| Size | Price | Stock | Qty |
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| 250mg |
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| 500mg |
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| 1g |
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| 2g |
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| 5g |
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| 10g |
| Targets |
muscarinic acetylcholine receptor/mAChR
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| ln Vitro |
In a CRE-luciferase assay using CRISPR-M3 HEK293T cells transfected with the human M4 mAChR, atropine inhibited carbachol (10 μM)-induced luminescence with an IC50 of 390 pM. The inhibitory constant (Ki) calculated from this data was 140 pM. [3]
In a CRE-luciferase assay using CRISPR-M3 HEK293T cells transfected with the chicken M4 mAChR (cM4), atropine inhibited carbachol (10 μM)-induced luminescence with an IC50 of 710 pM. The inhibitory constant (Ki) calculated from this data was 120 pM. [3] In a CRE-luciferase assay using CRISPR-M3 HEK293T cells transfected with the human alpha2A-adrenoceptor (hADRA2A), atropine inhibited clonidine (1 μM)-induced luminescence with an IC50 of 45 μM. The inhibitory constant (Ki) calculated from this data was 14 μM. [3] The study notes that at high concentrations (1-100 μM), atropine has been reported by others to have antagonist activity at alpha-adrenoceptors. [3] Acetylcholine-induced pulmonary vein dilatation in humans is inhibited by atropine sulfate (tropine; 1 μM; pulmonary veins and arteries) sulfate [4]. |
| ln Vivo |
The article discusses that topical atropine is effective against myopia in children, but the 1% dosage can induce side effects. A 0.01% concentration has been shown in recent studies to retain effectiveness for inhibiting myopia with reduced side effects. [3]
In the chick model of form-deprivation myopia (FDM), atropine is known to inhibit myopia, requiring estimated vitreal concentrations in the range of 0.1-10 mM to be effective. [3] The paper references findings that ablating cholinergic amacrine cells in chicks does not impair myopia inhibition by atropine, suggesting its site of action might be non-retinal. [3] The paper also notes that treatment with myopia-inhibiting concentrations of atropine in chick retina-RPE-choroid-sclera preparations causes a massive, nonspecific release of retinal neurotransmitters. [3] In in vitro preparations of mouse scleral fibroblasts, the inhibitory action of atropine on carbachol-induced proliferation was observed only at high concentrations (0.5-100 μM), which are 500-1000 fold higher than its Ki for mAChRs. [3] cardiac arrhythmias that usually happen during periods of anesthesia are suppressed by atropine (tropine; 10 mg/kg; intraperitoneally; once over 40 minutes; Peromyscus sp.) sulfate [2]. |
| Enzyme Assay |
This study did not perform direct enzyme assays. The primary assay used was a cell-based CRE-luciferase reporter assay to measure receptor activation and inhibition. [3]
CRE-Luciferase Assay for Receptor Antagonism: CRISPR-M3 HEK293T cells, which lack endogenous M3 muscarinic receptors, were co-transfected with a receptor clone (human M4, chicken cM4, or human ADRA2A), a cAMP response element luciferase vector (CRE-Luc), and a Renilla luciferase control vector (RLuc). 48 hours post-transfection, cells were incubated for 4 hours with a fixed, submaximal concentration of agonist (10 μM carbachol for M4/cM4, or 1 μM clonidine for ADRA2A) and increasing concentrations of the test antagonist, including atropine. After incubation, cells were lysed, and luciferase activity was measured sequentially using a Dual-Glo Luciferase Assay System. CRE-Luc activity (reflecting cAMP levels and receptor activation) was normalized to RLuc activity (a control for cell viability and transfection efficiency). Antagonist IC50 values were determined by nonlinear regression analysis of the normalized data. [3] |
| Cell Assay |
Cell Culture and Transfection: CRISPR-M3 HEK293T cells were cultured in DMEM with 10% FBS. For the assay, cells were seeded in 12-well plates and transfected at 30% confluency using Lipofectamine LTX. For each well, a mixture of Opti-MEM containing 160 ng of receptor DNA (e.g., human M4), 180 ng of CRE-Luc, and 160 ng of RLuc was combined with a Lipofectamine LTX solution. After a 5-minute incubation, the complexes were added to the cells. The medium was changed after 8 hours, and 24 hours post-transfection, cells were trypsinized and seeded into white, clear-bottomed 96-well plates at 7500 cells per well. [3]
CRE-Luc Luminescence Assay: 48 hours after the initial transfection, the medium in the 96-well plates was replaced with 50 μL of FluoroBrite DMEM containing a fixed concentration of agonist (10 μM carbachol for M4/cM4; 1 μM clonidine for ADRA2A) and various concentrations of the antagonist (e.g., atropine). Cells were incubated for 4 hours at 37°C. Subsequently, 50 μL of Dual-Glo Luciferase Reagent was added to each well. After a 10-minute incubation with shaking to ensure lysis, CRE-Luc luminescence was measured. Then, 50 μL of Dual-Glo Stop & Glo Reagent was added, and after another 10-minute incubation, Renilla luciferase luminescence was measured. CRE-Luc values were normalized to RLuc values to control for well-to-well variability. [3] |
| Animal Protocol |
The paper discusses animal models and protocols from the perspective of reviewed literature, rather than presenting new in vivo data for atropine. [3]
Chick Model of Myopia: In studies referenced by the paper, form-deprivation myopia (FDM) is induced in chicks. Atropine is administered to inhibit myopia, typically via intravitreal injection. Concentrations used range from 0.1 to 10 mM (estimated vitreal concentration), with a total amount of 20-2000 nmol per injection being common. [3] Rabbit Model for Ocular Distribution: The paper references studies where a single dose of 2% [3H]-atropine was delivered to the conjunctival sac of albino rabbits to study its distribution in ocular tissues. [3] Human Clinical Use: The paper discusses clinical protocols where atropine is delivered as daily eye drops at concentrations ranging from 0.01% to 1% for the treatment of childhood myopia. [3] Animal/Disease Models: White-footed mice (Peromyscus sp.) [2] Doses: 10 mg/kg Route of Administration: intraperitoneal (ip) injection; once, lasting 40 minutes. Experimental Results: increased heart rate and diminished arrhythmia. |
| ADME/Pharmacokinetics |
Ocular Distribution (from rabbit studies): One hour after a single topical dose of 2% [3H]-atropine to the conjunctival sac of albino rabbits, the concentration in ocular tissues was 0.09% of the original dose in the sclera, 0.05% in the choroid, and 0.008% in the retina. [3]
Another study cited found 0.01% in the retina and 0.38% in the choroid and sclera 30 minutes after topical application of [C14]-atropine. [3] Estimated Human Ocular Concentrations: Based on the rabbit data, a single topical dose of 0.01% to 1% atropine in humans is estimated to result in concentrations of 0.13-13 μM in the sclera, 0.07-7 μM in the choroid, and 0.01-1.1 μM in the retina. [3] Accumulation and Binding: The paper notes that serum levels of atropine can accumulate with repeated dosing, rising from undetectable levels after a single dose to 1.13-5.23 ng/μL when administered every 4-6 hours over 48 hours. [3] Furthermore, pigmented ocular tissues (RPE, iris, ciliary body, choroid, retina) are particularly adept at retaining atropine, which may lead to a prolonged effect. [3] |
| Toxicity/Toxicokinetics |
Clinical Side Effects: At the commonly prescribed 1% concentration for myopia treatment, atropine may induce allergic reactions and mAChR M3-mediated side effects such as mydriasis (pupil dilation), photophobia (light sensitivity), cycloplegia (loss of accommodation), and possibly early presbyopia. [3]
Rebound Effect: Upon cessation of 1% atropine treatment, a "rebound" phenomenon can occur, where myopia progresses at a faster rate than in untreated eyes. This is possibly due to desensitization of target receptors from the high drug concentration. [3] Side Effects at Low Dose: Even with the lower, more favored 0.01% concentration, some patients still complain of complications related to muscarinic receptor blockade, including photophobia and blurred vision. However, these symptoms are typically not severe enough to discontinue treatment. [3] In Vitro Cytotoxicity: In the cell-based assays of this study, high concentrations of atropine did not cause significant cell death in the CRISPR-M3 HEK293T cells, as determined by the Renilla luciferase control. [3] |
| References | |
| Additional Infomation |
Background: Atropine is a potent, non-selective muscarinic acetylcholine receptor (mAChR) antagonist. It has been the standard pharmacological treatment for myopia, but its mechanism of action in this context is unknown and is a central focus of the research paper. [3]
Mechanism of Action (Proposed): The paper hypothesizes that atropine's anti-myopia effects at the high concentrations used in vivo may be mediated through off-target binding to alpha2A-adrenoceptors (ADRA2A) rather than through its canonical target, the M4 mAChR. This is supported by the correlation between the drug's potency at hADRA2A and its reported ability to inhibit chick FDM, which is not seen for M4/cM4. [3] Clinical Use for Myopia: Topical atropine is effective in controlling myopia progression in children. Due to side effects at higher doses, 0.01% atropine eye drops are currently the most favored concentration prescribed by clinicians in Southeast Asia and North America. [3] Conclusion from the Study: The authors conclude from their data that the action of atropine via the mAChR M4 receptor is highly unlikely to be a factor in myopia control, and that non-mAChR targets, such as alpha-adrenoceptors, warrant further investigation. [3] |
| Molecular Formula |
2(C17H23NO3).H2SO4
|
|---|---|
| Molecular Weight |
676.82
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| Exact Mass |
676.302
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| Elemental Analysis |
C, 58.77; H, 7.25; N, 4.03; O, 25.33; S, 4.61
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| CAS # |
55-48-1
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| Related CAS # |
Atropine sulfate monohydrate;5908-99-6;Atropine;51-55-8;Atropine hydrobromide;6415-90-3
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| PubChem CID |
60196398
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| Appearance |
White to off-white solid powder
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| Boiling Point |
429.8ºC at 760 mmHg
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| Melting Point |
189-192 °C (A)(lit.)
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| Flash Point |
213.7ºC
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| LogP |
4.165
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
47
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| Complexity |
434
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| Defined Atom Stereocenter Count |
4
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| SMILES |
CN1[C@@H]2CC[C@H]1CC(C2)OC(=O)C(CO)C3=CC=CC=C3.CN1[C@@H]2CC[C@H]1CC(C2)OC(=O)C(CO)C3=CC=CC=C3.OS(=O)(=O)O
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| InChi Key |
HOBWAPHTEJGALG-JKCMADFCSA-N
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| InChi Code |
InChI=1S/2C17H23NO3.H2O4S/c2*1-18-13-7-8-14(18)10-15(9-13)21-17(20)16(11-19)12-5-3-2-4-6-12;1-5(2,3)4/h2*2-6,13-16,19H,7-11H2,1H3;(H2,1,2,3,4)/t2*13-,14+,15?,16?;
|
| Chemical Name |
[(1S,5R)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] 3-hydroxy-2-phenylpropanoate;sulfuric acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O : ~100 mg/mL (~295.50 mM)
DMSO : ~62.5 mg/mL (~184.69 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (6.15 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.15 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (6.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 100 mg/mL (295.50 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4775 mL | 7.3875 mL | 14.7750 mL | |
| 5 mM | 0.2955 mL | 1.4775 mL | 2.9550 mL | |
| 10 mM | 0.1477 mL | 0.7387 mL | 1.4775 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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