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| Targets |
(E)-4-Hydroxytamoxifen-d5 targets the estrogen receptor (ER) as it is a deuterated form of (E)-4-Hydroxytamoxifen, an estrogen receptor modulator. The parent compound (E)-4-Hydroxytamoxifen is a less active isomer of (Z)-4-hydroxytamoxifen, which is the active metabolite of tamoxifen. It binds to estrogen receptors, interfering with the normal binding of estrogen and thereby modulating estrogen-dependent gene expression. As an internal standard, its binding properties are identical to its non-deuterated counterpart but are not utilized for pharmacological effect.
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| ln Vitro |
Stable heavy isotopes of hydrogen, carbon, and other elements have been incorporated into drug molecules, largely as tracers for quantitation during the drug development process. Studies involving the human use of drugs labeled with deuterium suggest that these compounds may offer some advantages when compared with their nondeuterated counterparts. Deuteration has gained attention because of its potential to affect the pharmacokinetic and metabolic profiles of drugs. Deutetrabenazine is the first deuterated drug to receive Food and Drug Administration approval. This deuterated form of the drug tetrabenazine is indicated for the treatment of chorea associated with Huntington's disease as well as tardive dyskinesia. Ongoing clinical trials suggest that a number of other deuterated compounds are being evaluated for the treatment of human diseases and not merely as research tools.[1]
In vitro studies of the unlabeled parent compound show that (E/Z)-4-hydroxy Tamoxifen is cytotoxic to MCF-7 and MDA-MB-231 breast cancer cells, with IC50 values of 27 and 18 uM, respectively. It also stimulates LC3 lipidation and the formation of autophagic vesicles in MCF-7 cells in a superoxide-dependent manner. As the deuterated internal standard, (E)-4-Hydroxytamoxifen-d5 is not typically used to assess biological activity but to accurately quantify the concentration of its non-deuterated counterpart in complex biological matrices. |
| ln Vivo |
Deuterated compounds may, in some cases, offer advantages over nondeuterated forms, often through alterations in clearance. Deuteration may also redirect metabolic pathways in directions that reduce toxicities. The approval of additional deuterated compounds may soon follow. Clinicians will need to be familiar with the dosing, efficacy, potential side effects, and unique metabolic profiles of these new entities.
Unlabeled tamoxifen and its metabolites are active in vivo. Tamoxifen is a well-established anti-estrogen drug used for the treatment of breast cancer. It is also a potent activator of Hsp90 and enhances the Hsp90 molecular chaperone ATPase activity. For (E)-4-Hydroxytamoxifen-d5, in vivo activity data are not reported. This deuterated form is used as a tracer to study the pharmacokinetic and metabolic profiles of the active drug without being the primary pharmacological agent. |
| Enzyme Assay |
For competitive binding assays of estrogen receptors, a typical protocol involves incubating ERalpha or ERbeta protein (from baculovirus expression system) with 0.5-2 nM [3H]-estradiol in assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 0.1% BSA) at 4degC for 12-16 hours. Increasing concentrations of unlabeled competitor (0.1 nM to 10 uM) are added. Bound and free radioligand are separated by dextran-coated charcoal or filtration. Nonspecific binding is determined in the presence of 500-fold excess unlabeled estradiol. Scatchard analysis calculates Kd and Bmax. For MS-based quantification using the deuterated IS, the compound is spiked into samples prior to extraction.
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| Cell Assay |
Cell culture studies are typically performed on ER-positive MCF-7 cells. Cells are maintained in phenol red-free DMEM with 10% charcoal-stripped FBS for 48 hours. For cytotoxicity assays, cells are seeded in 96-well plates and treated with varying concentrations of compound for 72 hours. Cell viability is measured by MTT assay. For receptor binding, cells are lysed and protein extracts are analyzed. (E)-4-Hydroxytamoxifen-d5 would be used at tracer levels (1-100 ng/mL) as an internal standard in LC-MS analysis of cell lysates or culture medium, added prior to sample preparation to correct for analyte loss during extraction.
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| Animal Protocol |
Animal study protocols for the non-deuterated tamoxifen are well-established. For in vivo efficacy studies in breast cancer xenograft models, 5-6 week old female athymic nude mice are subcutaneously implanted with 1×10⁷ MCF-7 cells. When tumors reach 100-200 mm3, tamoxifen citrate (5-50 mg/kg) is administered daily via oral gavage for 4-6 weeks. Tumor volume is measured bi-weekly. For pharmacokinetic studies of tamoxifen, plasma is collected at various time points (0, 1, 2, 4, 6, 8, 12, 24 hours post-dose). A fixed amount of (E)-4-Hydroxytamoxifen-d5 (10-100 ng/mL) is added as internal standard for LC-MS quantification.
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| ADME/Pharmacokinetics |
As an internal standard, no dedicated PK data for (E)-4-Hydroxytamoxifen-d5 are available. However, the pharmacokinetics of tamoxifen and its active metabolite 4-hydroxytamoxifen are well characterized. Tamoxifen is well absorbed after oral administration (bioavailability ~100%), with peak plasma concentrations reached at 3-6 hours. It is highly protein bound (>99%). Tamoxifen is extensively metabolized by CYP3A4, CYP2D6, and CYP2C9 to active metabolites including 4-hydroxytamoxifen, which has a higher affinity for the estrogen receptor than the parent compound. The terminal half-life is long (5-7 days for tamoxifen, 14 days for 4-hydroxytamoxifen). Steady state is achieved after approximately 4 weeks.
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| Toxicity/Toxicokinetics |
No specific toxicity data for the deuterated compound (E)-4-Hydroxytamoxifen-d5 are available. The unlabeled parent drug tamoxifen has a well-established safety profile. Common adverse effects include hot flashes, vaginal discharge, and menstrual irregularities. Serious but less common side effects include increased risk of endometrial cancer, venous thromboembolism (deep vein thrombosis and pulmonary embolism), and stroke. Tamoxifen also causes cataracts and other ocular toxicities. Hepatotoxicity (fatty liver, steatohepatitis, and rare cases of cirrhosis) has been reported. Long-term use is associated with these risks.
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| References | |
| Additional Infomation |
(E)-4-Hydroxytamoxifen-d5 is for research use only, not for therapeutic or diagnostic use. It is a stable isotope-labeled compound primarily used as an internal standard for quantitative LC-MS analysis of tamoxifen and its active metabolites in pharmacokinetic studies, drug metabolism research, and clinical monitoring. The unlabeled (Z)-4-hydroxytamoxifen is a first-line drug for estrogen receptor-positive breast cancer treatment. Although the unlabeled parent has been approved by regulatory agencies (FDA), the deuterated form is strictly a research tool. The deuteration provides a mass increase of 5 Da, allowing precise quantification without interference from endogenous compounds.
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| Molecular Formula |
C26H24D5NO2
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| Molecular Weight |
392.54
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| Appearance |
Solid powder
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| Synonyms |
(E)-Afimoxifene-d5
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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) |
DMSO : 100 mg/mL (254.75 mM; with sonication)
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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 | 2.5475 mL | 12.7376 mL | 25.4751 mL | |
| 5 mM | 0.5095 mL | 2.5475 mL | 5.0950 mL | |
| 10 mM | 0.2548 mL | 1.2738 mL | 2.5475 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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