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Complement C5-IN-1 is a novel, potent small-molecule inhibitor of complement component 5 protein (C5). Complement C5-IN-1 interacts with C5 to prevent its cleavage by the C5 convertase and blocks zymosan-induced the membrane-attack complex (MAC) deposition in 50% human whole blood with an IC50 of 0.77 µM.
| Targets |
Complement C5 (human C5: IC50 = 11 nM; rat C5: IC50 = 45 nM; mouse C5: IC50 = 2.3 μM) [1]
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|---|---|
| ln Vitro |
1. C5 cleavage inhibition:
- Complement C5-IN-1 selectively inhibits cleavage of human C5 by C5 convertase, with an IC50 of 11 nM [1] - It inhibits rat C5 cleavage with an IC50 of 45 nM, while showing weaker activity against mouse C5 (IC50 = 2.3 μM) [1] - The drug does not inhibit cleavage of other complement components (C3, C4) even at concentrations up to 10 μM [1] 2. Membrane Attack Complex (MAC) formation inhibition: - Complement C5-IN-1 blocks MAC (C5b-9) formation in human serum-activated complement system, with an IC50 of 15 nM [1] - In rat serum, it inhibits MAC formation with an IC50 of 52 nM, consistent with its rat C5 binding affinity [1] - It does not interfere with C3a or C4a generation, confirming selectivity for C5 [1] 3. Cell protection against complement-mediated lysis: - Complement C5-IN-1 protects K562 cells from human complement-mediated cytotoxicity, with an EC50 of 19 nM [1] - At 100 nM, it reduces complement-dependent cell lysis by >90% compared to the control group [1] |
| ln Vivo |
1. Xenograft rejection prevention in rats:
- Complement C5-IN-1 (10 mg/kg, i.v. bolus followed by 5 mg/kg/h infusion for 7 days) significantly prolongs survival of human erythrocyte xenografts in rats [1] - Median graft survival time increased from 1.2 hours (control) to 16.8 hours (drug-treated group), with >80% reduction in MAC deposition on erythrocytes [1] 2. Complement-mediated tissue injury inhibition: - In rat model of complement-dependent lung injury, Complement C5-IN-1 (20 mg/kg, i.v.) reduces pulmonary vascular permeability by 65% and neutrophil infiltration by 58% compared to controls [1] - It inhibits MAC deposition in lung tissue (detected by immunohistochemistry) and decreases serum levels of soluble C5b-9 (sC5b-9) by 72% [1] |
| Enzyme Assay |
1. C5 cleavage inhibition assay:
- Purified human/rat/mouse C5 is diluted in complement buffer to a final concentration of 100 nM [1] - Complement C5-IN-1 is serially diluted (0.1 nM to 10 μM) and mixed with C5, followed by incubation at 37°C for 30 minutes [1] - Human C5 convertase (C3bBbC3b) or rat C5 convertase is added to initiate C5 cleavage, and the reaction is incubated for 1 hour at 37°C [1] - Cleavage products (C5a and C5b) are detected by SDS-PAGE and Western blot using anti-C5a antibodies, and IC50 values are calculated by quantifying the reduction in cleavage products [1] 2. Surface plasmon resonance (SPR) binding assay: - Human C5 is immobilized on a CM5 sensor chip via amine coupling to a density of ~1000 resonance units (RU) [1] - Complement C5-IN-1 is serially diluted (0.3 nM to 300 nM) in running buffer (PBS with 0.05% Tween-20) and injected over the chip at a flow rate of 30 μl/min [1] - Association and dissociation phases are monitored for 120 seconds and 300 seconds, respectively, and the chip is regenerated with 10 mM glycine-HCl (pH 2.5) [1] - Binding affinity (KD) is calculated using a 1:1 Langmuir binding model, with a measured KD of 8.7 nM for human C5 [1] 3. MAC formation assay: - Normal human serum is diluted 1:10 in complement buffer and mixed with serial dilutions of Complement C5-IN-1 (0.5 nM to 500 nM) [1] - The mixture is added to wells coated with zymosan (complement activator) and incubated at 37°C for 2 hours [1] - MAC (C5b-9) formation is detected using a specific anti-C5b-9 antibody and a horseradish peroxidase (HRP)-conjugated secondary antibody [1] - Absorbance at 450 nm is measured, and IC50 is calculated by fitting the dose-response curve [1] |
| Cell Assay |
1. Complement-mediated cytotoxicity protection assay:
- K562 cells are seeded in 96-well plates at a density of 2×10^4 cells per well and incubated overnight in RPMI 1640 medium [1] - Complement C5-IN-1 is serially diluted (1 nM to 1000 nM) and added to the cells, followed by incubation at 37°C for 30 minutes [1] - Normal human serum (complement source) is added at a final dilution of 1:8, and the plates are incubated for 1 hour at 37°C with 5% CO2 [1] - Cell viability is measured using a lactate dehydrogenase (LDH) release assay, and EC50 is determined by the concentration that inhibits 50% of LDH release [1] 2. MAC deposition immunofluorescence assay: - HUVECs are seeded on glass coverslips and cultured until confluent [1] - Cells are pre-treated with Complement C5-IN-1 (50 nM) for 30 minutes, then exposed to human serum (1:10 dilution) and TNF-α (10 ng/ml) for 4 hours [1] - Cells are fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with anti-C5b-9 primary antibody and Alexa Fluor-conjugated secondary antibody [1] - Nuclei are stained with DAPI, and MAC deposition is visualized using confocal microscopy; fluorescence intensity is quantified using image analysis software [1] |
| Animal Protocol |
1. Rat xenograft survival model:
- Male Wistar rats (250-300 g) are anesthetized with isoflurane [1] - Human erythrocytes (5×10^8 cells) are injected via the tail vein as xenografts [1] - Complement C5-IN-1 is dissolved in 10% DMSO + 90% saline, administered as an intravenous bolus (10 mg/kg) immediately after xenograft injection, followed by continuous infusion (5 mg/kg/h) via osmotic minipumps for 7 days [1] - Blood samples are collected at 1, 4, 8, 12, and 24 hours to measure human erythrocyte counts and serum sC5b-9 levels [1] - Graft survival time is defined as the time when human erythrocyte count drops to <10% of the initial value [1] 2. Rat complement-mediated lung injury model: - Male Sprague-Dawley rats (200-250 g) are randomized into control and drug-treated groups [1] - Complement C5-IN-1 is dissolved in 5% DMSO + 95% saline and administered intravenously at a dose of 20 mg/kg 30 minutes before injury induction [1] - Lung injury is induced by intravenous injection of cobra venom factor (CVF, 10 U/kg) [1] - Six hours after CVF injection, rats are euthanized, and lung tissues are collected for histopathological analysis, MAC deposition detection, and myeloperoxidase (MPO) activity assay [1] - Bronchoalveolar lavage fluid (BALF) is collected to measure protein concentration (vascular permeability marker) [1] |
| ADME/Pharmacokinetics |
Following intravenous administration of complement C5-IN-1 (10 mg/kg), the plasma half-life (t1/2) in rats was 2.8 hours [1]
- Volume of distribution (Vd) was 0.35 L/kg, and total clearance (CL) was 89 ml/kg/h [1] - Oral bioavailability was 12% (rat, oral 20 mg/kg), with peak plasma concentration (Cmax) of 320 ng/ml reached 1 hour after administration [1] - Plasma protein binding was 92% in human plasma and 88% in rat plasma [1] |
| References |
| Molecular Formula |
C24H32N2O6
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|---|---|
| Molecular Weight |
444.520687103271
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| Exact Mass |
444.226
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| CAS # |
2365402-67-9
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| PubChem CID |
138059708
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| Appearance |
White to off-white solid powder
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| LogP |
4.9
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
32
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| Complexity |
587
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| Defined Atom Stereocenter Count |
1
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| SMILES |
N(C1C=C(OCCCC(C)C)C(OC)=CC=1C(=O)O)C(=O)N[C@H](C1C=CC=CC=1OC)C
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| InChi Key |
UFAFCKVYKJIJTR-INIZCTEOSA-N
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| InChi Code |
InChI=1S/C24H32N2O6/c1-15(2)9-8-12-32-22-14-19(18(23(27)28)13-21(22)31-5)26-24(29)25-16(3)17-10-6-7-11-20(17)30-4/h6-7,10-11,13-16H,8-9,12H2,1-5H3,(H,27,28)(H2,25,26,29)/t16-/m0/s1
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| Chemical Name |
5-methoxy-2-[[(1S)-1-(2-methoxyphenyl)ethyl]carbamoylamino]-4-(4-methylpentoxy)benzoic 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) |
DMSO : ≥ 100 mg/mL (~224.96 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.62 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2496 mL | 11.2481 mL | 22.4962 mL | |
| 5 mM | 0.4499 mL | 2.2496 mL | 4.4992 mL | |
| 10 mM | 0.2250 mL | 1.1248 mL | 2.2496 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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