Acetylcholinesterase/AChE

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].

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].

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.

Absorption, Distribution and Excretion
Neostigmine bromide is poorly absorbed from the gastrointestinal tract following oral administration
NEOSTIGMINE...IS ABSORBED POORLY AFTER ORAL ADMINISTRATION, SUCH THAT MUCH LARGER DOSES ARE NEEDED THAN BY THE PARENTERAL ROUTE. ...THE EFFECTIVE PARENTERAL DOSE OF NEOSTIGMINE IN MAN IS 0.5 TO 2.0 MG, THE EQUIVALENT ORAL DOSE MAY BE 30 MG OR MORE. LARGE ORAL DOSES MAY PROVE TOXIC IF INTESTINAL ABSORPTION IS ENHANCED FOR ANY REASON.
...THE EXCRETION OF NEOSTIGMINE IS RETARDED IN PATIENTS WITH SEVERE KIDNEY DISEASE, MAKING THIS ANTICHOLINESTERASE DRUG AN ACCEPTABLE CHOICE IN PATIENTS WITH RENAL FAILURE.
The pharmacokinetics of neostigmine in patients with normal renal function were determined and compared with those of patients undergoing renal transplantation or bilateral nephrectomy. Ten to 15 min prior to the end of operation and anesthesia, d-tubocurarine infusion was terminated and neostigmine, 0.07 mg/kg and atropine 0.03 mg/kg were given by infusion over a 2-min period. In anephric patients the elimination half-life was prolonged. Total serum clearance was decr from 16.7 ml/kg/min in patients with normal renal function to 7.8 ml/kg/min in anephric patients. Neostigmine pharmacokinetics following renal transplantation were not different from those in patients with normal renal function. Renal excretion accounts for 50% of neostigmine clearance.

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Metabolism / Metabolites
Neostigmine undergoes hydrolysis by cholinesterase and is also metabolized by microsomal enzymes in the liver.
NEOSTIGMINE IS DESTROYED BY PLASMA ESTERASES, AND THE QUATERNARY ALCOHOL AND PARENT COMPOUND ARE EXCRETED IN THE URINE.
NEOSTIGMINE YIELDS 3-HYDROXYPHENYL TRIMETHYLAMMONIUM IN THE RAT. ROBERTS, JB ET AL; BIOCHEM PHARMAC 17: 9 (1968). /FROM TABLE/

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Biological Half-Life
The half-life ranged from 42 to 60 minutes with a mean half-life of 52 minutes.
The pharmacokinetics of neostigmine was evaluated in man after iv and oral admin. The mean plasma T/2 for neostigmine after iv admin was 0.89 hr. Following oral admin peak concn occurred 1-2 hr after intake, but biol availability was only 1-2% of the admin dose. In patients with myasthenia gravis, the decrement of the evoked electric muscle response of repetitive nerve stimulation correlated well with plasma concn of neostigmine.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Limited data indicate that use of neostigmine to treat myasthenia gravis may be acceptable during breastfeeding, although pyridostigmine may be preferred. Monitor newborns because abdominal cramps after each breastfeeding has been reported. Because of its short half-life, single doses of neostigmine to reverse neuromuscular blockade following surgery are unlikely to adversely affect the breastfed infant more than transiently.
◉ Effects in Breastfed Infants
Six infants of mothers treated with neostigmine for myasthenia gravis were reportedly breastfed successfully. One newborn infant appeared to have abdominal cramps after each breastfeeding, probably caused by neostigmine, although it could not be detected in the breastmilk of the infant's mother.
◉ Effects on Lactation and Breastmilk
Relevant published information in nursing mothers was not found as of the revision date. In animals, cholinergic drugs increase oxytocin release, and have variable effects on serum prolactin. The prolactin level in a mother with established lactation may not affect her ability to breastfeed.

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Protein Binding
Protein binding to human serum albumin ranges from 15 to 25 percent.

[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 comprising an anilinium ion core having three methyl substituents on the aniline nitrogen, and a 3-[(dimethylcarbamoyl)oxy] substituent at position 3. It is a parasympathomimetic which acts as a reversible acetylcholinesterase inhibitor. It has a role as an EC 3.1.1.7 (acetylcholinesterase) inhibitor and an antidote to curare poisoning.
A cholinesterase inhibitor used in the treatment of myasthenia gravis and to reverse the effects of muscle relaxants such as gallamine and tubocurarine. Neostigmine, unlike physostigmine, does not 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 agent that acts as a reversible acetylcholinesterase inhibitor.
A cholinesterase inhibitor used in the treatment of myasthenia gravis and to reverse the effects of muscle relaxants such as gallamine and tubocurarine. Neostigmine, unlike PHYSOSTIGMINE, does not cross the blood-brain barrier.
See also: Neostigmine Methylsulfate (has salt form).

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Drug Indication
Neostigmine is used for the symptomatic treatment of myasthenia gravis by improving muscle tone.

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Mechanism of Action
Neostigmine is a parasympathomimetic, specifically, a reversible cholinesterase inhibitor. The drug inhibits acetylcholinesterase which is responsible for the degredation of acetylcholine. So, with acetylcholinesterase inhibited, more acetylcholine is present By interfering with the breakdown of acetylcholine, neostigmine indirectly stimulates both nicotinic and muscarinic receptors which are involved in muscle contraction.. It does not cross the blood-brain barrier.
...PHARMACOLOGICAL EFFECTS OF ANTICHOLINESTERASE AGENTS ARE DUE PRIMARILY TO PREVENTION OF HYDROLYSIS OF ACH /ACETYLCHOLINE/ BY ACHE /ACETYLCHOLINESTERASE/ AT SITES OF CHOLINERGIC TRANSMISSION. TRANSMITTER THUS ACCUMULATES, AND THE ACTION OF ACH /ACETYLCHOLINESTERASE/ THAT IS LIBERATED BY CHOLINERGIC IMPULSES OR THAT LEAKS FROM THE NERVE ENDING IS ENHANCED.
Neostigmine increased both miniature end-plate potential and end-plate potential amplitudes but did not affect quantal content in isolated frog sciatic nerve-Sartorius muscle prepn. This suggests that cholinesterase inhibition was the only effect.
Long term (24-96 hr) treatment of a mouse-derived myogenic cell line (G8) with neostigmine markedly reduced binding of alpha-bungarotoxin (alpha-BuTx) to these cells. Protein synthesis in these cultures was markedly reduced and cell morphology degenerated. Myotubes maintained slightly hyperpolarized resting membrane potentials, and were able to respond to iontophoretic acetylcholine (Ach) application with overshooting action potentials. Degenerative changes at the neuromuscular junction associated with chronic neostigmine treatment in vivo are probably due to a direct action of the anticholinesterase on the muscle, rather than to altered intracleft ACh levels or to presynaptic effects of the anticholinesterase.
The intraluminal probe mounted with 2 electrode-strain gauge pairs, 4 cm apart, was used to study the effect of a neutral interview, a stressful interview, a meal (478.7 cal) and neostigmine (0.5 mg, im) on the contractile electrical complex, continuous electrical response activity and their associated contractions in 17 normal subjects. Neostigmine resulted in an incr in contractile electric complex & continuous electric response activity indexes 5-10 and 25-30 min after the injection, respectively. Both the meal and neostigmine incr the percentage of propagated contractile electric complexes during all of the recording periods.

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.)