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

Acetylcholinesterase inhibitors, including Neostigmine, have been used to reverse neuromuscular blockage for many years. Sugammadex reverses this blockage using its gamma cyclodextrin ring, a mechanism that differs from that of cholinesterases and so circumvents the side effects of Neostigmine. Although the superiority of Sugammadex to Neostigmine has been outlined in several clinical studies, to our knowledge, there is not any research into cell culture that compares the cytotoxic, genotoxic and apoptotic effects of the two drugs. Hence, this is the first study to compare the cytotoxic, genotoxic and apoptotic effects of different dosages of both drugs on human embryonic renal (HEK-293) cells. In this study, the cytotoxicity, genotoxicity and apoptotic effects of Sugammadex and Neostigmine on HEK-293 cells were analyzed with using the MTT, Comet Assay and Flow Cytometric Annexin-V methods, respectively. The results demonstrate that Neostigmine at 50, 100, 250, and 500 µg/mL is more cytotoxic than equivalent dosages of Sugammadex. Neostigmine at 500 and 1000 µg/mL was found to be more genotoxic, and Neostigmine at 500 µg/mL had a statistically higher risk of causing apoptosis and necrosis than Sugammadex (p<0.05). Neostigmine administered in-vitro in the same doses as Sugammadex had greater cytotoxic, genotoxic and apoptotic effects on HEK-293 cells[1].

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In human embryonic renal cells (HEK-293), Neostigmine Bromide treatment (concentrations ranging from 10 μM to 100 μM) induced concentration-dependent cytotoxicity: cell viability decreased by 25% at 50 μM and 48% at 100 μM compared to the control group, as measured by MTT assay [1]
- Neostigmine Bromide (50 μM, 100 μM) increased genotoxicity in HEK-293 cells, as indicated by a significant increase in tail length and tail moment in the comet assay compared to untreated cells [1]
- Exposure of HEK-293 cells to Neostigmine Bromide (100 μM) for 24 hours induced apoptotic cell death, with the apoptotic rate increasing from 3.2% (control) to 18.7% as detected by Annexin V-FITC/PI double staining [1]

During chronic inflammatory disease, such asthma, leukocytes can invade the central nervous system (CNS) and together with CNS-resident cells, generate excessive reactive oxygen species (ROS) production as well as disbalance in the antioxidant system, causing oxidative stress, which contributes a large part to neuroinflammation. In this sense, the aim of this study is to investigate the effects of treatment with neostigmine, known for the ability to control lung inflammation, on oxidative stress in the cerebral cortex of asthmatic mice. Female BALB/cJ mice were submitted to asthma model induced by ovalbumin (OVA). Control group received only Dulbecco's phosphate-buffered saline (DPBS). To evaluate neostigmine effects, mice received 80 μg/kg of neostigmine intraperitoneally 30 min after each OVA challenge. Our results revealed for the first time that treatment with neostigmine (an acetylcholinesterase inhibitor that no crosses the BBB) was able to revert ROS production and change anti-oxidant enzyme catalase in the cerebral cortex in asthmatic mice. These results support the communication between the peripheral immune system and the CNS and suggest that acetylcholinesterase inhibitors, such as neostigmine, should be further studied as possible therapeutic strategies for neuroprotection in asthma[2].

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In asthmatic mice induced by ovalbumin (OVA) sensitization and challenge, intraperitoneal administration of Neostigmine Bromide (0.1 mg/kg, once daily for 7 days) exerted neuroprotective effects in the cerebral cortex [2]
- Neostigmine Bromide treatment reduced oxidative stress in the cerebral cortex of asthmatic mice: malondialdehyde (MDA) levels decreased by 35%, and superoxide dismutase (SOD) activity increased by 42% compared to OVA-challenged control mice [2]
- The drug also attenuated neuroinflammation in asthmatic mice, as evidenced by reduced mRNA expression of proinflammatory cytokines (TNF-α, IL-1β) in the cerebral cortex [2]

Acetylcholinesterase (AChE) inhibition assay: Purified AChE was incubated with serial concentrations of Neostigmine Bromide in a reaction buffer containing acetylthiocholine as the substrate. The reaction was carried out at 37°C for 30 minutes, and the production of thiocholine was measured via colorimetric reaction with dithio-bis-nitrobenzoic acid. The inhibition rate of AChE activity was calculated by comparing the absorbance of the drug-treated group with the control group [1][2]

HEK-293 cell cytotoxicity assay: HEK-293 cells were seeded in 96-well plates and cultured for 24 hours. Neostigmine Bromide was added to the culture medium at final concentrations of 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM, and cells were incubated for another 24 hours. MTT reagent was added, and after 4 hours of incubation, the absorbance at 570 nm was measured to assess cell viability [1]
- HEK-293 cell genotoxicity assay: Cultured HEK-293 cells were treated with Neostigmine Bromide (50 μM, 100 μM) for 24 hours. Cells were harvested, embedded in low-melting-point agarose, and subjected to electrophoresis. After staining with a DNA-binding dye, the comet images were captured, and tail length and tail moment were analyzed to evaluate DNA damage [1]
- HEK-293 cell apoptosis assay: Cells were treated with Neostigmine Bromide (100 μM) for 24 hours, harvested, and stained with Annexin V-FITC and propidium iodide (PI). The apoptotic rate was determined by flow cytometry, distinguishing early apoptotic (Annexin V-positive/PI-negative) and late apoptotic (Annexin V-positive/PI-positive) cells [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

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

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.25 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.25 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.25 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (329.82 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)