Acetylcholinesterase/AChE
Acetylcholinesterase (AChE) [1][2]

乙酰胆碱酯酶抑制剂，包括新斯的明，已被用于拮抗神经肌肉阻滞多年。Sugammadex使用其γ-环糊精环逆转这种阻断，这种机制不同于胆碱酯酶，因此可以避免新斯的明的副作用。尽管在几项临床研究中已经概述了Sugammadex相对于新斯的明的优越性，但据我们所知，还没有任何关于细胞培养的研究来比较这两种药物的细胞毒性、遗传毒性和凋亡作用。因此，这是第一项比较不同剂量两种药物对人胚胎肾（HEK-293）细胞的细胞毒性、遗传毒性和凋亡作用的研究。在这项研究中，分别使用MTT、彗星试验和流式细胞术膜联蛋白-V方法分析了Sugammadex和新斯的明对HEK-293细胞的细胞毒性、遗传毒性和凋亡作用。结果表明，50、100、250和500µg/mL的新斯的明比同等剂量的Sugammadex具有更强的细胞毒性。发现500和1000µg/mL的新斯的明具有更高的遗传毒性，500µg/mL的新斯的明引起细胞凋亡和坏死的风险在统计学上高于Sugammadex（p＜0.05）。与Sugammadex相同剂量的新斯的明体外给药对HEK-293细胞具有更大的细胞毒性、遗传毒性和凋亡作用[1]。

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在人胚胎肾细胞（HEK-293）中，溴新斯的明（浓度范围10 μM至100 μM）诱导浓度依赖性细胞毒性：MTT法检测显示，50 μM浓度时细胞活力较对照组下降25%，100 μM浓度时下降48% [1]
- 溴新斯的明（50 μM、100 μM）可增加HEK-293细胞的遗传毒性，彗星实验显示，与未处理细胞相比，细胞尾长和尾矩显著增加 [1]
- HEK-293细胞经溴新斯的明（100 μM）处理24小时后，凋亡细胞比例升高，Annexin V-FITC/PI双染色检测显示凋亡率从对照组的3.2%增至18.7% [1]

在慢性炎症性疾病（如哮喘）期间，白细胞可以侵入中枢神经系统（CNS），并与CNS驻留细胞一起产生过量的活性氧（ROS）产生以及抗氧化系统失衡，导致氧化应激，这在很大程度上导致了神经炎症。从这个意义上讲，本研究的目的是研究以控制肺部炎症能力而闻名的新斯的明治疗对哮喘小鼠大脑皮层氧化应激的影响。将雌性BALB/cJ小鼠置于卵清蛋白（OVA）诱导的哮喘模型中。对照组仅接受杜尔贝科磷酸缓冲盐水（DPBS）。为了评估新斯的明的效果，小鼠在每次OVA攻击后30分钟腹腔注射80μg/kg的新斯的明。我们的研究结果首次表明，新斯的明（一种不穿过血脑屏障的乙酰胆碱酯酶抑制剂）治疗能够恢复哮喘小鼠大脑皮层中ROS的产生并改变抗氧化酶过氧化氢酶。这些结果支持外周免疫系统和中枢神经系统之间的沟通，并表明乙酰胆碱酯酶抑制剂，如新斯的明，应作为哮喘神经保护的可能治疗策略进行进一步研究[2]。

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在卵清蛋白（OVA）致敏和激发诱导的哮喘小鼠模型中，腹腔注射溴新斯的明（0.1 mg/kg，每日一次，连续7天）对大脑皮层具有神经保护作用 [2]
- 溴新斯的明处理可降低哮喘小鼠大脑皮层的氧化应激水平：与OVA激发对照组相比，丙二醛（MDA）水平下降35%，超氧化物歧化酶（SOD）活性升高42% [2]
- 该药物还可减轻哮喘小鼠的神经炎症，表现为大脑皮层中促炎细胞因子（TNF-α、IL-1β）的mRNA表达水平降低 [2]

乙酰胆碱酯酶（AChE）抑制实验：将纯化的AChE与系列浓度的溴新斯的明在含乙酰硫代胆碱（底物）的反应缓冲液中共同孵育。37°C孵育30分钟后，通过与二硫代双硝基苯甲酸的比色反应检测硫代胆碱的生成量，比较药物处理组与对照组的吸光度，计算AChE活性抑制率 [1][2]

HEK-293细胞毒性实验：将HEK-293细胞接种于96孔板，培养24小时后，向培养基中加入溴新斯的明，终浓度分别为10 μM、25 μM、50 μM、75 μM和100 μM，继续孵育24小时。加入MTT试剂孵育4小时后，检测570 nm处吸光度以评估细胞活力 [1]
- HEK-293细胞遗传毒性实验：培养的HEK-293细胞经溴新斯的明（50 μM、100 μM）处理24小时后收集，包埋于低熔点琼脂糖中进行电泳。经DNA结合染料染色后，捕获彗星图像并分析尾长和尾矩，以评估DNA损伤程度 [1]
- HEK-293细胞凋亡实验：细胞经溴新斯的明（100 μM）处理24小时后收集，用Annexin V-FITC和碘化丙啶（PI）染色，通过流式细胞术区分早期凋亡细胞（Annexin V阳性/PI阴性）和晚期凋亡细胞（Annexin V阳性/PI阳性），计算凋亡率 [1]

Sensitization, airway challenge and neostigmine treatment[2] The animals were sensitized by subcutaneous injections of 20 μg ovalbumin (OVA), diluted (200 μL) in Dulbecco’s phosphate-buffered saline (DPBS), on days 0 and 7, followed by three intranasal challenges with 100 μg of OVA, diluted in DPBS (50 μL), on days 14, 15, and 16 of the protocol. The control group received only DPBS in the sensitization and intranasal challenges. To evaluate neostigmine effects on the oxidative stress in the cerebral cortex, the mice received 80 μg/kg of neostigmine treatment intraperitoneally (Hofer et al. 2008) once a day during three consecutive days (14, 15, and 16) 30 min after of OVA challenge. On day 17 of the protocol, animals were anesthetized by intraperitoneal injection solution of ketamine (0.4 mg/g) and xylazine (0.2 mg/g) followed euthanasia by heart puncture exsanguination. Bronchoalveolar lavage (BAL), lung tissue and cerebral cortex for analyzes were collected. The study protocol is illustrated in Fig. 1.

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Asthmatic mouse neuroprotection model: Female mice were sensitized by intraperitoneal injection of OVA emulsified in adjuvant on day 0 and day 14, followed by OVA aerosol challenge once daily from day 21 to day 27 to induce asthma. Neostigmine Bromide was dissolved in normal saline and administered via intraperitoneal injection at a dose of 0.1 mg/kg once daily from day 21 to day 27. Control groups included non-asthmatic mice and OVA-challenged mice treated with normal saline. Mice were sacrificed on day 28, and cerebral cortex tissues were collected for oxidative stress and inflammatory parameter analysis [2]

Absorption, Distribution and Excretion
Neostigmine bromide is poorly absorbed in the gastrointestinal tract after oral administration. Neostigmine…is poorly absorbed orally, therefore requiring much larger doses than the parenteral route. …The effective parenteral dose of neostigmine in humans is 0.5 to 2.0 mg, with an equivalent oral dose of 30 mg or more. High oral doses may lead to toxicity if intestinal absorption is enhanced for any reason. …Neostigmine excretion is slowed in patients with severe renal disease, therefore this anticholinesterase drug is an acceptable option for patients with renal failure. We determined the pharmacokinetics of neostigmine in patients with normal renal function and compared them with those in patients who underwent kidney transplantation or bilateral nephrectomy. 10 to 15 minutes before the end of surgery and anesthesia, the d-tubocurarine infusion was stopped, and neostigmine 0.07 mg/kg and atropine 0.03 mg/kg were administered intravenously over 2 minutes. In patients without kidneys, the elimination half-life was prolonged. Total serum clearance decreased from 16.7 ml/kg/min in patients with normal renal function to 7.8 ml/kg/min in patients without kidneys. The pharmacokinetics of neostigmine were not different after kidney transplantation compared to patients with normal renal function. Renal excretion accounts for 50% of neostigmine clearance. Metabolism/Metabolites Neostigmine is hydrolyzed by cholinesterases and also metabolized by microsomal enzymes in the liver. Neostigmine is destroyed by plasma esterases, and quaternary ammonium alcohols and the parent compound are excreted in the urine. Neostigmine is converted to 3-hydroxyphenyltrimethylammonium in rats. ROBERTS, JB et al.; Biochemical Pharmacology 17: 9 (1968). /Excerpt from Table/ Biological Half-Life The half-life ranges from 42 to 60 minutes, with a mean half-life of 52 minutes. Pharmacokinetics of neostigmine were evaluated in humans after intravenous and oral administration. Following intravenous administration, the mean plasma half-life of neostigmine is 0.89 hours. After oral administration, peak plasma concentrations occur 1–2 hours post-administration, but bioavailability is only 1–2% of the administered dose. In patients with myasthenia gravis, the attenuation of repetitive nerve stimulation-induced muscle electrical responses correlated well with neostigmine plasma concentrations.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Limited data suggest that neostigmine may be acceptable for treating myasthenia gravis during lactation, but pyridostigmine may be preferred. Newborns should be closely monitored, as abdominal cramps have been reported after each feeding. Due to the short half-life of neostigmine, a single dose reversing postoperative neuromuscular blockade is unlikely to have any adverse effects other than transient effects on breastfed infants.
◉ Effects on Breastfed Infants
Infants born to six mothers who received neostigmine for myasthenia gravis were reported to be successfully breastfed. One newborn appeared to experience abdominal cramps after each feeding, possibly caused by neostigmine, although the drug was not detected in the mother's breast milk.
◉ Effects on Lactation and Breast Milk
As of the revision date, no published information was found regarding breastfeeding mothers. In animal experiments, cholinergic drugs can increase the release of oxytocin and have different effects on serum prolactin levels. Prolactin levels in established lactating mothers may not affect their lactation capacity. Protein binding: Protein binding to human serum albumin is between 15% and 25%. In vitro cytotoxicity: Neostigmine bromide induced concentration-dependent cytotoxicity, genotoxicity and apoptosis in HEK-293 cells, with significant effects observed at concentrations ≥50 μM [1]

[1].Comparison of the cytotoxic, genotoxic and apoptotic effects of Sugammadex and Neostigmine on human embryonic renal cell (HEK-293). Cell Mol Biol (Noisy-le-grand). 2018 Oct 30;64(13):74-78.
[2].Neostigmine treatment induces neuroprotection against oxidative stress in cerebral cortex of asthmatic mice. Metab Brain Dis. 2020 Jun;35(5):765-774.
[3]. Clin Colon Rectal Surg.2005 May;18(2):96-101.

Neostigmine is a quaternary ammonium ion compound with an aniline ion as its core structure. Three methyl substituents are attached to the aniline nitrogen atom, and a 3-[(dimethylcarbamoyl)oxy] substituent is attached at the 3-position. It is a parasympathomimetic drug and acts as a reversible acetylcholinesterase inhibitor. It can act as an EC 3.1.1.7 (acetylcholinesterase) inhibitor and as an antidote for curare poisoning. It is a cholinesterase inhibitor used to treat myasthenia gravis and to reverse the effects of muscle relaxants such as galamine and tubocurarine. Unlike physostigmine, neostigmine cannot cross the blood-brain barrier. Neostigmine is a cholinesterase inhibitor. The mechanism of action of neostigmine is as a cholinesterase inhibitor. Neostigmine is a parasympathomimetic drug and acts as a reversible acetylcholinesterase inhibitor. It is a cholinesterase inhibitor used to treat myasthenia gravis and to reverse the effects of muscle relaxants such as galamine and tubocurarine. Unlike physostigmine, neostigmine cannot cross the blood-brain barrier. See also: Neostigmine methyl sulfate (in salt form). Drug Indications Neostigmine treats the symptoms of myasthenia gravis by improving muscle tone. Mechanism of Action Neostigmine is a parasympathomimetic drug, specifically a reversible cholinesterase inhibitor. This drug inhibits acetylcholinesterase, which is responsible for the degradation of acetylcholine. Therefore, when acetylcholinesterase is inhibited, the level of acetylcholine increases. Neostigmine indirectly stimulates nicotinic and muscarinic receptors involved in muscle contraction by interfering with the breakdown of acetylcholine. It cannot cross the blood-brain barrier. …The pharmacological action of anticholinesterase drugs is primarily attributed to their ability to prevent the hydrolysis of acetylcholine by acetylcholinesterase at cholinergic transmission sites. Therefore, neurotransmitters accumulate, and the activity of acetylcholinesterase (ACH), released by cholinergic impulses or leaked from nerve endings, is enhanced. Neostigmine increased the amplitude of micro-endplate potentials and endplate potentials in isolated frog sciatic nerve-sartorius muscle complexes, but did not affect quantum content. This suggests that cholinesterase inhibition is the sole mechanism of action. Long-term (24–96 hours) treatment of mouse-derived myoblast cell lines (G8) with neostigmine significantly reduced the binding of α-bu-x venom (α-BuTx) to these cells. Protein synthesis in these cultures was significantly reduced, and cell morphology degenerated. Myotubes maintained a mildly hyperpolarized resting membrane potential and were able to produce overshoot action potential responses to iontophoretic acetylcholine (ACh). The in vivo chronic neostigmine treatment-related neuromuscular junction degenerative changes are likely due to the direct action of anticholinesterase on the muscle, rather than changes in interstitial acetylcholine levels or presynaptic effects of anticholinesterase. This study used an intraluminal probe equipped with two pairs of electrode-strain gauges spaced 4 cm apart to investigate the effects of neutral interviews, stress interviews, food intake (478.7 calories), and neostigmine (0.5 mg, intramuscular injection) on the contractile electrical complex, sustained electrical response activity, and related contractions in 17 normal subjects. Neostigmine injection resulted in increases in the contractile electrical complex and sustained electrical response activity indices at 5–10 minutes and 25–30 minutes post-injection, respectively. Both food intake and neostigmine increased the percentage of contractile electrical complex waves propagating throughout all recording periods.

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Neostigmine bromide (eustigmine; neoserine) is a reversible acetylcholinesterase inhibitor[1][2]
- Its mechanism of action is to inhibit acetylcholinesterase, thereby increasing the concentration of acetylcholine at cholinergic synapses and enhancing cholinergic neurotransmission[1][2]
- Clinically, it is used to reverse the effects of non-depolarizing neuromuscular blocking agents after anesthesia and to treat myasthenia gravis[1][2]
- In asthmatic mice, its neuroprotective effect may be achieved by reducing oxidative stress and neuroinflammation in the cerebral cortex[2]

C12H19N2O2.BR

303.2

302.062

C, 47.54; H, 6.32; Br, 26.35; N, 9.24; O, 10.55

114-80-7

Neostigmine methyl sulfate;51-60-5

4456

White to off-white solid powder

175-177 °C(lit.)

1.5

29.54

0

2

3

16

246

0

[Br-].O(C(N(C([H])([H])[H])C([H])([H])[H])=O)C1=C([H])C([H])=C([H])C(=C1[H])[N+](C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H]

LULNWZDBKTWDGK-UHFFFAOYSA-M

InChI=1S/C12H19N2O2.BrH/c1-13(2)12(15)16-11-8-6-7-10(9-11)14(3,4)5;/h6-9H,1-5H3;1H/q+1;/p-1

3-((dimethylcarbamoyl)oxy)-N,N,N-trimethylbenzenaminium bromide

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（例如氮气保护），避免吸湿/受潮和光照。

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

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.25 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (8.25 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (8.25 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 100 mg/mL (329.82 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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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。