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| Targets |
Boc-Val-Cit-OH is a cleavable ADC linker that targets the lysosomal protease cathepsin B. The Val-Cit peptide sequence is specifically recognized and cleaved by cathepsin B, which is predominantly active in the acidic environment of lysosomes and is often upregulated in malignant tissues. This enzymatic cleavage triggers the intracellular release of the conjugated payload, such as a cytotoxic drug, specifically within target cells. The Boc protecting group provides stability during synthesis and can be removed under acidic conditions for further functionalization. This linker enables targeted drug delivery by ensuring that the active pharmaceutical ingredient is released only after internalization into the target cell, thereby minimizing systemic toxicity.
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| ln Vitro |
An ADC cytotoxin is connected to an antibody by use of an ADC linker to form an ADC.
In vitro, Boc-Val-Cit-OH functions as a cleavable linker component in ADC constructs, enabling selective payload release upon cathepsin B-mediated cleavage. Studies have demonstrated that ADCs incorporating the Val-Cit linker exhibit potent cytotoxicity against cancer cell lines that overexpress cathepsin B, while showing reduced activity in cathepsin B-negative cells. The linker's stability in plasma and its efficient cleavage in the lysosomal compartment contribute to the selective killing of tumor cells. This in vitro selectivity is a critical factor in the design of effective ADCs, as it allows for the targeted delivery of highly potent cytotoxic agents directly to cancer cells while sparing normal tissues. |
| ln Vivo |
In vivo, Boc-Val-Cit-OH-based ADCs have shown significant antitumor efficacy in xenograft mouse models. The cleavable nature of the Val-Cit linker ensures that the cytotoxic payload is released preferentially within the tumor microenvironment following cathepsin B-mediated cleavage. This targeted release mechanism results in enhanced tumor growth inhibition and improved survival rates compared to non-cleavable linkers. Additionally, the use of this linker has been associated with a favorable safety profile, as the systemic release of the payload is minimized, thereby reducing off-target toxicities. These in vivo findings support the clinical utility of Val-Cit-based linkers in ADC development.
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| Enzyme Assay |
In vitro enzyme/receptor binding assays for Boc-Val-Cit-OH typically involve evaluating its cleavage by cathepsin B in a cell-free system. The assay is performed by incubating the linker or a linker-payload conjugate with purified recombinant cathepsin B in an acidic buffer (pH 5.5) at 37°C for varying time points. The reaction is quenched, and the extent of cleavage is analyzed by high-performance liquid chromatography (HPLC) or mass spectrometry to quantify the release of the payload. The specificity of cleavage can be confirmed by using cathepsin B inhibitors such as CA-074, which should block the cleavage, or by performing the assay in neutral pH where cathepsin B is inactive. This protocol is essential for validating the functionality of the linker in ADC design.
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| Cell Assay |
In vitro cellular assays for Boc-Val-Cit-OH involve the use of cancer cell lines to assess the cytotoxicity and specificity of ADCs containing this linker. Typically, cells are treated with varying concentrations of the ADC or a linker-payload conjugate for 72-96 hours, and cell viability is measured using an MTT or CellTiter-Glo assay. To confirm cathepsin B-dependent activity, parallel experiments can be performed using cathepsin B-knockout cells or in the presence of a cathepsin B inhibitor such as CA-074. The IC50 values are calculated to determine the potency of the conjugate. Additionally, intracellular localization of the payload can be monitored using fluorescently labeled analogues to confirm lysosomal uptake and cleavage.
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| Animal Protocol |
In vivo animal studies for Boc-Val-Cit-OH-based ADCs are commonly conducted using immunocompromised mice bearing subcutaneous human tumor xenografts. Tumor-bearing mice are randomized into treatment and control groups and administered the ADC via intravenous injection at various doses. Tumor volumes and body weights are measured twice weekly to monitor antitumor efficacy and toxicity. At the end of the study, tumors and major organs are collected for histopathological analysis and to determine the concentration of the released payload in tumor tissue versus plasma. Pharmacokinetic parameters such as half-life and clearance are also evaluated from plasma samples collected at multiple time points post-administration.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of Boc-Val-Cit-OH-based ADCs are influenced by the overall stability of the conjugate in circulation. The Val-Cit linker is designed to be stable in plasma, minimizing premature payload release and thereby prolonging the half-life of the ADC. Upon internalization into target cells and cleavage by cathepsin B, the payload is released intracellularly. The pharmacokinetic profile is typically characterized by a biphasic elimination curve, with an initial distribution phase followed by a slower elimination phase. The area under the curve (AUC) and clearance rates are determined by the physicochemical properties of the ADC, including the linker's hydrophilicity and the nature of the payload.
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| Toxicity/Toxicokinetics |
The toxicity profile of Boc-Val-Cit-OH-based ADCs is primarily associated with the off-target release of the cytotoxic payload. In preclinical studies, adverse effects such as weight loss, hepatotoxicity, and myelosuppression have been observed at high doses. However, the cleavable Val-Cit linker is designed to minimize systemic toxicity by ensuring that the payload is released predominantly within the tumor microenvironment. The use of this linker has been shown to improve the therapeutic index compared to non-cleavable linkers, as it reduces nonspecific uptake and release in normal tissues. Toxicity studies in animal models typically monitor body weight, clinical signs, and hematological parameters.
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| References | |
| Additional Infomation |
Boc-Val-Cit-OH is a widely used cleavable ADC linker that capitalizes on the tumor-specific overexpression of cathepsin B. It has been incorporated into multiple ADCs that have entered clinical trials, demonstrating its translational potential. The linker's stability in circulation and efficient cleavage in lysosomes make it a preferred choice for delivering potent payloads such as auristatins and maytansinoids. Its well-characterized properties and ease of synthesis have established it as a standard tool in the field of targeted cancer therapy. Ongoing research continues to explore novel applications of this linker in combination therapies and next-generation ADC designs.
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| Molecular Formula |
C16H30N4O6
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|---|---|
| Molecular Weight |
374.432604312897
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| Exact Mass |
374.216
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| CAS # |
870487-08-4
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| PubChem CID |
59841868
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
624.8±55.0 °C at 760 mmHg
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| Flash Point |
331.7±31.5 °C
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| Vapour Pressure |
0.0±3.9 mmHg at 25°C
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| Index of Refraction |
1.504
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| LogP |
0.66
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
26
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| Complexity |
515
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CC(C)[C@@H](C(=O)N[C@@H](CCCNC(=O)N)C(=O)O)NC(=O)OC(C)(C)C
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| InChi Key |
ZZLNUAVYYRBTOZ-QWRGUYRKSA-N
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| InChi Code |
InChI=1S/C16H30N4O6/c1-9(2)11(20-15(25)26-16(3,4)5)12(21)19-10(13(22)23)7-6-8-18-14(17)24/h9-11H,6-8H2,1-5H3,(H,19,21)(H,20,25)(H,22,23)(H3,17,18,24)/t10-,11-/m0/s1
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| Chemical Name |
(2S)-5-(carbamoylamino)-2-[[(2S)-3-methyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]butanoyl]amino]pentanoic acid
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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 | 2.6707 mL | 13.3536 mL | 26.7073 mL | |
| 5 mM | 0.5341 mL | 2.6707 mL | 5.3415 mL | |
| 10 mM | 0.2671 mL | 1.3354 mL | 2.6707 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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