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| 5mg |
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IAXO-102 is a novel and potent TLR4 antagonist with anti-inflammatory effects. It inhibits MAPK and p65 NF-kB phosphorylation involved in down regulation of the expression of TLR4 and TLR4 dependent proinflammatory proteins.
| Targets |
Toll-like receptor 4 (TLR4) [1]
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|---|---|
| ln Vitro |
IAXO-102 (1–10 μM) inhibits p65 NF-κB and MAPK phosphorylation for two hours in human vascular endothelial (HUVEC) cells [1]. IAXO-102 (10 μM) inhibits HUVEC for 17 hours.
IAXO-102 (up to 10 μM) did not affect HUVEC viability, but at 20 μM significantly reduced HUVEC viability (MTT assay). [1] Pre-treatment of HUVEC with IAXO-102 (1-10 μM for 1 hour) significantly inhibited LPS-stimulated (100 ng/ml for 1 hour) phosphorylation of JNK, p38, ERK and p65 NF-κB in a concentration-dependent manner, with the blocking effect evident at 10 μM and to a lesser extent at 1 μM (Western blot analysis). [1] In HUVEC pretreated with IAXO-102 (10 μM for 1 hour) and then exposed to LPS (100 ng/ml for 16 hours), semi-quantitative analysis on human Inflammation Antibody Array (40 proinflammatory proteins) showed that LPS upregulated 20/40 proteins, while IAXO-102 inhibited the expression of 17/20 LPS-driven proinflammatory proteins. [1] ELISA validation confirmed that IAXO-102 significantly blocked LPS-driven MCP-1 and IL-8 production in HUVEC. [1] |
| ln Vivo |
The aortic dilatation caused by angiotensin II is markedly postponed by IAXO-102 (3 mg/kg/day, administered subcutaneously for 28 days) [1].
In angiotensin II-infused ApoE deficient mice, IAXO-102 (3 mg/kg/day s.c. in Lipodisq™ for 72 hours) significantly inhibited angiotensin II-stimulated JNK, ERK and p65 NF-κB phosphorylation in thoracic, supra-renal and infra-renal aortic regions (Western blot analysis). [1] IAXO-102 treatment significantly down-regulated TLR4 expression in the supra-renal aortic region (where TLR4 up-regulation was evident after angiotensin II infusion) as shown by immunoblotting. [1] In the same model, IAXO-102 significantly inhibited production of MIP-1γ in all three regions of the mouse aorta (ELISA). [1] Semi-quantitative analysis on mouse Inflammation Antibody Array (40 proteins) showed that angiotensin II infusion upregulated 22/40 proinflammatory proteins, and IAXO-102 pretreatment inhibited 14/22 of these proteins. [1] In a 28-day study, IAXO-102 (3 mg/kg/day s.c.) significantly retarded angiotensin II-induced increase in aortic diameter: aortic diameter in IAXO-102 group was 1.055 ± 0.081 mm compared to 1.865 ± 0.501 mm in positive control group (p<0.001). IAXO-102 completely abolished aortic rupture (0% vs 30% in angiotensin II group), reduced aneurysm incidence to 30% (vs 86% in angiotensin II group), and increased mouse survival. [1] |
| Cell Assay |
Western Blot Analysis[1]
Cell Types: Human Umbilical Vein Endothelial (HUVEC) Cells Tested Concentrations: 1-10 µM Incubation Duration: 1 hour pretreatment, then 1] LPS Induced production of the pro-inflammatory proteins MCP-1 and IL-8. Then exposed to LPS (100 ng/mL) for 1 hour. Experimental Results: Significant inhibition of LPS-stimulated MAPK/p65nF-KB phosphorylation. HUVEC (passages 3-5) were maintained at 37°C with 5% CO2 in endothelial cell growth medium in 25 cm² flasks pre-coated with 1% attachment factor. For treatments, HUVEC were pre-treated with IAXO-102 (0-10 μM) for 1 hour, then exposed to LPS (100 ng/ml) for 1 or 16 hours. Cell viability was measured by MTT assay: cells (10,000/well in 96-well plate) were incubated with or without IAXO-102 (0-20 μM) and LPS (100 ng/ml) for 24 hours, and viability expressed as % of untreated control. [1] For Western blot analysis, cell lysates (20 μg protein) were separated on 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% skimmed milk in TBS/0.1% Tween-20 for 1 hour at room temperature, then incubated overnight at 4°C with primary antibodies against phospho-JNK, phospho-p38, phospho-ERK, phospho-p65 NF-κB, and TLR4 (1:1000 dilution in TBS with 1% milk). After washing, blots were incubated with HRP-conjugated antibody for 1 hour at room temperature, and immunoreactivity visualized with chemiluminescent substrate. Densitometric analysis was performed, with actin as loading control. [1] For Inflammation Antibody Array, tissue protein lysates (20 μg) from supra-renal aortic segment or cell lysates were semi-quantitatively analyzed on arrays containing 40 proinflammatory proteins according to manufacturer's instructions: membranes incubated with protein lysates, then with biotinylated antibody, then with streptavidin HRP-conjugated antibody, and immunoreactivity visualized. [1] For ELISA, human MCP-1 and IL-8 (from cell lysates) or mouse MIP-1γ (from tissue lysates, 20 μg protein) were measured using ELISA kits following manufacturer's instructions; absorbance measured at 450 nm using a microplate reader. [1] |
| Animal Protocol |
Animal/Disease Models: Six-month-old ApoE−/−/C57Bl6[1]
Doses: 3 mg/kg/day Route of Administration: SC 28 days Experimental Results: Dramatically delayed angiotensin II-induced increase in aortic diameter. All animal experiments were approved by the local Animal Research Work Ethical Review Board at St George's, University of London and conformed to British Home Office regulations (ASPA 1986; Project License PPL70/7560). Male ApoE homozygous deficient mice (6 months old, 37.2g ± 3.5) were used. AAA was induced by infusion of Angiotensin II (1 μg/min/kg) via osmotic pumps implanted under inhalation anaesthesia (isoflurane 2-3% mixed with 50% oxygen and 50% nitrous oxide). Two in vivo experiments were performed: animals sacrificed at 3 days (for protein analysis, n=4 per group) and at 28 days (for aortic diameter measurement, n=10 for Ang II groups, n=6 for sham). Mice were randomized into groups by random number allocation. IAXO-102 (3 mg/kg/day in 50 μl Lipodisq™) or vehicle control (50 μl Lipodisq™) was administered subcutaneously (s.c.) once daily at the same time (12:00 noon) for the duration of the experiments (3 or 28 days). For the 3-day experiment, tissue samples from thoracic, supra-renal and infra-renal aorta were prepared at 72 hours and soluble proteins analyzed. For the 28-day experiment, maximal aortic diameter was measured by computer-assisted micrometry. Mice were sacrificed using a lethal dose of anaesthesia (100 mg/0.2 mg per kg of Ketamine/Medetomidine), exsanguinated via cardiac puncture, and underwent cardiac reperfusion with protease inhibitor at 4°C (for 3-day experiment only). [1] |
| ADME/Pharmacokinetics |
Pharmacokinetic study using IAXO-102-FITC following s.c. injection (1 and 3 mg/kg) in ApoE deficient C57BL/6 mice showed that the signal was stable up to 24 hours for 1 mg/kg and up to 72 hours for 3 mg/kg. After 24 hours, IAXO-102-FITC was present in the bladder, small and large intestine, and to some extent in kidney or liver. FACS analysis of blood taken from animals demonstrated that IAXO-102-FITC was bound mainly to monocytes but not to lymphocytes or granulocytes. [1]
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| Toxicity/Toxicokinetics |
In HUVEC, IAXO-102 at concentrations up to 10 μM did not affect cell viability, but at 20 μM significantly reduced HUVEC viability as determined by MTT assay (24-hour exposure with or without LPS 100 ng/ml). [1]
|
| References | |
| Additional Infomation |
IAXO-102 was originally generated to block TLR4/CD14 interactions, and it may stimulate internalization and degradation of TLR4 rather than affect TLR4 gene expression. [1]
IAXO-102 blocked TLR4 signalling in response to distinct TLR4 ligands: in HUVEC it blocked TLR4 in response to LPS; it partially downregulated TLR4 signalling in response to minimally-oxidised LDL and small fragments of hyaluronan in HUVEC (unpublished data shown in the manuscript). [1] The fact that IAXO-102 was designed to block TLR4/CD14 interactions may explain its broad biochemical properties, as it blocks both human and mouse TLR4 signalling pathways, unlike species-specific TLR4 blocking antibodies. [1] This study demonstrates for the first time that IAXO-102, a TLR4 antagonist, is effective in blocking vascular inflammation leading to inhibition of experimental abdominal aortic aneurysm (AAA) development, suggesting its potential therapeutic use for pharmacological intervention of aneurysms. [1] |
| Molecular Formula |
C35H71NO5
|
|---|---|
| Molecular Weight |
585.941951990128
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| Exact Mass |
585.533
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| CAS # |
1115270-63-7
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| PubChem CID |
25222900
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| Appearance |
White to off-white solid powder
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| LogP |
12.1
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
30
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| Heavy Atom Count |
41
|
| Complexity |
537
|
| Defined Atom Stereocenter Count |
5
|
| SMILES |
O1[C@@H]([C@@H]([C@H]([C@@H]([C@H]1CN)O)OCCCCCCCCCCCCCC)OCCCCCCCCCCCCCC)OC
|
| InChi Key |
DJVFNDPRLMVKKO-KJQSSVQNSA-N
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| InChi Code |
InChI=1S/C35H71NO5/c1-4-6-8-10-12-14-16-18-20-22-24-26-28-39-33-32(37)31(30-36)41-35(38-3)34(33)40-29-27-25-23-21-19-17-15-13-11-9-7-5-2/h31-35,37H,4-30,36H2,1-3H3/t31-,32-,33+,34-,35+/m1/s1
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| Chemical Name |
(2R,3R,4S,5R,6S)-2-(aminomethyl)-6-methoxy-4,5-di(tetradecoxy)oxan-3-ol
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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) |
Ethanol : ~50 mg/mL (~85.33 mM)
DMSO : ~5 mg/mL (~8.53 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.83 mg/mL (1.42 mM) (saturation unknown) in 10% EtOH + 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 8.3 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.83 mg/mL (1.42 mM) (saturation unknown) in 10% EtOH + 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 8.3 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7067 mL | 8.5333 mL | 17.0666 mL | |
| 5 mM | 0.3413 mL | 1.7067 mL | 3.4133 mL | |
| 10 mM | 0.1707 mL | 0.8533 mL | 1.7067 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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