C, 41.19; H, 4.81; Br, 11.92; N, 6.27; O, 21.47; S, 14.34

1791396-46-7

Masupirdine free base;701205-60-9

118142730

Pale purple to purple solid powder

189

2

11

5

39

743

0

CN1CCN(CC1)CC2=CN(C3=C2C=C(C=C3)OC)S(=O)(=O)C4=CC=CC=C4Br.CS(=O)(=O)O.CS(=O)(=O)O

LOZVXBFXRPRECW-UHFFFAOYSA-N

InChI=1S/C21H24BrN3O3S.2CH4O3S/c1-23-9-11-24(12-10-23)14-16-15-25(20-8-7-17(28-2)13-18(16)20)29(26,27)21-6-4-3-5-19(21)22;2*1-5(2,3)4/h3-8,13,15H,9-12,14H2,1-2H3;2*1H3,(H,2,3,4)

1-(2-bromophenyl)sulfonyl-5-methoxy-3-[(4-methylpiperazin-1-yl)methyl]indole;methanesulfonic acid

SUVN-502 dimesylate; 1791396-46-7; Masupirdine mesylate; Masupirdine (mesylate); Masupirdine dimesylate; BFE1VOB95D; 1-(2-bromophenyl)sulfonyl-5-methoxy-3-[(4-methylpiperazin-1-yl)methyl]indole;methanesulfonic acid; SUVN502 mesylate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~37.28 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。