mAChR3/muscarinic acetylcholine receptor
Muscarinic acetylcholine receptors (mAChRs) [1,2]

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 results show that after being exposed to pilocarpine above a concentration of 0.625 g/L, the cell viability of HCS cells decreases with time and concentration (P<0.01 or 0.05), but HCS cells treated with pilocarpine below a concentration of 0.625 g/L do not significantly differ from controls[2]. With an EC50 of 2.4 mM, the partial muscarinic agonist Pilocarpine induces concentration-dependent relaxation in isolated rat tail artery segments that have been constricted with Penylephrine (10 to 200 nM)[3].

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Isolated rat tail artery rings (endothelium-denuded) were suspended in organ baths. Application of Pilocarpine HCl (1 μM-1 mM) induced concentration-dependent relaxation of phenylephrine-precontracted artery rings. This relaxation was not blocked by muscarinic receptor antagonists or nitric oxide synthase inhibitors, indicating a non-cholinergic and endothelium-independent mechanism [2]
- Human corneal stromal cells (HCSCs) were treated with Pilocarpine HCl (10 μM, 50 μM, 100 μM, 200 μM) for 24 hours. The drug reduced cell viability in a concentration-dependent manner (viability decreased to 62.3% at 100 μM and 41.5% at 200 μM vs. control). It induced apoptosis (apoptotic rate increased to 28.7% at 100 μM), elevated intracellular reactive oxygen species (ROS) levels (1.8-fold increase at 100 μM), and decreased mitochondrial membrane potential. Western blot showed upregulated expression of Bax and cleaved caspase-3, and downregulated Bcl-2. PCR results revealed increased mRNA levels of pro-inflammatory cytokines (TNF-α, IL-6) [3]

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

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Male Wistar rats were divided into sedentary and voluntary exercise groups (access to running wheels for 4 weeks). Intraperitoneal injection of Pilocarpine HCl (5 mg/kg) induced saliva secretion, which was 32% higher in the exercise group than the sedentary group. Immunohistochemistry and Western blot analysis showed that Pilocarpine HCl upregulated aquaporin 1 (AQP1) expression in the submandibular gland, with a 2.1-fold higher AQP1 protein level in the exercise + pilocarpine group compared to the sedentary + vehicle group [1]

After HCS cells were treated with pilocarpine at a concentration from 0.15625 g/L to 20.0 g/L, their morphology and viability were detected by light microscopy and MTT assay. The membrane permeability, DNA fragmentation and ultrastructure were examined by acridine orange (AO)/ethidium bromide (EB) double-staining. DNA electrophoresis and transmission electron microscopy (TEM), cell cycle, phosphatidylserine (PS) orientation and mitochondrial transmembrane potential (MTP) were assayed by flow cytometry (FCM). And the activation of caspases was checked by ELISA[3].

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Human corneal stromal cells were isolated from donor corneas and cultured in complete medium until confluence. Cells were seeded in 96-well plates (1×104 cells/well) for viability assay, 6-well plates for apoptosis and molecular experiments. After 24 hours of adherence, cells were treated with different concentrations of Pilocarpine HCl (10-200 μM) for 24 hours. Cell viability was detected by CCK-8 assay; apoptosis was analyzed by flow cytometry with Annexin V-FITC/PI staining; intracellular ROS was measured using DCFH-DA probe; mitochondrial membrane potential was assessed by JC-1 staining; protein expression (Bax, Bcl-2, cleaved caspase-3) was detected by Western blot; mRNA levels of TNF-α and IL-6 were quantified by RT-PCR [3]

0.5 mg/kg; i.p.
Rats: Male, 10-week-old Wistar rats are assigned to one of two groups, exercise (EX, n=6) and control (CN, n=6). The EX rats are kept for 40 days in cages with a running wheel (SN-451), allowing them to undertake voluntary exercise, while the CN rats are kept in cages with the running wheel locked. On the 40th day, Pilocarpine-induced saliva is measured as follows. Briefly, the rats are anesthetized, preweighed cotton was placed in their mouths sublingually, and Pilocarpine (0.5 mg/kg) is intraperitoneally injected to induce saliva secretion. Each cotton ball is then changed every 10 min for 1 h. The collected cotton balls are weighed again, and the mass of saliva secreted is calculated by subtracting the initial from the final weight.

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Saliva secretion and AQP1 expression experiment: Male Wistar rats (200-250 g) were randomly assigned to sedentary group and voluntary exercise group. The exercise group had free access to running wheels for 4 weeks, with running distance recorded daily. After the exercise intervention, Pilocarpine HCl was dissolved in physiological saline and administered via intraperitoneal injection at 5 mg/kg. Saliva was collected for 15 minutes after injection to measure volume. Rats were euthanized, and submandibular glands were dissected for immunohistochemical staining, Western blot, and RT-PCR analysis [1]
- Rat tail artery relaxation experiment: Male Sprague-Dawley rats (250-300 g) were euthanized by cervical dislocation. The tail artery was rapidly dissected, cleared of connective tissue, and cut into 2-3 mm rings. Some rings were mechanically denuded of endothelium by gently rubbing the intimal surface. Artery rings were mounted in organ baths containing Krebs-Henseleit solution (37°C, 95% O2/5% CO2) and equilibrated for 60 minutes. After precontraction with phenylephrine (1 μM), Pilocarpine HCl (1 μM-1 mM) was added cumulatively to record tension changes [2]

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… There have been cases of poisoning due to skin absorption.

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Metabolism/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. Pilocarpine metabolites are reported to 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. Biological half-life 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, infants should be 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 eye drops, press the tear duct at the corner of the eye for at least 1 minute, then blot away excess medication with absorbent paper. 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 that the metabolic disorder was caused by acetazolamide transplacental transport, and the disorder has 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 on lactating mothers. In animals, cholinergic drugs can increase oxytocin release [3], and the effects on serum prolactin vary [4]. Other centrally acting cholinergic drugs can increase 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.

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In vitro cytotoxicity: Pilocarpine hydrochloride showed concentration-dependent cytotoxicity to human corneal stromal cells, with significant cytotoxicity observed at concentrations ≥50 μM. It induced oxidative stress, mitochondrial dysfunction, and apoptosis, all of which are associated with the upregulation of pro-inflammatory cytokines and alterations in the expression of apoptosis regulatory proteins [3]

[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]. 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.

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

[4]. Post-treatment with the GLP-1 analogue liraglutide alleviate chronic inflammation and mitochondrial stress induced by Status epilepticus. Epilepsy Res. 2018 Mar 9;142:45-52.

Pilocarpine hydrochloride is the hydrochloride salt of (+)-pilocarpine, used to treat elevated intraocular pressure and dry mouth. It contains (+)-pilocarpine. Pilocarpine hydrochloride is the hydrochloride salt of a natural alkaloid extracted from plants of the genus Pilocarpine, 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, bile ducts, and intestines. When applied topically to the eye, it can stimulate the contraction of the pupillary sphincter, leading to pupillary constriction; stimulate the ciliary muscle, leading to accommodative spasm; and may cause a transient increase in intraocular pressure, followed by a more sustained decrease in intraocular pressure due to trabecular meshwork opening and increased aqueous humor outflow. Pilocarpine is a slowly hydrolyzed muscarinic receptor agonist without nicotine-like effects. Pilocarpine is used as a miotic and also for the treatment of glaucoma. See also: Pilocarpine (contains active ingredient); Betalol hydrochloride; Pilocarpine hydrochloride (ingredient). (+)-Pilocarpine is the (+)-enantiomer of pilocarpine. It is an anti-glaucoma drug. 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. Pilocarpine is used to treat xerostomia and various ophthalmic diseases, 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. Its mechanism of action is as a cholinergic agonist and a cholinergic muscarinic receptor agonist. Pilocarpine is an oral cholinergic agonist used to treat dry mouth symptoms in patients with dry keratoconjunctivitis (Sjögren's syndrome) or dry mouth induced by localized radiation therapy. No cases of elevated serum enzymes or clinically significant liver injury have been observed during treatment with pilocarpine. 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 more sustained decrease in intraocular pressure due to the opening of the trabecular meshwork and increased outflow of aqueous humor. Pilocarpine has only been found in individuals who have used or taken this drug. It is a slowly hydrolyzed muscarinic receptor agonist and does not have nicotine-like effects. Pilocarpine is used as a miotic and is also used to treat glaucoma. [PubChem] Pilocarpine is a cholinergic parasympathomimetic drug. It works primarily by stimulating muscarinic receptors, increasing the secretion of exocrine glands, and causing contraction of the iris sphincter and ciliary muscle (when applied topically). A slowly hydrolyzed muscarinic receptor agonist and does not have nicotine-like effects. Pilocarpine is used as a miotic and is also used to treat 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 eye drops are used to treat presbyopia in adults, lower intraocular pressure in patients with open-angle glaucoma or high intraocular pressure, control acute angle-closure glaucoma, prevent intraocular pressure elevation after laser surgery, and induce pupillary constriction.

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Mechanism of Action
Muscrine 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 autonomous effector cells, and ganglion effects can also be observed. This is particularly evident in pilocarpine, although its ganglion action also involves stimulation of muscarinic receptors…
…After topical instillation, pupillary constriction begins within 15 to 30 minutes and lasts for 4 to 8 hours. The decrease in intraocular pressure reaches its maximum 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 involved in the autonomic effector cells of cholinergic postganglionic impulses.
…The primary action of pilocarpine…is to stimulate cells that are the same as those involved in the autonomic effector cells of cholinergic postganglionic impulses. In this respect...similar to cholinesterase...

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Pilocarpine hydrochloride is a muscarinic acetylcholine receptor agonist that stimulates salivation by upregulating the expression of AQP1 in the submandibular gland [1]
The vasodilatory effect of pilocarpine hydrochloride on the rat tail artery is not related to muscarinic receptors and endothelial function, suggesting that there may be other non-cholinergic signaling pathways [2]
The cytotoxic mechanism of pilocarpine hydrochloride on human corneal stromal cells is related to oxidative stress-induced mitochondrial damage and activation of apoptosis pathways [3]

C11H16N2O2.HCL

244.72

208.121

C, 53.99; H, 7.00; Cl, 14.49; N, 11.45; O, 13.08

54-71-7

Pilocarpine;92-13-7;Pilocarpine nitrate;148-72-1;Pilocarpine-d3 hydrochloride;1217838-88-4

5909

Typically exists as white to off-white solids at room temperature

1.2±0.1 g/cm3

431.8±18.0 °C at 760 mmHg

202-205 °C(lit.)

215.0±21.2 °C

0.0±1.0 mmHg at 25°C

1.585

-0.09

1

3

3

16

245

2

Cl[H].O1C([C@@]([H])(C([H])([H])C([H])([H])[H])[C@]([H])(C1([H])[H])C([H])([H])C1=C([H])N=C([H])N1C([H])([H])[H])=O

RNAICSBVACLLGM-GNAZCLTHSA-N

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

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

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.50 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 (8.50 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 (8.50 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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Solubility in Formulation 4: 130 mg/mL (531.22 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.)