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| 1mg |
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| Other Sizes |
| Targets |
Duocarmycins
The Val-Cit (valine-citrulline) dipeptide linker is a substrate for cathepsin B, a lysosomal protease that is highly expressed in many cancer cells. Upon internalization of the ADC into cancer cells via receptor-mediated endocytosis, the linker is cleaved by cathepsin B in the lysosome, releasing the active Duocarmycin payload. Duocarmycin targets DNA in the nucleus, alkylating adenine residues and causing DNA damage, cell cycle arrest, and apoptosis. |
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| ln Vitro |
In vitro, Duocarmycin TM, the payload component, is a highly potent DNA alkylating agent with picomolar to low nanomolar IC50 values against various cancer cell lines. The Fmoc-Val-Cit-PAB linker is stable in plasma but is efficiently cleaved by cathepsin B in the acidic lysosomal environment. The full drug-linker conjugate is inactive until the linker is cleaved, ensuring targeted delivery. In ADC format, the conjugate shows potent cytotoxicity only in cells expressing the target antigen and containing active cathepsin B.
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| ln Vivo |
In vivo, Duocarmycin-based ADCs incorporating Val-Cit-PAB linkers have demonstrated potent antitumor efficacy in xenograft mouse models of various cancers. The Fmoc-Val-Cit-PAB-Duocarmycin TM conjugate, when attached to a tumor-targeting antibody, shows selective tumor accumulation, reduced systemic toxicity compared to the free payload, and dose-dependent tumor growth inhibition or regression. This linker-payload combination is used in preclinical ADC development studies.
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| Enzyme Assay |
Cell-free enzyme assays: Incubate purified cathepsin B (activated with DTT) with the Fmoc-Val-Cit-PAB-Duocarmycin TM conjugate (1-100 uM) in acetate buffer (pH 5.0) containing 1 mM EDTA and 5 mM DTT for 1-24 hours at 37degC. Quench the reaction, extract the released Duocarmycin TM, and analyze by LC-MS or HPLC to monitor linker cleavage. Measure the rate of payload release by quantifying Duocarmycin peak area over time. For stability studies, incubate the conjugate in human plasma (pH 7.4) for up to 48 hours and measure intact conjugate and released payload.
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| Cell Assay |
Culture antigen-positive cancer cell lines (e.g., HER2-positive cells for HER2-targeted ADCs, or a generic target of choice) in appropriate medium. For cytotoxicity assays, treat cells with increasing concentrations of antibody-Fmoc-Val-Cit-PAB-Duocarmycin ADC (0.1 pM-100 nM) or with the free Fmoc-Val-Cit-PAB-Duocarmycin TM conjugate (as a non-targeted control) for 72-120 hours. Assess cell viability using CellTiter-Glo (ATP quantification) or MTT. For internalization and processing studies, treat cells with fluorescently labeled ADC (e.g., Alexa Fluor 488-conjugated antibody) and visualize by confocal microscopy. Co-stain with lysotracker to confirm lysosomal localization. Treat cells with ADC (10-100 nM, 4-24 hours), wash, fix, permeabilize, and stain with anti-cathepsin B and anti-Duocarmycin antibodies to monitor linker cleavage and payload release. For DNA damage studies, treat cells with ADC for 4-24 hours, then stain for gamma-H2AX (a marker of DNA double-strand breaks) by immunofluorescence and quantify foci per nucleus. For apoptosis assays, treat cells for 48-72 hours and stain with Annexin V-FITC and propidium iodide for flow cytometry.
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| Animal Protocol |
For in vivo antitumor efficacy studies, use subcutaneous xenograft models in immunodeficient mice (e.g., NSG, nude mice). Inoculate antigen-positive cancer cells (e.g., 5 × 10^6 cells per mouse). When tumors reach approximately 100-150 mm3, randomize mice into treatment groups (n=8-10 per group). Administer the ADC (antibody conjugated to Fmoc-Val-Cit-PAB-Duocarmycin TM) via intravenous injection at doses of 1-10 mg/kg (based on antibody content), once weekly for 2-4 doses. Control groups include vehicle, isotype control ADC, and free Duocarmycin TM (administered at maximum tolerated dose). Measure tumor volume by caliper bi-weekly and calculate tumor growth inhibition (TGI %). Monitor body weight for toxicity. At the end of the study (typically when control tumors reach 1500-2000 mm3), collect tumors for ex vivo analysis: IHC for proliferation (Ki-67), apoptosis (cleaved caspase-3), and DNA damage (gamma-H2AX). Collect blood for PK analysis and serum cytokine assessment. For survival studies, continue dosing and monitoring until humane endpoints, and plot Kaplan-Meier survival curves. For biodistribution studies, use an ADC labeled with near-infrared dye (e.g., IRDye 800CW). Administer to mice, image at multiple time points (1, 24, 48, 72, 96, 120 hours post-injection) using an IVIS imaging system. At final time point, euthanize, harvest major organs and tumor, measure fluorescence ex vivo, and calculate % injected dose per gram of tissue (%ID/g).
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| ADME/Pharmacokinetics |
The Fmoc-Val-Cit-PAB linker has a molecular weight of approximately 553.6 g/mol (Fmoc-Val-Cit-PAB-OH). Duocarmycin TM is a highly potent DNA alkylator with a molecular weight of approximately 500-600 g/mol. The conjugate (Fmoc-Val-Cit-PAB-Duocarmycin TM) has a molecular weight of approximately 1050.59. The Val-Cit dipeptide linker is stable in circulation (plasma half-life >24 hours) but is cleaved efficiently (t1/2 < 1 hour) in the presence of cathepsin B at lysosomal pH (5.0). The linker's stability in circulation minimizes premature payload release and systemic toxicity. The drug-linker conjugate is typically soluble in DMSO. Storage: -20degC, protected from light and moisture, under inert gas. In solution, store at -80degC for extended periods.
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| Toxicity/Toxicokinetics |
Duocarmycin TM is a highly potent DNA alkylator with significant toxicity if released systemically. In ADC format, toxicity is reduced due to targeted delivery. The toxicity profile of the free payload includes myelosuppression, gastrointestinal toxicity, and hepatotoxicity at supratherapeutic doses. For the linker-payload conjugate alone (without antibody targeting), significant toxicity is expected due to non-specific uptake. Animal studies must be conducted with appropriate safety protocols and waste disposal. The compound is for research use only and not for human use. Use appropriate personal protective equipment (gloves, lab coat, safety glasses) and handle in a chemical fume hood. Duocarmycins are potent genotoxins; refer to institutional guidelines for handling highly potent compounds. Dispose of all materials contaminated with Duocarmycin as hazardous chemical waste. In case of skin contact, wash thoroughly with soap and water; if eye contact, rinse with water for 15 minutes and seek medical attention.
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| Additional Infomation |
Fmoc-Val-Cit-PAB-Duocarmycin TM is a drug-linker conjugate designed for the construction of antibody-drug conjugates (ADCs) for targeted cancer therapy research. The Duocarmycin family of natural products is derived from Streptomyces species. Duocarmycins exert their cytotoxic effect by binding to the minor groove of DNA and alkylating adenine at the N3 position, leading to cell death. The Fmoc-Val-Cit-PAB linker is a cathepsin-cleavable linker that has been widely used in ADCs. The Fmoc (9-fluorenylmethyloxycarbonyl) group protects the N-terminus during synthesis. The Val-Cit dipeptide is recognized and cleaved by cathepsin B in the lysosome. The PAB (para-aminobenzyl alcohol) spacer undergoes 1,6-elimination after cleavage to release the free payload.
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| Molecular Formula |
C58H60CLN7O10
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| Molecular Weight |
1050.59
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| Appearance |
Typically exists as solid at room temperature
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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 (~95.18 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 | 0.9518 mL | 4.7592 mL | 9.5185 mL | |
| 5 mM | 0.1904 mL | 0.9518 mL | 1.9037 mL | |
| 10 mM | 0.0952 mL | 0.4759 mL | 0.9518 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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