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| 1mg |
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| Targets |
Adenosine-15N5 is a stable isotope-labeled internal standard. Its unlabeled parent, adenosine, is an endogenous purine nucleoside that acts as a signaling molecule through four G protein-coupled receptors: A1, A2A, A2B, and A3 receptors. A1 receptors are coupled to Gi proteins, inhibiting adenylyl cyclase and activating K+ channels; A2A and A2B receptors are coupled to Gs proteins, activating adenylyl cyclase and increasing cAMP. In addition to receptor-mediated signaling, adenosine is a critical component of ATP, RNA, and DNA, serving as a building block for nucleic acid synthesis. It also functions as a regulator of coronary blood flow, a neurotransmitter in the central nervous system, and a modulator of immune responses. The ¹⁵N-labeled version is used as a tracer to study these pathways and as an internal standard for accurate quantification.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as tracers that influence measurement during the drug development process. It's possible that the drug's pharmacokinetics and functional range contribute to the concern for mutagenesis [3].
The in vitro biological activity of Adenosine-15N5 is presumed to be identical to its unlabeled parent, adenosine. Adenosine is a potent signaling molecule with EC₅0 values in the nanomolar to micromolar range for its receptors. In CHO cells expressing human A1 receptors, adenosine stimulates [3⁵S]GTPgammaS binding with an EC₅0 of approximately 300 nM. In rat cardiomyocytes, adenosine (0.1-100 uM) produces negative chronotropic and dromotropic effects via A1 receptor activation. In DDT1 MF-2 smooth muscle cells, adenosine (1-100 uM) stimulates cAMP accumulation via A2B receptors with an EC₅0 of approximately 12 uM. Adenosine also induces apoptosis in various cancer cell lines at concentrations of 100-1000 uM. The labeled Adenosine-15N5 is used as an internal standard in LC-MS analysis to accurately quantify adenosine levels in cell culture media, ensuring precise determination of endogenous and exogenous adenosine concentrations in these in vitro studies. |
| ln Vivo |
The in vivo activity of Adenosine-15N5 is not directly evaluated; it is used as a tracer and internal standard. Its unlabeled parent, adenosine, has potent and diverse physiological effects. In vivo, adenosine (0.1-10 mg/kg, i.v.) produces transient bradycardia, hypotension, and coronary vasodilation in animal models. In humans, adenosine is used clinically for the treatment of supraventricular tachycardia (6-12 mg i.v. bolus). Adenosine also acts as a neuromodulator in the central nervous system, inhibiting neurotransmitter release and promoting sleep. In models of inflammation, adenosine reduces pro-inflammatory cytokine production through A2A receptor activation. Due to its very short half-life (<10 seconds), endogenous adenosine levels are tightly regulated by rapid uptake via equilibrative nucleoside transporters (ENTs) and metabolism by adenosine deaminase and adenosine kinase. Adenosine-15N5 serves as a tracer to study these dynamics, allowing precise quantification of adenosine in plasma and tissues via LC-MS.
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| Enzyme Assay |
A generic non-cell-based assay for Adenosine-15N5 is its use as an internal standard in an LC-MS/MS method for quantifying adenosine in plasma. Prepare a standard stock solution of unlabeled adenosine in water (1 mg/mL). Prepare a separate stock solution of the internal standard Adenosine-15N5 at the same concentration. Prepare calibration standards by spiking the unlabeled analyte into a blank matrix (e.g., charcoal-stripped human plasma) to achieve concentrations ranging from 0.5 to 500 ng/mL. Add a fixed concentration of the internal standard (e.g., 50 ng/mL) to each calibration standard. Also prepare blank and double-blank samples. For sample preparation, add 200 uL of ice-cold acetonitrile to 100 uL of plasma to precipitate proteins. Vortex, incubate at -20degC for 10 minutes, and centrifuge at 12,000g for 10 minutes at 4degC. Transfer the supernatant to an autosampler vial. Analyze by LC-MS/MS in positive ion mode. Monitor the mass transitions: m/z 268 → 136 for adenosine (representing the loss of the ribose ring), and m/z 273 → 141 for Adenosine-15N5. Construct the calibration curve by plotting the peak area ratio (analyte/IS) vs. the nominal concentration. All procedures should be performed on ice to minimize enzymatic degradation of adenosine by adenosine deaminase.
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| Cell Assay |
A standard in vitro cell-based protocol for the unlabeled adenosine is used to study A2A receptor signaling in PC12 cells. Culture PC12 cells in RPMI 1640 medium supplemented with 10% horse serum, 5% FBS, and 1% penicillin/streptomycin at 37degC in a 5% CO2 incubator. Seed cells in 24-well plates at 5×10⁵ cells/well and allow to differentiate for 48 hours with 50 ng/mL nerve growth factor (NGF). On the day of the assay, wash cells twice with HBSS buffer. Pre-incubate with the phosphodiesterase inhibitor rolipram (10 uM) for 15 minutes. Then treat cells with increasing concentrations of unlabeled adenosine (1 nM to 100 uM) for 10-30 minutes. Lyse cells in 0.1 M HCl, neutralize, and measure intracellular cAMP levels using a competitive ELISA kit. Calculate the EC₅0 for cAMP accumulation. For validation, collect a separate set of culture media samples, add Adenosine-15N5 as the internal standard, and analyze by LC-MS/MS to quantify the exact concentration of adenosine in the medium during the experiment, accounting for potential degradation by adenosine deaminase in the culture medium.
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| Animal Protocol |
A typical in vivo animal protocol for Adenosine-15N5 is used as a tracer in a pharmacokinetic study of adenosine metabolism. Use male Sprague-Dawley rats (250-300 g, n = 4-6 per time point). Administer a single intravenous bolus dose of unlabeled adenosine (0.5 mg/kg) via the tail vein, along with a tracer dose of Adenosine-15N5 (0.05 mg/kg) to follow metabolic fate. Collect blood samples via jugular vein catheter at various time points (0, 0.5, 1, 2, 5, 10, 15, 20, 30, 60 seconds and 2, 5, 10, 20, 40, 60 minutes) into tubes containing erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, an adenosine deaminase inhibitor) to prevent ex vivo degradation. Immediately centrifuge at 4degC to obtain plasma. Also collect urine and tissue samples (liver, heart, brain) at terminal time points. For bioanalysis, spike plasma, urine, and tissue homogenates (50 uL) with additional internal standard if needed. Precipitate proteins with acetonitrile, centrifuge, and analyze the supernatant by LC-MS/MS. Calculate the pharmacokinetic parameters (Cmax, Tmax, AUC, t½, clearance, volume of distribution) and metabolic transformation rate from the tracer data. This protocol is used to study adenosine disposition in normal and disease states (e.g., ischemia, inflammation).
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| ADME/Pharmacokinetics |
The pharmacokinetics of Adenosine-15N5 are identical to its unlabeled parent, adenosine. Adenosine is one of the most rapidly cleared endogenous compounds in the human body. Following intravenous administration, plasma half-life in humans is extremely short (<10 seconds, typically 1-5 seconds) due to rapid cellular uptake via equilibrative nucleoside transporters (ENT1 and ENT2) and concentrative nucleoside transporters (CNTs), as well as extensive metabolism. The volume of distribution (Vd) is approximately 0.6 L/kg. Adenosine is metabolized by two primary pathways: (1) deamination by adenosine deaminase (ADA) to inosine (major pathway, >90%), which is further metabolized to hypoxanthine, xanthine, and uric acid; and (2) phosphorylation by adenosine kinase to AMP. The labeled Adenosine-15N5 allows for highly accurate quantification of these metabolic products, enabling detailed mapping of adenosine disposition and metabolic flux. As a tracer, it is critical for studying conditions associated with altered adenosine metabolism, including ischemia, inflammation, and cancer.
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| Toxicity/Toxicokinetics |
Adenosine-15N5 is a stable isotope-labeled research compound, not a drug. Its unlabeled parent, adenosine, is an endogenous nucleoside with known physiological effects and a very short half-life. Adenosine is generally considered safe for intravenous administration at therapeutic doses (6-12 mg bolus for tachycardia). Common side effects include flushing, chest pain, dyspnea, and transient heart block. Adenosine is contraindicated in patients with second- or third-degree AV block, sick sinus syndrome, or known hypersensitivity. No significant toxicity has been reported in animal studies at doses up to 100 mg/kg (i.v.). Adenosine has no known genotoxic or carcinogenic potential. For laboratory handling of Adenosine-15N5, standard safety precautions (gloves, lab coat) are sufficient. The compound should be stored as a powder at -20degC in a tightly sealed container, protected from light and moisture. When in solution, it should be used immediately or stored at -80degC to minimize degradation. It is for research use only and not for human diagnostic or therapeutic applications.
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| References | |
| Additional Infomation |
Adenosine-15N5 is the stable isotope-labeled version of adenosine, where all five nitrogen atoms in the adenine base have been replaced with nitrogen-15 (¹⁵N). It is intended for research use as an internal standard for the accurate quantification of adenosine and for metabolic flux studies in nucleotide metabolism and purinergic signaling using LC-MS or GC-MS. Adenosine is a ubiquitous endogenous nucleoside and an essential component of ATP, RNA, and DNA. It also acts as a signaling molecule via four adenosine receptors (A1, A2A, A2B, A3) that regulate numerous physiological processes including cardiac function, sleep, inflammation, and neurotransmission. Adenosine levels are tightly regulated and increase during stress or tissue injury. This labeled compound is a critical tool for researchers studying adenosine metabolism, purinergic signaling, and related disorders. For research use only.
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| CAS # |
168566-57-2
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| Related CAS # |
Adenosine;58-61-7;Adenosine-13C5;159496-13-6;Adenosine-1′-13C;201996-55-6;Adenosine-13C;54447-57-3;Adenosine-d2;82741-17-1;Adenosine-d;109923-50-4;Adenosine-2′-13C;714950-52-4;Adenosine-3′-13C;714950-53-5;Adenosine-d-1;119540-53-3;Adenosine-d-2;Adenosine-13C10,15N5;202406-75-5
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| Appearance |
White to off-white solid powder
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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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