M3 muscarinic receptor
The morphology and viability of human corneal stromal (HCS) cells are assessed using light microscopy and the MTT assay, respectively, in order to assess the cytotoxicity of pilocarpine. HCS cells exposed to Pilocarpine at concentrations between 0.625 and 20 g/L exhibit morphological abnormalities such as cellular shrinkage, cytoplasmic vacuolation, detachment from the culture matrix, and eventually death, as well as dose- and time-dependent proliferation retardation, according to morphological observations. However, there is no discernible difference between the controls and those exposed to Pilocarpine below the concentration of 0.625 g/L. The MTT assay's results show that, after being exposed to pilocarpine above a concentration of 0.625 g/L (P<0.01 or 0.05), the cell viability of HCS cells decreases with time and concentration[2]. In contrast, HCS cells treated with pilocarpine below a concentration of 0.625 g/L appear to be identical to controls. In separated sections of rat tail arteries that were constricted with penylephrine (10 to 200 nM), the partial muscarinic agonist pilocarpine elicits concentration-dependent relaxation with an EC50 of 2.4 mM[3].

The morphology and viability of human corneal stromal (HCS) cells are assessed using light microscopy and the MTT assay, respectively, in order to assess the cytotoxicity of pilocarpine. HCS cells exposed to Pilocarpine at concentrations between 0.625 and 20 g/L exhibit morphological abnormalities such as cellular shrinkage, cytoplasmic vacuolation, detachment from the culture matrix, and eventually death, as well as dose- and time-dependent proliferation retardation, according to morphological observations. However, there is no discernible difference between the controls and those exposed to Pilocarpine below the concentration of 0.625 g/L. The MTT assay's results show that, after being exposed to pilocarpine above a concentration of 0.625 g/L (P<0.01 or 0.05), the cell viability of HCS cells decreases with time and concentration[2]. In contrast, HCS cells treated with pilocarpine below a concentration of 0.625 g/L appear to be identical to controls. In separated sections of rat tail arteries that were constricted with penylephrine (10 to 200 nM), the partial muscarinic agonist pilocarpine elicits concentration-dependent relaxation with an EC50 of 2.4 mM[3].

Examined is the saliva secreted by the exercised (EX) and control (CN) rats in response to pilocarpine. Pilocarpine induces a significantly higher amount of saliva in the EX rats than in the CN rats (P<0.01). On the other hand, the EX rats' saliva has a significantly lower Na+ concentration than the CN rats' (P<0.05)[1].

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Intraperitoneal injection of pilocarpine (0.5 mg·kg⁻¹) induced saliva secretion in rats. The amount of pilocarpine-induced saliva was significantly greater in exercised rats compared to control rats. The sodium ion (Na⁺) concentration in the secreted saliva was significantly lower in exercised rats, while the protein concentration showed no significant change. The expression levels of M1 and M3 muscarinic receptor mRNA in the submandibular glands were not altered by voluntary exercise. [1]

The MTT assay is used to assess cell viability. In summary, HCS cells are cultivated and treated after being inoculated at a density of 1×104 cells/100 µL/well into a 96-well culture plate (Nunc). The medium containing the pilocarpine (0.625 to 20 g/L) is completely changed every 4 hours to 100 µL serum-free DMEM/F12 medium with 1.0 g/L MTT. The cells are then incubated for 4 hours at 37°C in the dark. Following the cautious disposal of the MTT-containing medium, 150 µL of DMSO is added to dissolve the formazan crystals that have formed. This is done at 37°C in the dark for 15 minutes, and a Multiskan GO microplate reader is used to measure the absorbance at 490 nm[2].

Rats: Male, aged ten weeks Two groups—exercise (EX, n = 6) and control (CN, n = 6)—of wistar rats are allocated. When the CN rats are housed in cages with the running wheel locked, the EX rats are kept in cages with a running wheel (SN-451) for 40 days, allowing them to engage in voluntary exercise. The following is the measurement of saliva produced by pilocarpine on day forty. To sum up, the rats are given anesthesia, sublingually given preweighed cotton, and then given an intraperitoneal injection of pilocarpine (0.5 mg/kg) to induce the production of saliva. And for one hour, every ten minutes, a new cotton ball is added. By deducting the initial weight from the final weight, the mass of saliva secreted is determined after the collected cotton balls are weighed once more.

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Male, 10-week-old Wistar rats were anesthetized. Pre-weighed cotton balls were placed sublingually in their mouths. Pilocarpine was then administered via intraperitoneal injection at a dose of 0.5 mg·kg⁻¹ body weight to induce saliva secretion. The cotton balls were replaced every 10 minutes for a total duration of 1 hour. The mass of secreted saliva was determined by weighing the cotton balls again and subtracting the initial weight. The collected saliva was then centrifuged for further analysis of Na⁺ and protein concentrations. [1]

Absorption, Distribution and Excretion
In healthy male subjects, after oral administration of 5 mg pilocarpine three times daily, the peak plasma concentration reached 15 μg/L after 1.25 hours. After oral administration of 10 mg pilocarpine three times daily, the peak plasma concentration reached 41 μg/L after 0.85 hours. Co-administration with food accelerates absorption. In healthy subjects, the overall median time to peak concentration (Tmax) after ocular administration was 2.2 hours. The mean (standard deviation) Cmax and AUC0-t were 897.2 (287.2) pg/mL and 2699 (741.4) hr × pg/mL, respectively. In patients with presbyopia, the mean Cmax and AUC0-t,ss values were 1.95 ng/mL and 4.14 ng × hr/mL, respectively. The median time to peak concentration (Tmax) after administration was 0.3 hours, ranging from 0.2 to 0.5 hours. Pilocarpine and its degradation products are mainly excreted in urine. No relevant information is available. No relevant information is available. There is currently no definitive information regarding the metabolism and excretion of pilocarpine. It is partially destroyed in the body, but most of it is excreted in urine in conjugated form. Pilocarpine can penetrate the eyes well; after topical eye drops… Poisoning due to skin absorption has been reported. Metabolites/Metabolites Limited information is available regarding the metabolism of pilocarpine in the human body. Inactivation of pilocarpine may occur at neuronal synapses or in the plasma. It has been reported that pilocarpine undergoes a CYP2A6-mediated 3-hydroxylation reaction to produce the stereoisomer of 3-hydroxypilocarpine. Pilocarpine can also be hydrolyzed in plasma and human liver by paraoxonase 1 (a calcium-dependent esterase). Pilocarpine acid may be a metabolite of this hydrolysis. It has been reported that the metabolites of pilocarpine have little or no pharmacological activity. Known metabolites of pilocarpine include 3-hydroxypilocarpine. It may occur at neuronal synapses and in plasma. Half-life: 0.76 hours. Following three daily doses of 5 mg or 10 mg, the elimination half-lives are 0.76 hours and 1.35 hours, respectively. After ocular administration in healthy subjects, the half-life is 3.96 hours.

Hepatotoxicity
Elevated serum enzyme levels were uncommon in clinical trials of pilocarpine, and the incidence was not significantly different from the placebo group. Although pilocarpine is widely used, there are no published reports of acute liver injury caused by pilocarpine. Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Limited information suggests that maternal use of ophthalmic pilocarpine does not have adverse effects on breastfed infants. If ophthalmic pilocarpine is used during lactation, the infant should be closely monitored for signs of cholinergic overdose (diarrhea, tearing, excessive salivation, or excessive urination), especially in younger, exclusively breastfed infants. To significantly reduce the amount of medication that enters breast milk after using the eye drops, press the tear duct at the corner of the eye for at least 1 minute, then blot away excess medication with absorbent tissue. Since there is currently no information regarding oral pilocarpine during lactation, alternative medications are recommended, especially for breastfed newborns or premature infants.
◉ Effects on breastfed infants
A woman with glaucoma used pilocarpine implant (Ocusert; concentration not specified) in one eye during 9 weeks of breastfeeding (breastfeeding time not specified). No adverse effects were observed in the infant. [1]
A mother used pilocarpine eye drops (concentration not specified) twice daily, along with 2 drops of 0.5% timolol eye drops twice daily and 250 mg of acetazolamide orally twice daily, and delivered a premature infant at 36 weeks of gestation. The infant was exclusively breastfed for 5 months starting 6 hours after birth. On day 2 after birth, the infant developed electrolyte disturbances, manifested as hypocalcemia, hypomagnesemia, and metabolic acidosis. The infant received oral calcium gluconate and a single intramuscular injection of magnesium sulfate. Despite continued breastfeeding and the mother's medication, the infant's mild metabolic acidosis resolved on day 4 after birth, and weight gain was normal at 1, 3, and 8 months, but mild hypotonia was present. The authors believe the metabolic disorder was caused by acetazolamide transplacental transport, and the disorder subsided despite continued breastfeeding. The infant gained weight well during breastfeeding, but had mild residual hypertonia in the lower extremities requiring physical therapy. [2] ◉ Effects on lactation and breast milk As of the revision date, no published information was found regarding lactating mothers. In animal studies, cholinergic drugs have increased oxytocin release [3] and have varying effects on serum prolactin levels [4]. Other centrally acting cholinergic drugs have increased serum prolactin levels in humans [5][6]. Prolactin levels in established lactating mothers may not affect their ability to breastfeed.

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Protein binding
Pilocarpine does not bind to human or rat plasma proteins in the concentration range of 5 to 25,000 ng/mL. The effects of pilocarpine on plasma protein binding of other drugs have not been evaluated.

[1]. Daily voluntary exercise enhances pilocarpine-induced saliva secretion and aquaporin 1 expression in rat submandibular glands. FEBS Open Bio. 2017 Dec 7;8(1):85-93.

[2]. Cytotoxicity of pilocarpine to human corneal stromal cells and its underlying cytotoxic mechanisms. Int J Ophthalmol. 2016 Apr 18;9(4):505-11.

[3]. Pilocarpine-induced relaxation of rat tail artery by a non-cholinergic mechanism and in the absence of an intact endothelium. Br J Pharmacol. 1994 Jun;112(2):525-32.

Pilocarpine is a slowly hydrolyzed muscarinic receptor agonist without nicotine-like effects. It is used as a miotic and to treat glaucoma. (+)-Pilocarpine is the (+)-enantiomer of pilocarpine and has anti-glaucoma activity. It is the enantiomer of (-)-pilocarpine. Pilocarpine is a naturally occurring alkaloid derived from plants of the genus Pilocarpine and is a muscarinic acetylcholine agonist. Pilocarpine produces a parasympathomimetic effect by selectively acting on muscarinic receptors. It is used to treat dry mouth and various ophthalmic conditions, including elevated intraocular pressure and glaucoma. The use of pilocarpine in the treatment of glaucoma dates back to 1875. Pilocarpine is a cholinergic receptor agonist. The mechanism of action of pilocarpine is as a cholinergic agonist and a cholinergic muscarinic receptor agonist. Pilocarpine is an orally effective cholinergic agonist used to treat dry mouth symptoms in patients with keratoconjunctivitis sicca (Sjögren's syndrome) or xerostomia induced by localized radiation therapy. No cases of elevated serum enzymes or clinically significant liver injury have been reported during pilocarpine treatment. Pilocarpine has been reported to be present in Pilocarpus microphyllus, Pilocarpus racemosus, and other organisms with relevant data. Pilocarpine is a natural alkaloid extracted from plants of the Pilocarpus genus, possessing cholinergic agonist activity. As a cholinergic parasympathomimetic drug, pilocarpine primarily binds to muscarinic receptors, thereby inducing exocrine gland secretion and stimulating the smooth muscle of the bronchi, urinary tract, biliary tract, and intestines. When applied topically to the eye, this drug can stimulate the contraction of the pupillary sphincter, leading to pupillary constriction; stimulate the contraction of the ciliary muscle, leading to accommodative spasm; and may cause a transient increase in intraocular pressure, followed by a sustained decrease in intraocular pressure due to the opening of the trabecular meshwork and increased outflow of aqueous humor. Pilocarpine is only present in individuals who have used or taken this drug. It is a slowly hydrolyzed muscarinic agonist and does not have nicotine-like effects. Pilocarpine is used as a miotic and also for the treatment of glaucoma. [PubChem] Pilocarpine is a cholinergic parasympathomimetic drug. It primarily works by stimulating muscarinic receptors, increasing the secretion of exocrine glands, and causing contraction of the iris sphincter and ciliary muscle (when applied topically). It is a slowly hydrolyzed muscarinic agonist and does not have nicotine-like effects. Pilocarpine can be used as a miotic and also for the treatment of glaucoma. See also: Pilocarpine hydrochloride (in salt form).

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Drug Indications
Pilocarpine oral tablets are indicated for the treatment of dry mouth caused by Sjögren's syndrome or radiotherapy for head and neck tumors. Pilocarpine ophthalmic preparations are used to treat presbyopia in adults, lower intraocular pressure in patients with open-angle glaucoma or high intraocular pressure, treat acute angle-closure glaucoma, prevent increased intraocular pressure after laser surgery, and induce pupillary constriction.

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Mechanism of Action
Muscari M3 receptors are expressed in various endocrine and exocrine glands, including gastric and salivary glands. They are also present in the smooth muscle cells of the pupillary sphincter and ciliary body. The M3 receptor is a Gq protein-coupled receptor that activates phospholipase C and upregulates inositol triphosphate and intracellular calcium ion levels. Activation of the M3 receptor is associated with smooth muscle contraction and salivary gland stimulation.
Pilocarpine is an agonist of M1 and M2 receptors, as well as a complete and partial agonist of the M3 receptor.
…It primarily acts on muscarinic receptors on autonomic effector cells, and ganglion effects can also be observed. This is especially true of pilocarpine, although its ganglion action also involves stimulation of muscarinic receptors…
…After topical instillation, the pupil begins to constrict within 15 to 30 minutes and lasts for 4 to 8 hours. Intraocular pressure reaches its maximum decrease within 2 to 4 hours, which is associated with the maximum decrease in aqueous humor outflow resistance. The effect on intraocular pressure lasts longer than the effect on aqueous humor outflow…pilocarpine…may reduce aqueous humor production.
…The primary action of pilocarpine is to stimulate cells that are the same as those that act as autonomic effector cells acting as cholinergic postganglionic nerve impulses.
…The primary action of pilocarpine is to stimulate cells that are the same as those that act as autonomic effector cells acting as cholinergic postganglionic nerve impulses. In this respect…similar to cholinesterases…
pilocarpine is an M3 muscarinic receptor agonist with sympathetic-like effects. Studies have shown that this method can be used to assess the effects of stress response on salivation. This study used it as a pharmacological tool to stimulate salivation in rats in order to assess the effects of chronic voluntary exercise on salivary gland function. [1]

C₁₁H₁₇N₃O₅

271.27

271.117

C, 50.89; H, 6.08; N, 18.26; O, 19.55; S, 5.22

148-72-1

Pilocarpine Hydrochloride; 54-71-7; Pilocarpine; 92-13-7

657349

White to off-white solid powder

520.5ºC at 760 mmHg

173,5-174°C

268.6ºC

1.16E-11mmHg at 25°C

81 ° (C=2, H2O)

1.337

1

6

3

19

270

2

[O-][N+](O)=O.O=C1OC[C@H](CC2=CN=CN2C)[C@@H]1CC

PRZXEPJJHQYOGF-GNAZCLTHSA-N

InChI=1S/C11H16N2O2.HNO3/c1-3-10-8(6-15-11(10)14)4-9-5-12-7-13(9)2;2-1(3)4/h5,7-8,10H,3-4,6H2,1-2H3;(H,2,3,4)/t8-,10-;/m0./s1

(3S,4R)-3-ethyl-4-[(3-methylimidazol-4-yl)methyl]oxolan-2-one;nitric acid

Glycylpressin; Pilagan; Pilocarpine (nitrate); Pilocarpine mononitrate; Pilocarpini nitras; Pilocarpine nitrate salt; Pilocarpinum nitricum; Terlipressin; Remestyp

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product is not stable in solution, please use freshly prepared working solution for optimal results.  (2). Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: ~250 mg/mL (~921.6 mM)
H2O: ~100 mg/mL (~368.6 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.67 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 20.8 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: ≥ 2.08 mg/mL (7.67 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 20.8 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: ≥ 2.08 mg/mL (7.67 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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