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| 1mg |
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| Targets |
The target of TK216-d4 is the same as that of the parent compound, TK216. It is designed to bind to the EWS-FLI1 fusion protein, a well-known oncogenic driver in Ewing's sarcoma and other cancers characterized by ETS gene rearrangements. Specifically, TK216 directly binds to EWS-FLI1 and inhibits its interaction with RNA helicase A. By blocking this protein-protein interaction, the compound prevents the EWS-FLI1 transcription factor complex from activating the expression of pro-oncogenic genes. The deuterated version retains this binding ability but is not used pharmacologically; instead, it serves as a tracer for the quantification of the parent drug.
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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.
TK216-d4 possesses the same in vitro biochemical activity as the parent drug TK216. It directly binds to the EWS-FLI1 fusion protein and disrupts its interaction with the RNA helicase A (RHA) complex. This inhibition occurs through direct binding to the EWS-FLI1 protein, blocking a critical protein-protein interaction required for transcriptional activity. The IC50 for this inhibitory effect is in the low micromolar range for the unlabeled parent. The deuterated version serves as the analytical counterpart, used as an internal standard in cell-based experiments to quantify the uptake and retention of TK216 in cancer cells following treatment. It is not used as a primary screening tool due to its intended function as an internal standard. |
| 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.
The in vivo activity is demonstrated by the parent compound TK216, not the labeled d4 standard. TK216 is an orally active compound that has shown potent anti-tumor efficacy in preclinical xenograft models of Ewing's sarcoma and other ETS-driven cancers. In these models, oral administration of TK216 leads to significant tumor growth inhibition (TGI) and prolonged survival. The mechanism is validated by demonstrating reduced expression of EWS-FLI1 target genes within the tumor tissue. The labeled internal standard (TK216-d4) is used to support these studies by providing accurate quantification of the drug concentrations in the plasma and tumor tissue of the treated animals, enabling PK/PD relationship analysis. |
| Enzyme Assay |
A specific in vitro binding assay for EWS-FLI1 can be used to characterize the interaction. For TK216, an ELISA-based competition assay is typically performed. Recombinant EWS-FLI1 protein is immobilized on a plate. The plate is incubated with a fixed concentration of a fluorescently labeled probe that binds to EWS-FLI1, along with varying concentrations of TK216 or TK216-d4. After washing, the fluorescence signal is measured. A decrease in signal indicates that the test compound displaces the probe from the protein, confirming direct binding. The IC50 of TK216-d4 is expected to be nearly identical to that of the parent compound due to the minimal kinetic isotope effect of deuterium on such a large protein-protein interaction interface. A typical concentration range for such an assay is 0.1 nM to 100 microM.
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| Cell Assay |
TK216-d4 is not typically used as a treatment in cell-based activity assays; rather, the parent TK216 is the active agent. For evaluating the cellular activity of TK216, Ewing's sarcoma cell lines (e.g., A673, SK-ES-1) that harbor the EWS-FLI1 fusion gene are seeded in 96-well plates. Cells are treated with varying concentrations of the test compound (0.1 nM to 10 microM) for 72 hours. Cell viability is measured by CellTiter-Glo or MTT assay. Apoptosis is assessed by flow cytometry using Annexin V/PI staining. RT-qPCR is used to measure the expression of EWS-FLI1 target genes (e.g., NR0B1, PPP1R1A). These assays demonstrate the anti-proliferative and pro-apoptotic effects of the parent drug. The labeled version is added to the lysates as an internal standard for compound quantification.
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| Animal Protocol |
In vivo animal studies are performed with the parent TK216 to assess efficacy, while TK216-d4 is used for bioanalysis. For a xenograft model, female NOD/SCID or athymic nude mice (6-8 weeks old) are injected subcutaneously with 5 × 10⁶ A673 cells. When tumors reach a volume of 100-200 mm3, mice are randomized into treatment groups (n=8-10 per group). TK216 is formulated in a vehicle like 0.5% methylcellulose or 10% DMSO/40% PEG300/50% water. The compound is administered orally at doses ranging from 10-50 mg/kg once or twice daily for 3-4 weeks. Tumor volumes are measured bi-weekly, and blood samples are collected for PK analysis. For PK, TK216-d4 is added to the plasma samples as an internal standard before LC-MS/MS analysis. This allows for the generation of plasma concentration-time curves and calculation of key PK parameters like Cmax, Tmax, and AUC.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of TK216-d4 are expected to be identical to those of the parent drug TK216 due to the minimal isotope effect. TK216 is an orally bioavailable compound with moderate to good absorption in preclinical species. Following oral administration in rodents, it exhibits a Tmax of approximately 1-2 hours and a terminal half-life (t1/2) of 3-6 hours. The compound undergoes hepatic metabolism, primarily via CYP3A4. As a stable isotope-labeled version, TK216-d4 is not administered alone for PK studies; it is spiked into the matrix as an internal standard. However, when incorporated into a drug, deuterium can sometimes alter the metabolism rate (i.e., a kinetic isotope effect), though this is often negligible for pharmaceuticals. In this context, it primarily serves as a bioanalytical tool.
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| Toxicity/Toxicokinetics |
TK216-d4 is a labeled chemical and is not used as a drug, so its toxicological profile is directly extrapolated from the parent drug, TK216. In preclinical safety studies, TK216 was generally well-tolerated at efficacious doses. Potential toxicities are expected to be mechanism-based, relating to the inhibition of ETS transcription factors which play normal roles in various biological processes. Off-target effects may include mild gastrointestinal disturbances. No specific toxicity data is published for the deuterated version, but as a stable isotope, the label does not introduce new toxicities. Standard laboratory safety practices should be followed when handling this compound. It is intended for research use only and is not for human consumption. The parent compound is a potential anticancer drug.
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| Additional Infomation |
TK216-d4 has CAS number 3034816-36-6. Its molecular formula is C19H11D4Cl2NO3, and its molecular weight is 380.26 g/mol. It is a deuterium-labeled version of the research compound TK216 . It is typically supplied as a white to off-white solid powder. The compound is stored at -20degC in a sealed, dry container, protected from moisture and light. It is soluble in DMSO, and the concentration of stock solutions should be verified by UV-Vis spectroscopy or LC-MS. The compound is intended for research use only, specifically as an analytical internal standard for mass spectrometry.
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| Molecular Formula |
C19H11D4CL2NO3
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| Molecular Weight |
380.26
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| CAS # |
3034816-36-6
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| Related CAS # |
TK216; (-)-TK216; (+)-TK216
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| Appearance |
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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6298 mL | 13.1489 mL | 26.2978 mL | |
| 5 mM | 0.5260 mL | 2.6298 mL | 5.2596 mL | |
| 10 mM | 0.2630 mL | 1.3149 mL | 2.6298 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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