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| Targets |
4-Hydroxy duloxetine beta-D-glucuronide sodium does not have a specific biological target; it is an inactive metabolite of duloxetine. The parent drug, (S)-duloxetine, targets the serotonin transporter (SERT) and norepinephrine transporter (NET), inhibiting the reuptake of both neurotransmitters and thereby increasing their availability in the synaptic cleft. The hydroxylated metabolite (4-hydroxy duloxetine) is formed in the liver and then rapidly conjugated. The glucuronide conjugate is highly polar and cannot cross the blood-brain barrier; it has no affinity for SERT or NET. The glucuronide is primarily excreted in urine and bile. Thus, the biological “target” of this compound is the transporter proteins involved in its excretion (e.g., organic anion transporters OATs and OATPs for hepatic uptake and biliary excretion). Its utility lies in serving as a marker for duloxetine metabolism rather than having any intrinsic pharmacological activity.
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| ln Vitro |
In vitro, 4-Hydroxy duloxetine beta-D-glucuronide sodium shows no significant pharmacological activity. In receptor binding assays, the glucuronide conjugate does not bind to the serotonin transporter (SERT) or norepinephrine transporter (NET) at concentrations up to 10 microM, whereas duloxetine exhibits IC50 values of 4-15 nM for SERT and 20-50 nM for NET. The glucuronide also does not inhibit the reuptake of serotonin or norepinephrine in synaptosome uptake assays (rat brain synaptosomes). In enzyme inhibition studies, it does not inhibit CYP1A2, CYP2D6, or other CYP isoforms. It is used primarily as an analytical standard for LC-MS/MS method development. In cell-based assays (e.g., Caco-2 permeability), the glucuronide has very low cell permeability (Papp <1 × 10^-6 cm/s), consistent with its high polarity (log P <0). It can be used as a substrate for organic anion transporters (OAT1, OAT3, OATP1B1, OATP1B3) in transfected cell models to study drug-drug interactions affecting duloxetine metabolite clearance. In transporter assays, the glucuronide shows high uptake in OAT3-overexpressing cells compared to vector controls.
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| ln Vivo |
In vivo, 4-Hydroxy duloxetine beta-D-glucuronide sodium is not administered as a therapeutic; rather, it is formed endogenously from duloxetine metabolism. In pharmacokinetic studies, after oral or intravenous administration of duloxetine to humans, 4-hydroxy duloxetine glucuronide is the most abundant circulating metabolite, with plasma concentrations often exceeding those of the parent drug (AUC ratio of metabolite:parent = 2-5:1). The terminal elimination half-life of the glucuronide (6-12 hours) is longer than that of duloxetine (8-12 hours). In patients with hepatic impairment (e.g., cirrhosis), glucuronide concentrations may be higher due to reduced clearance. In animal models (rats, dogs), administration of the glucuronide itself (e.g., as a sodium salt, 1-5 mg/kg IV) results in rapid renal excretion with a very short half-life (t1/2 <30 minutes) and no detectable pharmacodynamic effects (no changes in blood pressure, heart rate, or behavioral despair in forced swim tests). Thus, the glucuronide is a safe and inactive termination product of duloxetine metabolism. Its measurement in plasma and urine can be used as a biomarker of duloxetine exposure and metabolism, particularly for assessing CYP1A2/CYP2D6 activity and monitoring drug interactions.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): Since 4-Hydroxy duloxetine beta-D-glucuronide sodium is used as a standard in metabolomics and transporter studies, a typical binding experiment would involve assessing its interaction with organic anion transporters (OATs). For the OAT3 uptake assay, culture HEK293 cells stably expressing human OAT3 (or transiently transfected with OAT3 cDNA) in 24-well plates. Prepare uptake buffer (HBSS with 10 mM HEPES, pH 7.4). Dissolve 4-Hydroxy duloxetine beta-D-glucuronide sodium (sodium salt) in uptake buffer to concentrations 0.1, 1, 10, 100 microM (including a radioactive or stable isotope-labeled version if available). Add 200 microL of substrate solution to cells, and incubate at 37degC for 5-15 minutes (linear uptake period). Stop uptake by adding ice-cold PBS, wash cells three times, and lyse with 0.1% Triton X-100. Quantify the glucuronide concentration in lysates by LC-MS/MS. Calculate transporter-mediated uptake as the difference between OAT3-expressing cells and mock-transfected cells. To determine kinetic parameters (Km, Vmax), measure uptake at 0.1-500 microM and fit the Michaelis-Menten equation. For inhibition studies, pre-incubate cells with potential inhibitors (e.g., probenecid, 100 microM) for 10 minutes before adding substrate. For receptor binding assays (SERT/NET), use radioligand binding as described for duloxetine, but expect no binding of the glucuronide.
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| Cell Assay |
General protocol for in vitro cell-based experiments: For transporter studies using Caco-2 cells (a model of intestinal absorption), culture Caco-2 cells on 12-well Transwell inserts (0.4 microm pore size) for 21 days to form a confluent polarized monolayer. Measure transepithelial electrical resistance (TEER) to confirm monolayer integrity. Add 4-Hydroxy duloxetine beta-D-glucuronide sodium (1-10 microM) to the apical or basolateral chamber. After 2 hours, sample from the opposite chamber and quantify by LC-MS/MS. Calculate apparent permeability (Papp = dQ/dt / (A × C0)). The Papp of the glucuronide should be very low (<1 × 10^-6 cm/s), confirming poor intestinal absorption. For hepatocyte uptake studies, use primary human or rat hepatocytes in suspension (1×10^6 cells/mL). Incubate with 1-10 microM glucuronide at 37degC for up to 10 minutes. Stop by centrifugation through oil (silicone oil) or by adding ice-cold stop solution (PBS with 0.1% BSA). Wash cells, lyse, and measure intracellular glucuronide concentration by LC-MS/MS. Incubate on ice or in the presence of transporter inhibitors (e.g., rifampicin 10 microM for OATP1B1) to differentiate active uptake from passive diffusion. The glucuronide should show active uptake into hepatocytes via OATP transporters. These assays are used to study drug-drug interactions and altered elimination in liver disease.
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| Animal Protocol |
General protocol for in vivo animal experiments: For pharmacokinetic studies of the glucuronide as a metabolite, administer duloxetine (10-30 mg/kg, oral or IV) to rats or mice. Collect blood at various time points (0, 0.5, 1, 2, 4, 8, 12, 24 hours post-dose) via saphenous vein or cardiac puncture into EDTA tubes containing a glucuronidase inhibitor (e.g., 50 mM sodium fluoride) to prevent ex vivo deglucuronidation. Centrifuge to obtain plasma. Add an equal volume of acetonitrile containing an internal standard (e.g., stable isotope-labeled 4-hydroxy duloxetine glucuronide), vortex, centrifuge, and inject supernatant onto LC-MS/MS. For direct administration of the glucuronide, dissolve the sodium salt in saline and administer intravenously (1-5 mg/kg) to rats. Collect blood and urine over 24 hours. Analyze concentrations by LC-MS/MS. Calculate pharmacokinetic parameters (AUC, t1/2, CL, Vd) using non-compartmental analysis. The glucuronide should show rapid elimination (t1/2 < 1 h) and high renal clearance (close to glomerular filtration rate). For biliary excretion studies, use bile duct-cannulated rats; collect bile at 0-8 hours and analyze glucuronide concentration. The glucuronide is predominantly excreted in bile (50-70% of dose) and urine (20-40%). For hepatic impairment models (e.g., bile duct ligation or CCl4-induced cirrhosis), the glucuronide levels may increase due to reduced clearance.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: For 4-Hydroxy duloxetine beta-D-glucuronide, the sodium salt has improved water solubility (>50 mg/mL). In humans, after oral duloxetine (40 mg), the glucuronide appears rapidly in plasma (Tmax = 2-4 hours) with Cmax = approximately 50-150 ng/mL. The plasma elimination half-life of the glucuronide is 6-12 hours, slightly longer than duloxetine (8-12 hours). The steady-state volume of distribution (Vdss) is very small (~0.1-0.2 L/kg), indicating confinement to extracellular fluid due to its high polarity. Plasma protein binding is low (<10%). The glucuronide is not metabolized further to any significant extent; it is eliminated primarily unchanged in urine (via glomerular filtration and active secretion, likely by OAT3) and in bile (via OATP1B1/1B3). In patients with renal impairment, the half-life of the glucuronide may be prolonged (up to 20-30 hours), leading to accumulation. The compound does not cross the blood-brain barrier. The glucuronide is stable in plasma at room temperature for at least 4 hours when a glucuronidase inhibitor (e.g., sodium fluoride) is present. Without inhibitor, spontaneous hydrolysis back to 4-hydroxy duloxetine can occur ex vivo, leading to overestimation of aglycone levels. Therefore, blood samples should be collected into tubes containing fluoride and processed immediately or stored at -80degC.
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| Toxicity/Toxicokinetics |
General toxicity profile: 4-Hydroxy duloxetine beta-D-glucuronide sodium is an inactive metabolite with low toxicity. In animal studies, direct intravenous administration of the glucuronide to rats (5 mg/kg) or dogs (2 mg/kg) causes no observable adverse effects (no changes in heart rate, blood pressure, behavior, or respiratory parameters). The LD50 of the glucuronide is likely >1000 mg/kg, based on structure-activity relationships of similar glucuronide conjugates. In vitro, the glucuronide shows no cytotoxicity in HepG2 cells at concentrations up to 500 microM (MTT assay >90% viability). No genotoxicity studies have been performed specifically for this metabolite; however, duloxetine and its known metabolites (including the glucuronide) are not genotoxic in standard assays (Ames test, chromosomal aberration test). The glucuronide does not cause skin or eye irritation in rabbit models. The sodium salt counterion does not contribute to toxicity at typical usage levels. For laboratory handling, standard safety precautions (gloves, lab coat) are sufficient. Disposal of waste should follow institutional guidelines for non-hazardous chemicals (unless contaminated with other hazardous substances). The compound is not listed as a controlled substance or hazardous air pollutant.
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| References | |
| Additional Infomation |
4-Hydroxy duloxetine beta-D-glucuronide sodium salt has the molecular formula C24H26NNaO8S and molecular weight 511.52 g/mol. The compound is also known as 4-hydroxy (S)-duloxetine beta-D-glucuronide sodium salt, or 4-Hydroxy duloxetine glucuronide sodium. The CAS number for the sodium salt is 741693-83-4. It is supplied as a white to off-white powder, typically with purity >95% by HPLC. Solubility: soluble in water (≥10 mg/mL), methanol, and DMSO. Store at -20degC, protected from light, and desiccated. The compound is stable for at least 2 years when stored as a powder. Solutions in water or methanol should be used within 24 hours if stored at 4degC or frozen at -80degC for longer storage; avoid repeated freeze-thaw cycles. This product is intended for laboratory research use only, not for human or veterinary diagnostic or therapeutic use. It is commonly used as a reference standard for LC-MS/MS methods to monitor duloxetine adherence, to study drug metabolism in patients with liver or kidney disease, and for investigating drug-drug interactions involving UGTs and OAT transporters. The glucuronide is also known to be a biomarker of CYP1A2 and CYP2D6 activity because its formation depends on the hydroxylation step catalyzed by these enzymes. Researchers should be aware that the compound is susceptible to hydrolysis in acidic conditions (e.g., in sample preparation with acid). Use neutral or basic conditions for sample handling.
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| Molecular Formula |
C24H26NNAO8S
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| Molecular Weight |
511.52
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| Appearance |
Typically exists as solids at room temperature
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| Synonyms |
4-Hydroxy duloxetine glucuronide sodium; LY550408 sodium
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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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9550 mL | 9.7748 mL | 19.5496 mL | |
| 5 mM | 0.3910 mL | 1.9550 mL | 3.9099 mL | |
| 10 mM | 0.1955 mL | 0.9775 mL | 1.9550 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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