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| Targets |
The primary target of this peptide is lipid A, the conserved membrane anchor of LPS in Gram-negative bacterial outer membranes. By binding directly to lipid A, the peptide effectively detoxifies LPS and prevents its interaction with Toll-like receptor 4 (TLR4)/MD-2 complex on immune cells, thereby blocking the downstream activation of NF-kappaB and MAPK signaling pathways. This reduces the production of pro-inflammatory cytokines including TNF-alpha, IL-1beta, IL-6, and other mediators of septic shock.
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| ln Vitro |
In vitro, endotoxin inhibitor TFA binds to lipid A with high affinity, inhibiting LPS-induced cytokine release from immune cells. It is a synthetic peptide that effectively neutralizes LPS bioactivity, thereby detoxifying endotoxin and preventing the downstream inflammatory cascade that leads to septic shock. The peptide shows high specificity for LPS and minimal cross-reactivity with other bacterial components.
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| ln Vivo |
Endotoxin inhibitor (ip; 0.5 mg/kg) TFA can also significantly reduce heat exposure-induced hypothermia and plasma in elderly rats, as well as mRNA increases in IL-1β, COX-2, IκB-β, and LPS activity. Reactions between ACTH and catecholamines [1].
In vivo, endotoxin inhibitor administered intraperitoneally (i.p.; 0.5 mg/kg) significantly reduces the heat exposure-induced increase in IL-1beta, COX-2, IkappaB-beta, and LPS activity-related mRNA levels, as well as the responses to heat exposure-induced hypothermia and plasma ACTH and catecholamine levels in aged rats. The compound also effectively inhibits the febrile response to LPS while demonstrating very low toxicity and lethality. It can suppress the febrile response to LPS and has a favorable toxicity profile. |
| Enzyme Assay |
For LPS binding assays, coat 96-well plates with LPS (1-10 ug/well) in 50 mM carbonate-bicarbonate buffer (pH 9.6) overnight at 4degC. Block with 1% BSA in PBS. Add biotinylated endotoxin inhibitor TFA (1 nM-1 uM) in binding buffer (PBS with 0.05% Tween-20) for 2 hours at 25degC. Wash, add HRP-conjugated streptavidin, incubate for 30 minutes, wash, add TMB substrate, and measure absorbance at 450 nm. For competitive binding assays, add unlabeled endotoxin inhibitor at varying concentrations (1 pM-10 uM) along with a fixed concentration of biotinylated peptide. Calculate IC50 and binding affinity (Kd) by nonlinear regression. Use surface plasmon resonance (SPR) for real-time binding kinetics to lipid A or LPS.
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| Cell Assay |
Culture RAW 264.7 mouse macrophages or THP-1 human monocytes in DMEM or RPMI-1640 with 10% FBS and 1% penicillin/streptomycin at 37degC with 5% CO2. Seed cells in 96-well plates (1 × 10^5 cells/well). Pre-treat cells with endotoxin inhibitor TFA at concentrations of 0.1-100 uM for 30-60 minutes. Stimulate with LPS (1-100 ng/mL) for 4-24 hours. Collect supernatant and measure pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6, IL-8) by ELISA or multiplex bead-based assays. Assess cell viability by MTT or LDH assay to exclude non-specific cytotoxicity. For NF-kappaB activation assays, perform Western blot for IkappaB-alpha degradation and p65 nuclear translocation. Alternatively, use NF-kappaB reporter cell lines with luciferase readout. For intracellular signaling, measure MAPK phosphorylation (p38, ERK, JNK) by Western blot.
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| Animal Protocol |
For endotoxemia/sepsis models, use C57BL/6 mice or Sprague-Dawley rats. Administer LPS (1-10 mg/kg) via intraperitoneal (i.p.) or intravenous (i.v.) injection to induce endotoxemia. Co-administer endotoxin inhibitor TFA i.p. at 0.1-5 mg/kg before or after LPS challenge. Monitor animal survival (typically 24-72 hours). Collect blood for measurement of plasma cytokines (TNF-alpha, IL-1beta, IL-6) by ELISA. Harvest tissues (liver, lung, kidney) for histopathological assessment of organ injury. For fever studies, monitor body temperature by telemetry or rectal probe. For aged rat models (18-24 months old), administer endotoxin inhibitor TFA i.p. at 0.5 mg/kg prior to heat exposure or LPS challenge; measure plasma catecholamines, ACTH, and cytokine levels. For survival studies, administer a single dose of LPS (lethal dose, 15-25 mg/kg i.p.) and treat with endotoxin inhibitor TFA; record mortality every 6-12 hours for 72 hours.
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| ADME/Pharmacokinetics |
The peptide is a synthetic decapeptide (MW 1908.72 for TFA salt). As a peptide, it is subject to proteolytic degradation in circulation. The TFA salt improves solubility and formulation stability. When administered intraperitoneally at 0.5 mg/kg in rats, the peptide is expected to have a short plasma half-life (minutes to <1 hour), rapid distribution, and renal clearance. Bioavailability is moderate via i.p. administration, but the compound is likely not orally bioavailable due to gastric degradation. For in vivo formulation, dissolve in DMSO first (for stock) and then dilute in appropriate vehicle (e.g., 5% DMSO + 30% PEG300 + 5% Tween 80 + 60% saline/PBS).
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| Toxicity/Toxicokinetics |
Endotoxin inhibitor TFA has a favorable safety profile with very low toxicity and lethality reported in animal studies. At doses that effectively neutralize LPS (0.5 mg/kg i.p.), no significant acute toxicity is observed. The compound does not cause significant organ toxicity or mortality when administered alone. The peptide is not hemolytic and has minimal immunogenicity. The major adverse effect of endotoxin itself (sepsis, fever, shock) is effectively prevented by the compound. No genotoxicity or carcinogenicity data are available, but no concerns are expected for a short synthetic peptide. Avoid inhalation of aerosolized powder. Standard laboratory precautions (gloves, lab coat, safety glasses) should be used.
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| References |
[1]. Toshihiko Katafuchi, et al. Endotoxin inhibitor blocks heat exposure-induced expression of brain cytokine mRNA in aged rats. Brain Res Mol Brain Res. 2003 Oct 21;118(1-2):24-32.
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| Additional Infomation |
The TFA salt form contains trifluoroacetate as a counterion, which enhances peptide solubility and stability. The molecular weight is 1908.72, formula C67H103F18N15O24S2. Sequence: Lys-Thr-Lys-Cys-Lys-Phe-Leu-Lys-Lys-Cys with disulfide bridge between Cys4 and Cys10 (forming a cyclic peptide structure). This compound is also known as Reltecimod TFA or AB-103 TFA in some sources. It is exclusively for research use and not for human therapeutic applications.
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| Molecular Formula |
C67H103F18N15O24S2
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| Molecular Weight |
1908.72
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| Related CAS # |
Endotoxin inhibitor;147396-10-9
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| Appearance |
Typically exists as solid at room temperature
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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) |
H2O :~100 mg/mL (~52.39 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 | 0.5239 mL | 2.6196 mL | 5.2391 mL | |
| 5 mM | 0.1048 mL | 0.5239 mL | 1.0478 mL | |
| 10 mM | 0.0524 mL | 0.2620 mL | 0.5239 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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