NET and SERT[1]

开发了一种液相色谱-电喷雾电离质谱（LC-ESI-MS）方法，用于同时测定六种合成掺杂物，即芬氟拉明、酚酞、N-二去甲基西布曲明、N-单去甲基西布曲明和奥利司他。该方法被应用于分析草药减肥膳食补充剂。使用溶剂a：乙腈和溶剂B：20mM甲酸铵水溶液的梯度洗脱，在C（8）反相柱上实现了分析物的色谱分离。西地那非被用作定量的内标。质谱检测器在正电喷雾电离模式下运行。对m/z 232、319、252、266、280、496和475分别进行了芬氟拉明、酚酞、N-二甲基西布曲明、N-单甲基西布曲明、西布曲明、奥利司他和西地那非的选择性离子监测（SIM）。该方法的准确度、精密度、线性和选择性得到了验证。六种合成掺杂物的检测限范围为0.0018至0.73微克/克（-1）。所提出的方法用于对22种膳食补充剂进行小型调查，其中11种样品掺入了酚酞、N-单脱甲基西布曲明和西布曲明，掺入量为0.212至96.2 mg g（-1）[3]。

西布曲明是一种中枢作用的单胺再摄取抑制剂，在肥胖管理中被用作食欲抑制剂。其作用主要归因于血清素和去甲肾上腺素转运体（分别为SERT和NET）对其强效代谢产物去甲基西布曲明（M1）和去甲基西布曲明的抑制。然而，关于临床疗效和少数患者观察到的剂量无关无反应的机制，人体体内数据很少。12名健康男性患者（平均年龄41岁）完成了一项双盲、安慰剂对照、受试者内交叉研究，研究稳定状态下每天服用15mg西布曲明对大脑SERT的占用情况。测量了占用率与（i）西布曲明、M1和M2的血浆浓度之间的相关性；（ii）食欲抑制。（11） 在HRRT相机上进行C-DASB PET扫描。结合电位（BP（ND））通过Logan参考组织（小脑）方法计算。SERT占用率适中（平均30+/-10%），各脑区相似，但受试者之间差异很大（15-46%）。占用率与M2浓度呈正相关（p=0.09），但与西布曲明或M1无关。在<25%的入住率下没有明显的食欲抑制，最大的抑制与最高入住率（25-46%）有关。然而，一些占用率较高（36-39%）的受试者没有食欲抑制。临床剂量西布曲明对SERT的占用程度适中，可能主要由M2介导。5-羟色胺再摄取抑制可能是必要的，但对于西布曲明在人体中的疗效来说还不够，支持临床前数据表明，低剂量效应需要SERT和NET的共同抑制[1]。

Metabolism / Metabolites
Desmethylsibutramine is a known human metabolite of (S)-Sibutramine.

[1]. Brain serotonin transporter occupancy by oral sibutramine dosed to steady state: a PET study using (11)C-DASB in healthy humans. Neuropsychopharmacology. 2010 Feb;35(3):741-51.

[2]. The contribution of metabolites to the rapid and potent down-regulation of rat cortical beta-adrenoceptors by the putative antidepressant sibutramine hydrochloride. Neuropharmacology. 1989 Feb;28(2):129-34.

[3]. Analysis of six synthetic adulterants in herbal weight-reducing dietary supplements by LC electrospray ionization-MS. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2008 Jul;25(7):822-30.

[4]. Quantification of sibutramine and its two metabolites in human plasma by LC-ESI-MS/MS and its application in a bioequivalence study. J Pharm Anal. 2012 Aug;2(4):249-257.

Sibutramine HCl is an inhibitor of the reuptake of monoamines with a pharmacological profile in rodents indicative of antidepressant activity. The secondary (BTS 54 354) and primary (BTS 54 505) amine metabolites of the tertiary amine sibutramine HCl exhibit similar in vivo pharmacological activity to the parent compound. Thus, each compound displays potent activity in acute behavioural models predictive of antidepressant effects and a comparable ability to inhibit the uptake of monoamines in vivo. In addition, BTS 54 354 and BTS 54 505 induce an equally rapid and potent down-regulation of cortical beta-adrenoceptors in the rat as sibutramine HCl. The secondary and primary amines are, however, considerably more active than sibutramine HCl as inhibitors of the uptake of noradrenaline, dopamine and 5-hydroxytryptamine in vitro. The potent inhibition of the reuptake of noradrenaline by the secondary and primary amine metabolites probably contributes to the rapid and potent down-regulation of beta-adrenoceptors in the rat, induced by the putative antidepressant sibutramine HCl.[2]

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Obesity can be considered as a chronic illness of epidemic proportion and its incidents have increased exponentially in recent years. The use of anti-obesity drugs such as sibutramine is somewhat helpful. There is a need to quantify such drugs in biological samples, which is generally quite difficult. In this report, we developed and validated a simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of sibutramine (SB) and its two metabolites N-des methyl sibutramine (DSB) and N-di desmethyl sibutramine (DDSB) in human plasma. Zorbax SB-C18 (4.6 mm×75 mm, 3.5 μm, 80 Å) analytical column and 5 mM ammonium formate:acetonitrile (10:90, v/v) mobile phase were used for chromatographic separation of SB, DSB and DDSB. Multiple reaction monitoring (MRM) in the positive mode was used to detect SB, DSB and DDSB at m/z 280.3/124.9, 266.3/125.3 and 252.2/124.9, respectively. Liquid-liquid extraction was used for the extraction of analytes and internal standard from human plasma. This method was validated over a linear concentration range of 10.0-10,000.0 pg/mL for SB, DSB and DDSB with correlation coefficients (r) of ≥0.9997. The drug and the two metabolites were stable in plasma samples. The validated method was successfully applied in a bioequivalence and pharmacokinetic study with human volunteers under fasting condition.[4]

C16H24CLN

265.82

265.16

168835-59-4

Desmethyl Sibutramine hydrochloride;84467-94-7

10199199

White to off-white solid powder

4.786

12.03

1

1

5

18

252

0

PLXKZKLXYHLWHR-UHFFFAOYSA-N

InChI=1S/C16H24ClN/c1-12(2)11-15(18-3)16(9-4-10-16)13-5-7-14(17)8-6-13/h5-8,12,15,18H,4,9-11H2,1-3H3

1-[1-(4-chlorophenyl)cyclobutyl]-N,3-dimethylbutan-1-amine

Desmethylsibutramine; Desmethyl Sibutramine; 168835-59-4; N-Desmethylsibutramine; 1-[1-(4-chlorophenyl)cyclobutyl]-N,3-dimethylbutan-1-amine; 889I657R9P; {1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutyl}(methyl)amine; Desmethyl Sibutramine (hydrochloride);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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。