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| 1mg |
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| Targets |
(rac)-3-O-Methyl DOPA-d3 is a stable isotope-labeled internal standard. Its unlabeled parent, 3-O-Methyl DOPA (3-OMD), is not a pharmacologically active drug itself but is the primary and major circulating metabolite of the anti-Parkinson's medication L-DOPA. The target of this metabolite is not a classical receptor; rather, it is implicated as a potential competitor for L-DOPA transport into the brain. 3-OMD shares the same large neutral amino acid transporter (LAT1) that transports L-DOPA across the blood-brain barrier. Elevated plasma levels of 3-OMD can accumulate in patients on long-term L-DOPA therapy (especially those on high doses without a COMT inhibitor) and may reduce the amount of L-DOPA that enters the brain by competitive inhibition, potentially contributing to the “wearing-off” phenomenon. 3-OMD is also a marker of peripheral COMT activity. The deuterated version is used as a tracer to study these transport mechanisms and to accurately quantify 3-OMD levels in biological matrices. 3-OMD has no intrinsic dopaminergic activity.
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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 effect on a drug's pharmacokinetics and metabolic properties, it is a cause for concern [1].
The in vitro biological activity of (rac)-3-O-Methyl DOPA-d3 is not directly studied; it is used as an internal standard. Its unlabeled parent, 3-O-Methyl DOPA (3-OMD), has been studied in brain capillary endothelial cell lines (e.g., RBE4 cells, a model of the blood-brain barrier). In uptake assays using radiolabeled [3H]-L-DOPA or [14C]-3-OMD, unlabeled 3-OMD (10-500 uM) inhibits the uptake of L-DOPA in a concentration-dependent manner, with an IC₅0 of approximately 50-100 uM. This is because both L-DOPA and 3-OMD compete for the same large neutral amino acid transporter (LAT1) on the luminal and abluminal membranes of the BBB. In COMT enzyme assays using purified rat liver COMT and S-adenosylmethionine (SAM) as the methyl donor, 3-OMD is not a substrate itself (it is the methylated product of L-DOPA). However, it can be further methylated to 3,4-dimethoxyphenylalanine (DOPAC) by COMT at a much slower rate. In neuronal cell cultures (e.g., SH-SY5Y), 3-OMD (up to 500 uM) does not activate dopamine receptors and shows no neurotoxic or neuroprotective effects on its own. The labeled (rac)-3-O-Methyl DOPA-d3 is used as an internal standard to accurately quantify 3-OMD levels in cell culture media. |
| ln Vivo |
The in vivo activity of (rac)-3-O-Methyl DOPA-d3 is not evaluated; it is an internal standard. Its unlabeled parent, 3-O-Methyl DOPA (3-OMD), is an endogenous compound produced from L-DOPA by COMT. In animal models of Parkinson's disease (e.g., 6-OHDA-lesioned rats), co-administration of L-DOPA with unlabeled 3-OMD is used to study the effect of high 3-OMD levels on L-DOPA's central efficacy. Studies have shown that increasing plasma 3-OMD levels (by co-administering entacapone, a COMT inhibitor, which paradoxically can alter 3-OMD) can affect brain L-DOPA uptake. Specifically, high peripheral 3-OMD levels (achieved by administering large doses of exogenous 3-OMD along with L-DOPA) compete with L-DOPA for brain uptake, reducing striatal dopamine levels and L-DOPA's motor effect. This observation supports the hypothesis that 3-OMD accumulation contributes to the loss of L-DOPA efficacy over time. 3-OMD itself has no intrinsic motor activity. (rac)-3-O-Methyl DOPA-d3 is used as an internal standard to accurately quantify the concentrations of both L-DOPA and 3-OMD in plasma and brain tissue in these studies.
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| Enzyme Assay |
A generic non-cell-based assay for (rac)-3-O-Methyl DOPA-d3 involves its use as an internal standard in an LC-MS/MS method for quantifying 3-O-Methyl DOPA in human plasma. Prepare a standard stock solution of unlabeled (rac)-3-O-Methyl DOPA in 0.1 M HCl (1 mg/mL). Prepare a separate stock solution of the internal standard (rac)-3-O-Methyl DOPA-d3 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 5 to 2,000 ng/mL. Add a fixed concentration of the internal standard (e.g., 200 ng/mL) to each calibration standard. For sample preparation, add 200 uL of acetonitrile containing 0.1% formic acid to 50 uL of plasma to precipitate proteins. Vortex and centrifuge at 12,000g for 10 minutes. Transfer the supernatant to an autosampler vial. Analyze by LC-MS/MS in positive ion mode. Monitor the mass transitions: m/z 212 → 166 (loss of CH2O from the methoxy group) for 3-O-Methyl DOPA, and m/z 215 → 169 for the deuterated internal standard. Alternatively, monitor m/z 212 → 180 (loss of CH3OH from the carboxylic acid) and m/z 215 → 183 for the labeled analog. Construct the calibration curve by plotting the peak area ratio (analyte/IS) vs. the nominal concentration. This method is routinely used in clinical pharmacology studies of L-DOPA.
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| Cell Assay |
A standard in vitro cell-based protocol for the unlabeled 3-O-Methyl DOPA is used to study its effect on L-DOPA uptake across the blood-brain barrier using the RBE4 cell line (rat brain endothelial cells). Culture RBE4 cells in DMEM/F12 supplemented with 10% FBS, 1% penicillin/streptomycin, and 1 ng/mL bFGF, at 37degC in a 5% CO2 incubator, in collagen-coated flasks. Seed cells on collagen-coated 12-well Transwell filter inserts (0.4 um pore size) at a density of 1×10⁵ cells/well to form a confluent monolayer. Allow the cells to differentiate for 5-7 days until transepithelial electrical resistance (TEER) reaches >150 omega·cm2, indicating formation of a tight barrier. On the day of the assay, wash the inserts and wells with HBSS buffer. Add HBSS containing 10 uM L-DOPA (unlabeled) to the apical (upper) chamber, and add buffer with or without varying concentrations (10, 50, 100, 250 uM) of unlabeled 3-O-Methyl DOPA to both chambers. Incubate at 37degC for 30-60 minutes. Sample the basolateral (lower) chamber at various time points. Quantify L-DOPA transport by LC-MS/MS using (rac)-3-O-Methyl DOPA-d3 as internal standard. Calculate the apparent permeability coefficient (Papp, cm/s) and the percentage inhibition of L-DOPA transport by 3-OMD.
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| Animal Protocol |
A typical in vivo animal protocol for (rac)-3-O-Methyl DOPA-d3 is used in a rat model to study L-DOPA pharmacokinetics. Use male Sprague-Dawley rats (250-300 g, n = 5-6 per group). Administer a single oral dose of unlabeled L-DOPA (10 mg/kg) combined with carbidopa (2.5 mg/kg, a peripheral DOPA decarboxylase inhibitor) via gavage. In the treatment group, co-administer unlabeled (rac)-3-O-Methyl DOPA (50 mg/kg) to elevate plasma 3-OMD levels, simulating chronic L-DOPA therapy. Include a control group receiving L-DOPA + carbidopa + vehicle. Collect blood samples via tail vein at various time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24 h) into heparinized tubes. Immediately centrifuge to obtain plasma. At the terminal time point (e.g., 2 or 4 hours post-dose for brain sampling), euthanize separate groups (n = 3-4) and collect whole brain, dissect the striatum, and homogenize in 0.4 M perchloric acid. For bioanalysis, spike plasma and striatal homogenate samples (50 uL) with a fixed amount of (rac)-3-O-Methyl DOPA-d3 internal standard. Precipitate proteins with acetonitrile, centrifuge, and analyze the supernatant by LC-MS/MS. Quantify both L-DOPA and 3-OMD levels. Calculate the striatal-to-plasma concentration ratio of L-DOPA in each group. This protocol is used to investigate the impact of elevated 3-OMD on central L-DOPA delivery.
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| ADME/Pharmacokinetics |
(rac)-3-O-Methyl DOPA-d3 is an analytical internal standard. Its unlabeled parent, 3-O-Methyl DOPA (3-OMD), is the major circulating metabolite of L-DOPA. In humans, following oral administration of L-DOPA (with carbidopa), plasma 3-OMD levels reach peak concentrations (Cmax) of 2-5 uM (approximately 400-1,000 ng/mL) within 2-4 hours, which is typically 5-10 times higher than the Cmax of the parent L-DOPA (0.5-1 uM). The half-life of 3-OMD in plasma is 2-4 hours in healthy subjects. In patients with Parkinson's disease on chronic L-DOPA therapy, 3-OMD may accumulate to higher steady-state concentrations (up to 10-20 uM) due to long-term dosing and potential reductions in renal clearance with age. 3-OMD is primarily eliminated via renal excretion; approximately 60-80% of a radiolabeled dose is recovered in urine as 3-OMD and its conjugates. The labeled (rac)-3-O-Methyl DOPA-d3 is critical for the accurate quantification of 3-OMD in clinical and preclinical PK studies.
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| Toxicity/Toxicokinetics |
(rac)-3-O-Methyl DOPA-d3 is a research-grade stable isotope-labeled compound, not a therapeutic agent. Its unlabeled parent, 3-O-Methyl DOPA (3-OMD), is an endogenous metabolite of L-DOPA and is generally considered safe at physiological levels. In normal subjects, plasma 3-OMD concentrations are usually below detection or very low (<0.1 uM). In Parkinson‘s patients on chronic L-DOPA therapy, 3-OMD can accumulate to much higher concentrations (up to 10-20 uM). Although some studies have suggested that high 3-OMD levels may compete with L-DOPA for transport into the brain, direct toxicity from 3-OMD is not well-documented. Animal studies involving high-dose 3-OMD administration (200 mg/kg i.p.) did not produce significant behavioral or biochemical signs of toxicity (e.g., no alterations in locomotor activity or striatal dopamine levels). No carcinogenicity or genotoxicity data are available for 3-OMD. For laboratory handling, standard safety precautions (gloves, lab coat, eye protection) are sufficient. (rac)-3-O-Methyl DOPA-d3 should be stored as a powder at -20degC in a tightly sealed container, protected from light and moisture. It is for research use only, not for human consumption.
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| References | |
| Additional Infomation |
(rac)-3-O-Methyl DOPA-d3 is the stable isotope-labeled version of (rac)-3-O-Methyl DOPA (3-OMD), the primary circulating metabolite of L-DOPA (levodopa), the standard of care for the symptomatic treatment of Parkinson's disease. It is intended for research use as an internal standard for the accurate quantification of 3-OMD and L-DOPA in biological samples by LC-MS/MS. 3-OMD is formed by the action of catechol-O-methyltransferase (COMT) on L-DOPA in the periphery. Elevated levels of 3-OMD have been hypothesized to compete with L-DOPA for transport across the blood-brain barrier, potentially contributing to the loss of L-DOPA efficacy over time (the “wearing-off” phenomenon). The labeled compound is a critical analytical tool for pharmacokinetic studies of L-DOPA, for assessing COMT activity, and for research into the optimization of Parkinson's disease therapy. 3-OMD is also a marker of COMT activity and is used in studies involving COMT inhibitors like entacapone. For research use only, not for diagnostic or therapeutic applications.
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| Molecular Formula |
C10H13NO4
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|---|---|
| Molecular Weight |
214.233008146286
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| Exact Mass |
214.103
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| CAS # |
1219173-95-1
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| Related CAS # |
3-O-Methyldopa-d3;586954-09-8;3-O-Methyldopa-d3 hydrate
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| PubChem CID |
46782235
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
-2.4
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
15
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| Complexity |
222
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(O)(=O)C(N)CC1=CC=C(O)C(OC([2H])([2H])[2H])=C1
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| InChi Key |
PFDUUKDQEHURQC-FIBGUPNXSA-N
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| InChi Code |
InChI=1S/C10H13NO4/c1-15-9-5-6(2-3-8(9)12)4-7(11)10(13)14/h2-3,5,7,12H,4,11H2,1H3,(H,13,14)/i1D3
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| Chemical Name |
2-amino-3-[4-hydroxy-3-(trideuteriomethoxy)phenyl]propanoic 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: Please store this product in a sealed and protected environment (e.g. under nitrogen), 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) |
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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.6679 mL | 23.3394 mL | 46.6788 mL | |
| 5 mM | 0.9336 mL | 4.6679 mL | 9.3358 mL | |
| 10 mM | 0.4668 mL | 2.3339 mL | 4.6679 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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