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| Targets |
The target of NH2-C6-ARC186 sodium is complement component C5, a central protein in the complement cascade. ARC186 functions by binding to C5 and blocking its proteolytic activation by the C5 convertase enzyme complex. By inhibiting the cleavage of C5, it prevents the downstream formation of the potent anaphylatoxin C5a and the membrane attack complex (MAC, C5b-9). C5a is a powerful inflammatory mediator that recruits and activates immune cells, while the MAC is directly responsible for lysing pathogens and cells. Inhibiting C5 is a validated strategy for treating complement-mediated diseases such as paroxysmal nocturnal hemoglobinuria (PNH). The NH2-C6 linker provides a conjugation handle, allowing the aptamer to be linked to targeting moieties (e.g., antibodies, peptides) for site-specific delivery or to surfaces for biosensing applications.
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| ln Vitro |
In vitro, ARC186 is a highly potent inhibitor of complement activation. Classical pathway hemolytic assays using antibody-sensitized sheep red blood cells (EA cells) and human serum complement demonstrate that ARC186 can inhibit hemolysis with an IC50 in the low nanomolar range. The aptamer effectively prevents the formation of the membrane attack complex (MAC) on the surface of target cells. It also blocks the generation of the anaphylatoxin C5a in a concentration-dependent manner. The NH2-C6 modification at the 5‘ end of the aptamer does not interfere with its binding to C5 or its inhibitory activity, as the modification is introduced at a site that is typically not involved in target recognition. This makes it an ideal tool for creating conjugates that retain full biological activity.
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| ln Vivo |
No specific in vivo data for this conjugated aptamer is provided in the search results, but the in vivo activity of the parent ARC186 is known. In models of complement-mediated disease, such as the reverse passive Arthus reaction in rats or the hemolytic anemia model in mice, systemic administration of ARC186 has been shown to significantly reduce tissue damage, inflammation, and hemolysis. It demonstrates a dose-dependent inhibition of complement activity (measured by CH50 or hemolytic assays) in the serum of treated animals. Unlike small molecule inhibitors, aptamers are subject to nuclease degradation, but modifications like the C6 linker can enhance their stability in serum. The NH2-C6 linker provides a site for conjugating to PEG or other moieties to extend its circulation half-life, making it a versatile research tool.
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| Enzyme Assay |
A cell-free hemolysis assay is the standard protocol to measure C5 inhibition. In a 96-well plate, antibody-sensitized sheep red blood cells (EA cells, 1×10⁸ cells/mL) are prepared in gelatin veronal buffer (GVB++). For the assay, 50 uL of EA cells are mixed with 50 uL of a diluted human serum complement source (e.g., 1:10 dilution in GVB++) and 50 uL of serial dilutions of NH2-C6-ARC186 sodium (0.01-1000 nM). The plate is incubated at 37degC for 30 minutes with gentle shaking. The reaction is stopped by placing the plate on ice and adding 200 uL of cold PBS. The plate is then centrifuged at 1500g for 5 minutes. 100 uL of the supernatant is transferred to a new 96-well plate, and the absorbance of the released hemoglobin is measured at 541 nm using a spectrophotometer. The percentage of hemolysis is calculated relative to a control (water, 100% lysis). The IC50 is determined by plotting the percent inhibition versus the log concentration of the inhibitor.
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| Cell Assay |
A cell-based complement activation assay can be performed using human whole blood or isolated cells. For a C5a generation assay, human whole blood is collected in sodium citrate tubes. The blood is diluted 1:1 with PBS. 200 uL of diluted blood is added to each well of a 96-well plate. NH2-C6-ARC186 sodium (0.1-1000 nM) is added to the wells and incubated for 15 minutes at 37degC. Complement activation is then triggered by adding an activator, such as LPS (lipopolysaccharide) or zymosan (10-100 ug/mL). The plate is further incubated at 37degC for 30-60 minutes. The reaction is stopped by adding EDTA (final 10 mM) to chelate calcium and stop complement activation. The samples are centrifuged to pellet cells. The supernatant is collected, and the concentration of C5a is quantified using a human C5a ELISA kit according to the manufacturer's instructions. A reduction in C5a levels in the presence of the aptamer indicates inhibition of C5 cleavage.
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| Animal Protocol |
A mouse model of complement-mediated injury, such as the reverse passive Arthus reaction, can be used to evaluate the in vivo efficacy of this conjugate. In this model, 6-8 week old male C57BL/6 mice are used. On day 0, the dorsal skin is shaved. Mice are injected intravenously with the test compound (NH2-C6-ARC186 sodium, 0.1-10 mg/kg) or a control. Fifteen minutes later, an intradermal injection of 50 ug of an anti-ovalbumin (anti-OVA) antibody in 50 uL PBS is administered into the dorsal skin. Immediately after, the mice are injected intravenously with 2 mg of OVA antigen (to form immune complexes). After 4-6 hours, the mice are euthanized, and the skin is removed. The local inflammatory response is quantified by measuring the wet weight of the skin lesions (edema) and by measuring myeloperoxidase (MPO) activity in the skin homogenates as an index of neutrophil infiltration. The NH2-C6-ARC186 group is expected to show a significant reduction in edema and MPO activity compared to controls.
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| ADME/Pharmacokinetics |
NH2-C6-ARC186 sodium is an aptamer (nucleic acid) with an approximate molecular weight of 12881.00 g/mol. It is provided as a lyophilized powder and should be stored at -20degC, sealed, and protected from moisture, where it is stable for up to 3 years. It is soluble in water at 100 mg/mL (7.76 mM) and in PBS. It is anionic and soluble in aqueous solutions. As a nucleic acid, it is sensitive to nuclease degradation; therefore, it is recommended to use nuclease-free water and to minimize repeated freeze-thaw cycles. For in vivo studies, solutions are typically prepared in saline or PBS. The compound has a high molecular weight and negative charge, which reduces renal filtration and can prolong its half-life compared to small molecules, though it will eventually be cleared by the liver and kidneys.
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| Toxicity/Toxicokinetics |
This product is for research use only and is not for human diagnostic or therapeutic use. No specific toxicity data is available for this modified aptamer. The sodium salt form is generally considered safe in research settings. However, as with all nucleic acids, standard laboratory safety precautions should be followed to avoid inhalation or skin contact. The complement system is a crucial part of innate immunity, and systemic inhibition of C5 can increase susceptibility to certain bacterial infections (particularly Neisseria meningitidis). Therefore, while this is a research tool, these potential on-target effects should be kept in mind when designing in vivo experiments. The NH2-C6 linker is a standard chemical modification that is generally non-toxic.
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| References | |
| Additional Infomation |
ARC186 is an aptamer therapeutic that has been developed for the treatment of complement-related disorders. The NH2-C6 modification provides a versatile conjugation site for coupling the aptamer to other functional molecules, such as polyethylene glycol (PEG) to improve pharmacokinetics, targeting ligands for cell-specific delivery, or fluorophores for imaging. This makes it a valuable modular platform for research. The parent aptamer (ARC186) specifically binds to C5 and blocks its activation by convertases. The WO2006088888A2 patent describes the use of aptamer therapeutics for treating complement-related disorders.
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| Molecular Weight |
12881.00
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| Appearance |
White to off-white 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, avoid exposure to moisture. |
| 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) |
H2O :~100 mg/mL (~7.76 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 | 0.0776 mL | 0.3882 mL | 0.7763 mL | |
| 5 mM | 0.0155 mL | 0.0776 mL | 0.1553 mL | |
| 10 mM | 0.0078 mL | 0.0388 mL | 0.0776 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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