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Purity: ≥98%
MMAD (also known as Monomethyl auristatin D), an auristatin analog, is a novel and highly potent tubulin inhibitor/antimitotic agent that is often used as a warhead or a toxin payload in antibody drug conjugates (ADCs). Antibodies covalently linked to extremely powerful drugs through a range of conjugation technologies are known as antibody-drug conjugates (ADCs). When used as therapeutics, they combine the cytotoxic drug's capacity to kill cells with the exceptional specificity of antibodies, which allows for the differentiation between healthy and diseased tissue. With two US Food and Drug Administration-approved ADCs now on the market (Adcetris and Kadcyla) and about 40 more undergoing clinical evaluation, this potent and exciting class of targeted therapy has shown considerable promise in the treatment of various cancers. The majority of these ADCs, however, are heterogeneous mixtures, which can have significant pharmacokinetic effects and lead to a limited therapeutic window. Sophisticated site-specific conjugation technologies to link the drug and antibody are essential for ADCs to perform to the best of their abilities.
| Targets |
Auristatin; The primary target of MMAD is tubulin. As a microtubule-disrupting agent, MMAD inhibits tubulin polymerization, disrupting microtubule dynamics and preventing microtubule formation, thereby inhibiting cell division. This action leads to G2/M cell cycle arrest and induces rapid apoptosis in cancer cells. As a member of the auristatin family, MMAD's specific binding to tubulin makes it an ideal choice as a highly potent toxin payload in ADC drug development.
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| ln Vitro |
MMAD (Monomethyl Dolastatin 10) is joined via a steady oxime-ligation procedure to produce a number of nearly homogenous drug-to-antibody conjugates (ADCs) with an approximate drug-to-antibody ratio of 2.0. The resultant conjugates exhibit strong in vitro cytotoxic activity against HER2+ cancer cells along with good pharmacokinetic characteristics. Site-specific unnatural amino acid-based ADCs are demonstrated to exhibit higher in vitro cytotoxicity in comparison to ADCs made by cysteine alkylation after native interchain disulfide reduction[1].
MMAD exhibits extremely potent cytotoxic activity against various cancer cell lines in vitro. In HER2-positive breast cancer cells, MMAD demonstrates picomolar GI₅₀ values: 0.09 nM against MDA-MB-361 cells and 0.12 nM against BT-474 cells. In benchmark comparisons with novel auristatin analogs, MMAD shows a GI₅₀ of 0.12 nM in BT474 cells, approximately 1.7-fold more potent than PF-06380101. When conjugated via site-specific conjugation technologies, MMAD-conjugated ADCs demonstrate increased in vitro cytotoxicity compared to those prepared by conventional cysteine alkylation methods. |
| ln Vivo |
In rodents, the resultant antibody-drug conjugates (ADCs) show total tumor regression. Additionally, they have a better toxicology profile in rats [1].
MMAD-conjugated ADCs demonstrate significant therapeutic efficacy in animal models. Studies have shown that MMAD-ADCs prepared via site-specific conjugation technologies induce complete tumor regression in rodents. When using multivalent drug linker technologies, MMAD achieves drug-to-antibody ratios of up to 10 with low aggregation, and the in vitro potency of the resulting ADCs scales proportionally with drug loading. However, it should be noted that MMAD undergoes site-dependent C-terminal degradation in mouse plasma, which may impact ADC potency in vivo; degradation is minimal in rat plasma and not detected in cynomolgus monkey or human plasma. |
| Enzyme Assay |
The binding affinity of MMAD to tubulin can be assessed using tubulin polymerization inhibition assays. Purified tubulin (bovine brain-derived or recombinant human) is mixed with various concentrations of MMAD in tubulin polymerization buffer containing GTP (e.g., 80 mM PIPES pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP). Tubulin polymerization is continuously monitored at 37°C using a fluorescence microplate reader (excitation 360-380 nm, emission 440-460 nm) or by measuring the increase in absorbance at 340 nm. The polymerization inhibition rate is calculated, and IC₅₀ values are obtained by non-linear regression fitting. Colchicine or paclitaxel can be used as positive controls.
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| Cell Assay |
Exponentially growing HER2-positive breast cancer cells (e.g., BT-474, MDA-MB-361, or NCI-N87 cells) are seeded into 96-well culture plates at appropriate densities (5,000-10,000 cells/well) in medium containing 10% fetal bovine serum and cultured overnight. The following day, various concentrations of MMAD or MMAD-ADC (0.0001-100 nM) are added and incubated for 72-96 hours. Cell viability is assessed using MTS or CellTiter-Glo luminescent assays, with absorbance measured at 490 nm or 570 nm using a microplate reader. Cell viability percentages are calculated, and GI₅₀ values are obtained by fitting concentration-response curves using GraphPad Prism. Each experiment includes three replicate wells and is independently repeated three times.
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| Animal Protocol |
Six-to-eight-week-old female immunodeficient mice (e.g., BALB/c nude or NSG mice) are subcutaneously inoculated with HER2-positive tumor cells (e.g., BT-474 or NCI-N87 cells, 5×10⁶ cells/100 μL PBS). When tumor volumes reach approximately 100-150 mm³, animals are randomly assigned to treatment groups (6-8 mice per group). MMAD-ADCs are administered via tail vein injection at doses ranging from 1-10 mg/kg, once weekly for 2-3 consecutive weeks. Tumor volume (length × width²/2) and body weight are measured 2-3 times per week. At the end of the experiment (approximately 28 days or when tumor volume reaches 2000 mm³), animals are euthanized, and tumor tissues are collected for histopathological analysis and pharmacodynamic evaluation.
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| ADME/Pharmacokinetics |
As a toxin payload for ADCs, the pharmacokinetic properties of MMAD depend on the conjugation method and linker design. When ADCs are prepared via stable oxime-ligation processes, MMAD-ADCs demonstrate good pharmacokinetic properties. MMAD undergoes site-dependent C-terminal degradation in mouse plasma, which may impact ADC potency in vivo; however, this degradation is much less pronounced in rat plasma and is not detected in cynomolgus monkey or human plasma. This degradation can be eliminated by selecting an appropriate antibody conjugation site or modifying the C-terminus of MMAD. The high hydrophobicity of MMAD enables the achievement of drug-to-antibody ratios of up to 10 using multivalent drug linker technologies.
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| Toxicity/Toxicokinetics |
Toxicological data on MMAD are primarily derived from ADC studies. In rodent models, MMAD-ADCs prepared via site-specific conjugation technologies demonstrate improved toxicology profiles. The C-terminal degradation of MMAD in mouse plasma represents an important species difference consideration: this degradation occurs in mice but is minimal in rats and not detected in non-human primates. In in vitro cytotoxicity assays, MMAD exhibits picomolar GI₅₀ values in HER2-positive breast cancer cells, indicating potent cytotoxicity at extremely low concentrations. Unlike other hydrophobic payloads, MMAD maintains low aggregation even at high drug-to-antibody ratios (up to 10) when using multivalent drug linkers. This compound is intended for research use only and is not for human therapeutic applications.
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| References |
| Molecular Formula |
C41H66N6O6S
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|---|---|
| Molecular Weight |
771.0643
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| Exact Mass |
770.476
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| Elemental Analysis |
C, 63.87; H, 8.63; N, 10.90; O, 12.45; S, 4.16
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| CAS # |
203849-91-6
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| Related CAS # |
MMAD-d8
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| PubChem CID |
10723894
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| Appearance |
White to off-white solid powder
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| Density |
1.1±0.1 g/cm3
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| Boiling Point |
906.1±65.0 °C at 760 mmHg
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| Flash Point |
501.8±34.3 °C
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| Vapour Pressure |
0.0±0.3 mmHg at 25°C
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| Index of Refraction |
1.537
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| LogP |
5.82
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
21
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| Heavy Atom Count |
54
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| Complexity |
1190
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| Defined Atom Stereocenter Count |
9
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| SMILES |
S1C([H])=C([H])N=C1[C@]([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C([C@]([H])(C([H])([H])[H])[C@]([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@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])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)OC([H])([H])[H])=O
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| InChi Key |
BLUGYPPOFIHFJS-UUFHNPECSA-N
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| InChi Code |
InChI=1S/C41H66N6O6S/c1-12-27(6)36(46(9)41(51)35(26(4)5)45-39(50)34(42-8)25(2)3)32(52-10)24-33(48)47-21-16-19-31(47)37(53-11)28(7)38(49)44-30(40-43-20-22-54-40)23-29-17-14-13-15-18-29/h13-15,17-18,20,22,25-28,30-32,34-37,42H,12,16,19,21,23-24H2,1-11H3,(H,44,49)(H,45,50)/t27-,28+,30-,31-,32+,34-,35-,36-,37+/m0/s1
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| Chemical Name |
(2S)-N-[(2S)-1-[[(3R,4S,5S)-3-methoxy-1-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino]propyl]pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]-3-methyl-2-(methylamino)butanamide
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| Synonyms |
Monomethylauristatin D; MMAD; 203849-91-6; Monomethylauristatin D; Demethyldolastatin 10; W4ZIA40FZ9; DTXSID101010182; Demethyldolastatin 10; Monomethyl Dolastatin 10
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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: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. (2). 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: ~24.5 mg/mL (~31.8 mM)
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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 | 1.2969 mL | 6.4846 mL | 12.9692 mL | |
| 5 mM | 0.2594 mL | 1.2969 mL | 2.5938 mL | |
| 10 mM | 0.1297 mL | 0.6485 mL | 1.2969 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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