Acetylcholinesterase/AChE
Acetylcholinesterase (AChE)[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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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].
Neostigmine treatment (80 μg/kg, intraperitoneal) for three consecutive days in asthmatic mice (OVA-induced) significantly decreased total cell count in bronchoalveolar lavage (BAL) (P<0.001 vs OVA group) and reduced absolute counts of macrophages (P<0.05), lymphocytes (P<0.01), neutrophils (P<0.001), and eosinophils (P<0.001) in BAL compared to asthma group.[2]
Neostigmine treatment reduced peribronchial (P<0.05) and perivascular (P<0.001) leukocyte infiltrate in lung tissue of asthmatic mice as quantified by histology.[2]
Neostigmine administration reverted ROS production in the cerebral cortex of asthmatic mice as measured by DCF fluorescence (P<0.01 vs OVA group).[2]
Neostigmine treatment significantly decreased catalase (CAT) activity in the cerebral cortex of asthmatic mice (P<0.001 vs OVA group) and increased the SOD/CAT ratio (P<0.05 vs OVA group), indicating improved antioxidant system balance. SOD and GPx activities were not altered between groups.[2]
Neostigmine did not alter Na+,K+-ATPase activity in the cerebral cortex of asthmatic mice (no significant difference vs OVA group).[2]
Neostigmine did not reduce AChE activity in the cerebral cortex of asthmatic mice (no significant difference vs OVA group), possibly due to the time of tissue collection (24 hours after last administration) and the drug's short half-life.[2]

Acetylcholinesterase (AChE) activity assay: The cerebral cortex was homogenized in 0.1 mM potassium phosphate buffer (pH 7.5) and centrifuged at 1000g for 10 minutes. The supernatant was used for enzymatic analysis. Hydrolysis rates were measured at an acetylcholine concentration of 0.8 mM in a 300 μL assay solution containing 30 mM phosphate buffer (pH 7.5) and 1.0 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) at 25°C. Hydrolysis was monitored by the formation of the thiolate dianion of DTNB at 412 nm for 2-3 minutes at 30-second intervals.[2]
Catalase (CAT) activity assay: CAT activity was measured based on the disappearance of H2O2 at 240 nm. The cerebral cortex supernatant was incubated in 20 mM H2O2, 0.1% Triton X-100, 10 mM potassium phosphate buffer pH 7.0, and 0.1-0.3 mg protein/ml. One CAT unit is defined as 1 μmol of hydrogen peroxide consumed per minute.[2]
Superoxide dismutase (SOD) activity assay: SOD activity is based on the capacity of pyrogallol to autoxidize, a process highly dependent on superoxide. The cerebral cortex supernatant was mixed in a solution containing 1 mM EDTA, 50 mM Tris, 80 U/ml catalase, and 0.8 mM pyrogallol. Activity was indirectly measured at 420 nm.[2]
Glutathione peroxidase (GPx) activity assay: GPx activity was measured using tert-butyl-hydroperoxide as substrate. The cerebral cortex supernatant was incubated in medium containing 2 mM glutathione, 0.15 U/mL glutathione reductase, 0.4 mM azide, 0.5 mM tert-butyl-hydroperoxide, and 0.1 mM NADPH. NADPH disappearance was monitored at 340 nm. One GPx unit is defined as 1 μmol of NADPH consumed per minute.[2]
Reactive species production (ROS) assay: ROS production was measured based on the oxidation of 2',7'-dichlorofluorescein (H2DCF). The sample was incubated in medium containing 100 μM of 2',7'-dichlorofluorescein diacetate (H2DCF-DA). The reaction produces the fluorescent compound dichlorofluorescein (DCF), measured at λem = 488 nm and λex = 525 nm. Results were represented as nmol DCF/mg protein.[2]
Na+,K+-ATPase activity assay: The cerebral cortex was homogenized in 0.32 mM sucrose solution containing 5 mM HEPES and 1 mM EDTA (pH 7.4). Homogenates were centrifuged at 3000g for 10 minutes at 4°C. The reaction mixture contained 5.0 mM MgCl2, 80.0 mM NaCl, 20.0 mM KCl, and 40.0 mM Tris-HCl (pH 7.4) in a final volume of 170 μL. The reaction was initiated by addition of ATP. Controls were carried out with 1.0 mM ouabain. Released inorganic phosphate (Pi) was measured. Specific activity was expressed as nmol Pi released per minute per mg of protein.[2]

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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Animal model: Female BALB/cJ mice (6-8 weeks old) were sensitized by subcutaneous injections of 20 μg ovalbumin (OVA) diluted in 200 μL DPBS on days 0 and 7, followed by three intranasal challenges with 100 μg OVA diluted in 50 μL DPBS on days 14, 15, and 16. Control group received only DPBS. To evaluate neostigmine effects, mice received neostigmine at 80 μg/kg intraperitoneally once daily for three consecutive days (days 14, 15, and 16) 30 minutes after each OVA challenge. On day 17, animals were anesthetized with intraperitoneal ketamine (0.4 mg/g) and xylazine (0.2 mg/g) and euthanized by heart puncture exsanguination.[2]

Neostigmine has a short half-life of approximately one hour. The drug does not cross the blood-brain barrier (BBB).[2]

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 on breastfed infants other than transient ones.
◉ Effects on Breastfed Infants
Six infants born to mothers receiving neostigmine for myasthenia gravis have been 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 lactating mothers. In animal studies, cholinergic drugs increased oxytocin release, and their effects on serum prolactin levels varied. Prolactin levels in established lactating mothers may not affect their ability to breastfeed.

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[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 methyl sulfate is an aryl ammonium sulfate. It is an EC 3.1.1.8 (cholinesterase) 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 (containing the active moiety); Glyceryl bromide; Neostigmine methyl sulfate (ingredient).

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Neostigmine is an acetylcholinesterase inhibitor that acts by inhibiting acetylcholinesterase and thus upregulating acetylcholine (ACh). ACh is a key component of the cholinergic anti-inflammatory pathway (CAP) that leads to inflammation control. Neostigmine cannot cross the blood-brain barrier. This study demonstrates for the first time that neostigmine treatment provides neuroprotection against oxidative stress in the cerebral cortex of asthmatic mice by reverting ROS production and modulating the antioxidant enzyme catalase (CAT). These results support communication between the peripheral immune system and the CNS, suggesting that acetylcholinesterase inhibitors such as neostigmine should be further studied as possible therapeutic strategies for neuroprotection in asthma.[2]

C13H22N2O6S

334.387

334.119

C, 46.70; H, 6.63; N, 8.38; O, 28.71; S, 9.59

51-60-5

Neostigmine Bromide;114-80-7; 114-80-7 (bromide); 1212-37-9 (iodide); 59-99-4 (cation); 51-60-5 (methylsulfate); 588-17-0 (hydroxide)

5824

White to off-white solid powder

457ºC

175-177 °C(lit.)

2.117

0

6

3

22

337

0

S(=O)(=O)([O-])OC([H])([H])[H].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]

3-((dimethylcarbamoyl)oxy)-N,N,N-trimethylbenzenaminium methyl sulfate

OSZNNLWOYWAHSS-UHFFFAOYSA-M SMILES

Neostigmine, Prostigmin, Vagostigmin, Polstigmine Neostigmine methylsulfate

Prostigmin; Neostigmine; Neostigmine methyl sulfate; 51-60-5; NEOSTIGMINE METHYLSULFATE; Syntostigmin; Hodostin; Neostigmine methylsulphate; Neostigmeth; Synstigmine; Polstigmine; Neostigmine methylsulfate; Vagostigmin

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)

DMSO : ≥ 100 mg/mL (~299.05 mM)
H2O : ~100 mg/mL (~299.05 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.48 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 (7.48 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 (7.48 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 (299.05 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.)