![NH2-bicyclo[1.1.1]pentane-7-MAD-MDCPT hydrochloride](/upload/1124/V76704.png)
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| Targets |
The primary target of this compound is topoisomerase I, a nuclear enzyme that is essential for DNA replication and transcription. It acts as a topoisomerase I poison/stabilizer. By binding to and stabilizing the transient topoisomerase I-DNA cleavage complex, it prevents the re-ligation of the DNA strand. This results in the accumulation of persistent DNA single-strand breaks. During replication, these single-strand breaks are converted into lethal double-strand breaks, leading to cell cycle arrest and ultimately apoptosis. It is structurally related to the camptothecin class of topoisomerase I inhibitors. The rigid bicyclo[1.1.1]pentane linker is designed to improve the drug-to-antibody ratio (DAR) and to prevent premature release of the payload in circulation, thereby reducing off-target toxicity.
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| ln Vitro |
In vitro, the free payload (NH2-bicyclo[1.1.1]pentane-7-MAD-MDCPT) is a potent topoisomerase I inhibitor. Its activity is typically measured in cancer cell proliferation assays. When delivered as part of an ADC (conjugated to a targeting antibody), it exhibits potent and specific cytotoxicity against cells that express the target antigen. IC50 values for the ADC are often in the low nanomolar or picomolar range on antigen-positive cancer cells, while being significantly less toxic (10 to 100-fold higher IC50) to antigen-negative cells, demonstrating the 'bystander effect‘ or targeted delivery. The payload itself, in its free (unconjugated) form, is highly cytotoxic to all dividing cells. Its in vitro activity is independent of the antigen, making the ADC a potent tool.
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| ln Vivo |
The in vivo antitumor activity of NH2-bicyclo[1.1.1]pentane-7-MAD-MDCPT has been validated when used as an ADC payload. In mouse xenograft models, ADCs incorporating this payload have been shown to induce complete and durable tumor regressions at well-tolerated doses. For example, in gastric cancer (NCI-N87) or breast cancer (SK-BR-3) xenograft models, treatment with the ADC (1-10 mg/kg, administered intravenously once a week for 2-3 cycles) results in significant tumor growth inhibition (TGI) and improved survival compared to control groups. The rigid bicyclo[1.1.1]pentane linker contributes to the stability of the ADC in circulation, ensuring that the payload is released preferentially within the target tumor cells. The ADC activity is dose-dependent and specific to the antigen expression.
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| Enzyme Assay |
A standard cell-free topoisomerase I inhibition assay is used to confirm the direct activity of the payload. The reaction mixture (20 uL) contains 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 15 ug/mL BSA, 0.5 ug of supercoiled pBR322 plasmid DNA, 1 unit of recombinant human topoisomerase I, and varying concentrations of NH2-bicyclo[1.1.1]pentane-7-MAD-MDCPT hydrochloride (0.001-100 uM). The reaction is incubated at 37degC for 30 minutes. The reaction is stopped by adding 5 uL of a stop buffer containing 5% SDS and 2.5 mg/mL proteinase K, followed by further incubation at 37degC for 15 minutes. The DNA samples are then mixed with 5 uL of 6X DNA loading dye and separated on a 1% agarose gel by electrophoresis. The gel is stained with ethidium bromide and visualized under UV light. The presence of nicked or linear DNA (indicative of cleavage) versus supercoiled DNA (no cleavage) is assessed. The IC50 is the concentration at which 50% of the supercoiled DNA is converted to the nicked form.
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| Cell Assay |
A typical in vitro ADC cytotoxicity assay uses a 96-well plate format. Antigen-positive and antigen-negative cell lines are seeded at 5,000-10,000 cells per well and allowed to adhere overnight. The ADC (e.g., antibody conjugated to this payload) is serially diluted (10-point, 1:5 dilution, from 100 nM to 0.005 nM) and added to the cells. The plates are incubated for 72-96 hours at 37degC. Cell viability is then assessed using a CellTiter-Glo® Luminescent Cell Viability Assay according to the manufacturer's protocol. Luminescence is read on a plate reader. The percentage of viable cells is calculated relative to untreated control wells. The IC50 is determined by plotting the log concentration of the ADC vs. the cell viability using non-linear regression. Specificity is confirmed when the IC50 is at least 100-fold lower on antigen-positive cells compared to antigen-negative cells. The free payload (unconjugated) should be similarly cytotoxic to both cell lines.
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| Animal Protocol |
The in vivo efficacy is typically evaluated using a subcutaneous xenograft model in immunodeficient mice (e.g., BALB/c nude or NSG mice, 6-8 weeks old). 5×10⁶ tumor cells (e.g., NCI-N87 gastric cancer or SK-BR-3 breast cancer cells) are injected into the flank. Once tumors reach an average volume of 150-250 mm3, the mice are randomized into groups (n=8-10 per group). They receive the ADC (e.g., conjugated to trastuzumab for HER2-targeting) at various dose levels (e.g., 1, 3, 10 mg/kg) by intravenous injection via the tail vein. The dosing schedule is typically a single dose or once weekly for 2-3 weeks. Tumor volumes are measured twice a week with calipers and calculated as (length × width2)/2. Body weight is monitored as an indicator of tolerability. At the end of the study, tumors are harvested and analyzed for target expression, proliferation markers (Ki-67), and apoptosis (cleaved caspase-3) by IHC. The anti-tumor activity is measured as Tumor Growth Inhibition (TGI%) and/or tumor regression events. A TGI of >100% indicates tumor regression.
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| ADME/Pharmacokinetics |
NH2-bicyclo[1.1.1]pentane-7-MAD-MDCPT hydrochloride has a molecular formula of C26H24ClN3O6 and a molecular weight of 509.94 g/mol. The lyophilized powder should be stored at -20degC, where it is stable for up to 3 years. For solution storage, it should be stored at -80degC for up to 1 year. It is recommended to prepare stock solutions in DMSO. The hydrochloride salt form is used to enhance the aqueous solubility of the basic payload. The compound is soluble in DMSO (e.g., 10-20 mg/mL) and is intended for conjugation to antibodies via the primary amine group. Once conjugated, the ADC is formulated in a buffered solution (e.g., PBS) for in vivo administration. The compound is a potent cytotoxin and should be handled with extreme care.
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| Toxicity/Toxicokinetics |
This product is for research use only and is strictly not for human use. As a highly potent topoisomerase I inhibitor, it is a cytotoxic agent and should be handled as a hazardous chemical. Direct exposure must be avoided. The hydrochloride salt may cause skin and eye irritation. Use appropriate personal protective equipment (PPE), including gloves, lab coat, and safety glasses, and work in a certified chemical fume hood. In vivo, the on-target toxicity of the free payload includes myelosuppression (decrease in white blood cells, platelets), alopecia (hair loss), and gastrointestinal damage, similar to other topoisomerase I inhibitors. The ADC format is designed to deliver the payload specifically to cancer cells, thereby significantly reducing these off-target toxicities at therapeutic doses. The compound is an ADC cytotoxin and a topoisomerase I inhibitor.
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| References | |
| Additional Infomation |
This compound is classified as an ADC Cytotoxin and a Topoisomerase I inhibitor. It is a derivative of camptothecin, incorporating a novel rigid bicyclo[1.1.1]pentane spacer (7-MAD-MDCPT) to optimize the ADC‘s stability and activity. The NH2 group allows for efficient conjugation to antibodies via standard amide bond formation (using NHS esters or similar coupling chemistry). This payload can be used to construct ADCs for a wide range of cancer targets. It is an important research tool for advancing the field of targeted cancer therapy and for evaluating the ADC's potential for the bystander effect, where the payload can diffuse out of the target cell to kill neighboring cancer cells. It is not a clinically approved drug and is intended for research purposes only.
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| Molecular Formula |
C26H24CLN3O6
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| Molecular Weight |
509.94
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| Related CAS # |
NH2-bicyclo[1.1.1]pentane-7-MAD-MDCPT;2857037-69-3
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| Appearance |
Yellow to brown solid 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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 (~196.10 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 | 1.9610 mL | 9.8051 mL | 19.6102 mL | |
| 5 mM | 0.3922 mL | 1.9610 mL | 3.9220 mL | |
| 10 mM | 0.1961 mL | 0.9805 mL | 1.9610 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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