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| ln Vitro |
Targeting antibodies with strong small-molecule payloads are combined to form antibody-drug conjugates (ADC). On the anaplastic large cell lymphoma line L-82, ADCs are designed to target distinct receptors. However, they all deliver the same cytotoxic payload, monomethyl auristatin E, or MMAE. Independent of target expression or drug:antibody ratios, the intracellular concentration of released MMAE correlated with in vitro ADC-mediated cytotoxicity. Using a parallel cohort of L-82 tumors under the same treatment protocol, the concentration of MMAE is measured by LC-MS. Two patterns are seen in the intratumoral MMAE measurement three days after dosage, despite the fact that tumor volume is not varied between treatment groups. In order to achieve more anticancer effect, the intratumoral MMAE concentration first rises in direct proportion to the ADC dose. Furthermore, it was observed that the tumor responded equally to both ADCs[1] and that the intratumoral MMAE concentration derived from Treatment with both cOKT9-vcMMAE and cAC10-vcMMAE is similar at each dose.
In vitro studies of D8-MMAE are typically focused on its use as an analytical standard rather than its biological activity. However, as a deuterated analog of MMAE, it is expected to exhibit the same potent antimitotic activity as the parent compound. MMAE is a potent inhibitor of tubulin polymerization and induces cell cycle arrest and apoptosis in cancer cells. In ADC applications, MMAE is released from the antibody-drug conjugate upon internalization into target cells, where it exerts its cytotoxic effects. D8-MMAE can be used in in vitro assays to study the mechanism of action of MMAE and to quantify MMAE levels in biological samples. |
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| ln Vivo |
In tumor xenograft models, intramoral MMAE concentrations are reliably correlated with the degree of tumor growth inhibition. Because nonbinding control treatments do not kill either CD30+ or CD30-Karpas 299 cells, IHC analysis shows that tumors treated with these treatments contain both CD30+ and CD30- cells. Tumors treated with cAC10-vcMMAF only contain CD30- cells, indicating that the majority of CD30+ cells are eliminated by cAC10-vcMMAF. It's interesting to note that by the time the study is over, both of the tumors that relapsed after receiving cAC10-vcMMAE treatment were also discovered to be CD30-, suggesting that some CD30- cells may have managed to elude bystander death in these two surviving tumors[1].
In vivo studies of D8-MMAE are primarily focused on its use as an internal standard for pharmacokinetic studies of MMAE and MMAE-based ADCs. The deuterium labeling allows for accurate quantification of MMAE in plasma, tissues, and other biological matrices using mass spectrometry. D8-MMAE can be co-administered with MMAE or ADC formulations to track drug distribution and metabolism. The compound's biological activity in vivo is expected to be similar to that of MMAE, as the deuterium labeling does not significantly alter its pharmacological properties. D8-MMAE is also used in studies of ADC pharmacology and toxicology. |
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| Enzyme Assay |
For in vitro enzyme/receptor binding assays, D8-MMAE is primarily used as an analytical standard rather than in functional assays. However, the parent compound MMAE can be evaluated in tubulin polymerization assays to measure its ability to inhibit microtubule assembly. These assays typically involve incubating purified tubulin with various concentrations of MMAE and measuring polymerization by turbidimetry or fluorescence. MMAE's binding to tubulin can also be studied using surface plasmon resonance or isothermal titration calorimetry. D8-MMAE may be used in these assays as a control or for method development. Standard assay conditions include physiological buffer systems with appropriate concentrations of GTP, which is required for tubulin polymerization.
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| Cell Assay |
For in vitro cellular experiments, D8-MMAE is used as an analytical standard to quantify MMAE levels in cell culture experiments. The parent compound MMAE is tested in cancer cell lines to evaluate its cytotoxicity, typically using cell viability assays such as MTT or CellTiter-Glo. Cells are cultured in appropriate media and treated with various concentrations of MMAE (typically ranging from picomolar to micromolar). Cell cycle analysis by flow cytometry and apoptosis assays (Annexin V staining, caspase activation) are used to characterize the mechanism of cell death. D8-MMAE may be spiked into cell culture samples as an internal standard for LC-MS/MS analysis of MMAE uptake and metabolism.
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| Animal Protocol |
For in vivo animal experiments, D8-MMAE is primarily used as an internal standard for pharmacokinetic studies of MMAE and MMAE-based ADCs. The compound is administered to animals (typically rodents) along with MMAE or ADC formulations, and blood samples are collected at various time points. Plasma concentrations of MMAE are measured using LC-MS/MS with D8-MMAE as the internal standard. Tissue distribution studies may also be performed. The deuterium-labeled compound allows for accurate quantification of MMAE levels without interference from endogenous compounds. D8-MMAE itself is not typically used as a therapeutic agent in vivo, but rather as a tool for bioanalytical method development.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of D8-MMAE are not separately characterized, as it is used as an internal standard for MMAE pharmacokinetics. MMAE, the parent compound, has a molecular weight of approximately 718 g/mol and is highly potent. As a cytotoxic agent, MMAE is typically administered as part of an ADC to achieve targeted delivery to tumor cells. When released from the ADC, MMAE has a relatively short half-life in circulation. The deuterium labeling of D8-MMAE does not significantly alter its pharmacokinetic properties compared to unlabeled MMAE. The compound is primarily used for analytical purposes to enable accurate quantification of MMAE in biological samples.
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| Toxicity/Toxicokinetics |
Toxicological data for D8-MMAE are limited, as it is primarily used as an analytical standard. The parent compound MMAE is a potent cytotoxic agent that inhibits tubulin polymerization and induces cell death. As such, MMAE has significant toxicity, including myelosuppression, peripheral neuropathy, and gastrointestinal toxicity, which are typical of antimitotic agents. In ADC formulations, the toxicity of MMAE is mitigated by targeted delivery to tumor cells, but off-target toxicity remains a concern. D8-MMAE, as a deuterated analog, would be expected to have similar toxicity to MMAE. Appropriate safety precautions should be taken when handling this compound, including the use of personal protective equipment and adherence to institutional safety guidelines for handling potent cytotoxic agents.
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| References | ||
| Additional Infomation |
D8-MMAE is a deuterium-labeled isotopologue of MMAE used primarily as an internal standard for mass spectrometry-based quantification of MMAE in pharmacokinetic and bioanalytical studies. No clinical trials or regulatory approvals have been reported for D8-MMAE as a therapeutic agent. It is available from various chemical suppliers for research purposes only. The compound is a potent mitotic inhibitor and tubulin inhibitor, and is used as a cytotoxic warhead in ADCs. The deuterium labeling allows for accurate quantification of MMAE in biological samples without interference from endogenous compounds. D8-MMAE is an important tool for ADC development and pharmacokinetic studies.
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| Molecular Formula |
C39H67N5O7
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|---|---|
| Molecular Weight |
726.0279
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| Exact Mass |
725.554
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| CAS # |
2070009-72-0
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| Related CAS # |
Monomethyl auristatin E;474645-27-7
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| PubChem CID |
78357789
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| Appearance |
White to off-white solid powder
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| LogP |
4.1
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
20
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| Heavy Atom Count |
51
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| Complexity |
1100
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| Defined Atom Stereocenter Count |
10
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| SMILES |
O(C([H])([H])[H])[C@]([H])([C@]([H])(C(N([H])[C@]([H])(C([H])([H])[H])[C@]([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])O[H])=O)C([H])([H])[H])[C@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C(C([H])([H])[C@]([H])([C@]([H])([C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])N(C([H])([H])[H])C([C@]([2H])(C([2H])(C([2H])([2H])[2H])C([2H])([2H])[2H])N([H])C([C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C([H])([H])[H])=O)=O)OC([H])([H])[H])=O
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| InChi Key |
DASWEROEPLKSEI-CMHCZSPYSA-N
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| InChi Code |
InChI=1S/C39H67N5O7/c1-13-25(6)34(43(10)39(49)33(24(4)5)42-38(48)32(40-9)23(2)3)30(50-11)22-31(45)44-21-17-20-29(44)36(51-12)26(7)37(47)41-27(8)35(46)28-18-15-14-16-19-28/h14-16,18-19,23-27,29-30,32-36,40,46H,13,17,20-22H2,1-12H3,(H,41,47)(H,42,48)/t25-,26+,27+,29-,30+,32-,33-,34-,35+,36+/m0/s1/i4D3,5D3,24D,33D
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| Chemical Name |
(2S)-2,3,4,4,4-pentadeuterio-N-[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-3-[[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-2-[[(2S)-3-methyl-2-(methylamino)butanoyl]amino]-3-(trideuteriomethyl)butanamide
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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: This product is not stable in solution, please use freshly prepared working solution for optimal results. |
| 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 (~137.74 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.44 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (3.44 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (3.44 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3774 mL | 6.8868 mL | 13.7735 mL | |
| 5 mM | 0.2755 mL | 1.3774 mL | 2.7547 mL | |
| 10 mM | 0.1377 mL | 0.6887 mL | 1.3774 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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