| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
Human mAChR M4: Atropine acts as an antagonist with an IC50 of 390 pM and a calculated Ki(CP) of 140 pM in a CRE-luciferase assay using transfected HEK293T cells. [3]
Chicken mAChR cM4: Atropine acts as an antagonist with an IC50 of 710 pM and a calculated Ki(CP) of 120 pM in a CRE-luciferase assay using transfected HEK293T cells. [3] Human alpha2A-adrenoceptor (hADRA2A): Atropine acts as an antagonist with an IC50 of 45 μM and a calculated Ki(CP) of 14 μM in a CRE-luciferase assay using transfected HEK293T cells. [3] |
|---|---|
| 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 (tropine; 1 μM; pulmonary veins and arteries) hydrobromide [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] Atropine hydrobromide (peromyscus sp.; intraperitoneal injection; 10 mg/kg; once over 40 minutes) Cardiac arrhythmias that usually happen during anesthesia are suppressed by hydrobromide [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 |
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. 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] |
| 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 |
C17H23NO3.HBR
|
|---|---|
| Molecular Weight |
370.28136
|
| Exact Mass |
369.094
|
| Elemental Analysis |
C, 55.14; H, 6.53; Br, 21.58; N, 3.78; O, 12.96
|
| CAS # |
6415-90-3
|
| Related CAS # |
Atropine;51-55-8;Atropine sulfate;55-48-1
|
| PubChem CID |
21090485
|
| Appearance |
Typically exists as solid at room temperature
|
| LogP |
2.826
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
4
|
| Rotatable Bond Count |
5
|
| Heavy Atom Count |
22
|
| Complexity |
353
|
| Defined Atom Stereocenter Count |
2
|
| SMILES |
Br.OCC(C(OC1CC2CCC(N2C)C1)=O)C1C=CC=CC=1
|
| InChi Key |
VZDNSFSBCMCXSK-ZZJGABIISA-N
|
| InChi Code |
InChI=1S/C17H23NO3.BrH/c1-18-13-7-8-14(18)10-15(9-13)21-17(20)16(11-19)12-5-3-2-4-6-12;/h2-6,13-16,19H,7-11H2,1H3;1H/t13-,14+,15?,16?;
|
| Chemical Name |
[(1R,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] 3-hydroxy-2-phenylpropanoate;hydrobromide
|
| Synonyms |
Atropine hydrobromide; 6415-90-3; Atropine (hydrobromide); 6VKW7R97ZU; 1alphaH,5alphaH-Tropan-3alpha-ol (+-)-tripate (ester), hydrobromide;
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
|
|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7007 mL | 13.5033 mL | 27.0066 mL | |
| 5 mM | 0.5401 mL | 2.7007 mL | 5.4013 mL | |
| 10 mM | 0.2701 mL | 1.3503 mL | 2.7007 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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