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| 1mg |
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| Other Sizes |
| Targets |
Pirenzepine-d8 dihydrochloride, like its non-deuterated parent, selectively targets the M1 subtype of muscarinic acetylcholine receptors (mAChRs), a family of G-protein-coupled receptors (GPCRs). It acts as a competitive antagonist, blocking the binding of the endogenous neurotransmitter acetylcholine to M1 receptors. M1 receptors are predominantly expressed in the central nervous system (cortex, hippocampus) and in the peripheral nervous system (gastric parietal cells, salivary glands, and smooth muscle). By blocking M1 receptors in the stomach, Pirenzepine inhibits gastric acid secretion. The deuterium labeling does not alter the receptor binding affinity or pharmacological mechanism but is used for analytical purposes.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as quantitative tracers while the drugs were being developed. Because deuteration may have an impact on a drug's pharmacokinetics and metabolic profile, it has drawn attention [1].
In vitro, Pirenzepine-d8 dihydrochloride exhibits the same pharmacological profile as Pirenzepine, acting as a selective M1 muscarinic receptor antagonist. The binding affinity (Ki) for the M1 receptor is typically in the low nanomolar range (e.g., 5-20 nM). The compound shows lower affinity for M2, M3, M4, and M5 receptors, conferring its functional selectivity. In receptor binding assays using membranes from cells expressing specific mAChR subtypes, the deuterated version would compete with radioligands such as [3H]N-methylscopolamine ([3H]NMS) with the same potency as the unlabeled compound. In functional assays, Pirenzepine inhibits acetylcholine-induced calcium mobilization or phosphoinositide hydrolysis in M1-expressing cells. The deuterated version is typically used as an internal standard in bioanalytical assays rather than in pharmacological studies. |
| Enzyme Assay |
A competition binding assay is performed using membranes from CHO or HEK293 cells stably expressing the human M1 muscarinic receptor. Membranes (10-20 microg protein) are incubated with 0.5-1 nM of a radiolabeled antagonist, such as [3H]N-methylscopolamine ([3H]NMS) or [3H]quinuclidinyl benzilate (QNB), in binding buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM EDTA, 0.1% BSA) in the presence of increasing concentrations (0.01-10,000 nM) of Pirenzepine (unlabeled) or Pirenzepine-d8 (as a test compound if needed). Nonspecific binding is determined in the presence of 10 microM atropine. The reaction is incubated at 25-30degC for 60 min. Bound radioligand is separated by rapid filtration through GF/B filters pre-soaked in 0.3% polyethyleneimine. The filters are washed 3 times with ice-cold buffer, and the retained radioactivity is measured by liquid scintillation counting. The IC50 is determined using nonlinear regression, and the Ki is calculated using the Cheng-Prusoff equation. The Ki value for Pirenzepine is typically 5-20 nM for M1 receptors.
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| Cell Assay |
Pirenzepine-d8 dihydrochloride is not typically used in cell-based assays for its biological activity; it is used as an internal standard in drug quantification. However, a functional cellular assay can be used to confirm the antagonistic activity. Chinese hamster ovary (CHO) or HEK293 cells stably expressing the human M1 receptor are seeded in 96-well plates (2×10⁴ cells/well). The cells are loaded with a calcium-sensitive dye (e.g., Fluo-4 AM, 4 microM) for 30-60 min at 37degC. The cells are washed and incubated with varying concentrations of Pirenzepine (0.1-1000 nM) for 10-15 min. The M1 agonist carbachol (10-30 microM, approximate EC80) is then injected, and the increase in intracellular calcium is measured using a FlexStation or FLIPR (ex/em = 485/535 nm). The IC50 for inhibition of the calcium response is calculated using nonlinear regression. The EC50 for inhibition is typically in the low nanomolar range. Pirenzepine-d8 dihydrochloride would produce identical results.
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| Animal Protocol |
Pirenzepine-d8 dihydrochloride is primarily used as an internal standard in PK studies of Pirenzepine in animals or humans. The following protocol describes a rat PK study: Male Sprague-Dawley rats (200-300 g, n=5 per time point) are administered a single oral (p.o., 10-30 mg/kg in 0.5% methylcellulose) or intravenous (i.v., 1-5 mg/kg in saline) dose of Pirenzepine. Blood samples (200-300 microL) are collected via the jugular vein at pre-dose and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h post-dose. Plasma is separated by centrifugation at 4degC. A fixed concentration of Pirenzepine-d8 dihydrochloride (e.g., 10-50 ng/mL) is added to each plasma sample as an internal standard. The samples are processed by liquid-liquid extraction (e.g., with ethyl acetate) or solid-phase extraction (SPE). The organic layer is evaporated to dryness, reconstituted in the mobile phase, and injected into the LC-MS/MS system. Chromatographic separation is achieved on a C18 column (50 mm x 2.1 mm, 1.7 microm) with a mobile phase of 0.1% formic acid in water and acetonitrile (gradient elution). Detection is performed in positive ion mode (ESI+) using multiple reaction monitoring (MRM): for Pirenzepine, m/z 352 → m/z 113 (or appropriate transition); for the d8 internal standard, m/z 360 → m/z 113. The peak area ratio (analyte/IS) is used for quantification against a standard curve prepared in blank plasma. PK parameters are calculated using non-compartmental analysis.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for Pirenzepine itself are well-known. Pirenzepine is a hydrophilic, positively charged molecule with limited oral bioavailability (approximately 10-20% in humans and rats). The elimination half-life (t½) in humans is 10-12 hours, while in rats it is 1-2 hours. After oral administration, Tmax is 2-4 hours. The compound is primarily excreted unchanged in the urine, with minimal metabolism. The deuterated version is expected to have nearly identical PK properties as the non-deuterated compound, but it is used only as an internal standard in analytical methods. The small changes due to the deuterium isotope effect (if any) are generally considered negligible for bioanalytical purposes.
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| Toxicity/Toxicokinetics |
Pirenzepine is generally well-tolerated in both research and clinical settings. The most common adverse effects are due to its anticholinergic action (M1 antagonism) and include dry mouth, blurred vision, constipation, and urinary retention. These effects are dose-dependent. No specific toxicity data for the d8-labeled version are available, as it is used only as an analytical standard in trace amounts. The labeled compound should be handled according to standard safety procedures for pharmaceutical compounds.
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| References |
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| Additional Infomation |
Pirenzepine-d8 dihydrochloride is a stable isotope-labeled compound used as an internal standard for LC-MS/MS quantification of Pirenzepine in biological samples. The parent compound, Pirenzepine, is a selective M1 muscarinic receptor antagonist. It exhibits significant biological activity by inhibiting gastric acid secretion and alleviating muscle spasms. The deuterated version is not intended for therapeutic use; it is strictly an analytical tool for research and development. Store as a light yellow to yellow solid powder at -20degC, protected from moisture and light.
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| Molecular Formula |
C19H15D8CL2N5O2
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| Related CAS # |
Pirenzepine dihydrochloride;29868-97-1;Pirenzepine;28797-61-7;Pirenzepine-d8
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| Appearance |
Typically exists as solid at room temperature
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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 |
| 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) |
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
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| 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.) |
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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