| Targets |
ECL1i TFA targets the C-C chemokine receptor 2 (CCR2), a G protein-coupled receptor (GPCR) expressed on monocytes, macrophages, and other immune cells. CCR2 is the primary receptor for the chemokine CCL2 (monocyte chemoattractant protein-1, MCP-1). The CCL2/CCR2 axis is a critical pathway mediating the recruitment of inflammatory monocytes and macrophages to sites of inflammation and injury. ECL1i TFA acts as an allosteric inhibitor, binding to a site distinct from the orthosteric CCL2 binding pocket, thereby selectively inhibiting CCL2/CCR2-mediated chemotaxis without blocking CCR2 binding to other chemokines. By interfering with CCR2-positive cell recruitment, ECL1i TFA attenuates the progression of inflammatory diseases.
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| ln Vitro |
ECL1i TFA is a specific, allosteric inhibitor of CCL2/CCR2-mediated chemotaxis. It demonstrates potent selectivity for CCR2 over other chemokine receptors, including CCR5 and CXCR4. In vitro, using transwell chemotaxis assays, ECL1i TFA (0.1-10 uM) inhibits CCL2-induced migration of primary human monocytes and CCR2-expressing cell lines (e.g., THP-1 cells) in a concentration-dependent manner. The peptide does not affect baseline cell viability at concentrations up to 50 uM, as measured by MTT assays. ECL1i TFA also inhibits CCL2-induced calcium flux and ERK phosphorylation in CCR2-expressing cells. IC₅0 values for inhibition of chemotaxis are typically in the low micromolar range (1-5 uM).
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| ln Vivo |
In vivo, ECL1i TFA has demonstrated efficacy in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Administration of ECL1i TFA (e.g., 10-30 mg/kg, intraperitoneally) in EAE-induced mice significantly reduces the infiltration of CCR2-positive monocytes into the central nervous system (CNS). This results in a marked attenuation of disease progression, as measured by a decrease in clinical scores and reduced demyelination in spinal cord sections. ECL1i TFA also shows activity in models of atherosclerosis and tumor metastasis by modulating the tumor microenvironment. The compound interferes with the recruitment of CCR2-positive myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), potentially reducing tumor growth and enhancing the efficacy of immunotherapy.
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| Enzyme Assay |
In vitro CCR2 binding and functional assays are used to characterize ECL1i TFA. For chemotaxis inhibition assays, THP-1 cells (2×10⁵ cells) are loaded into the upper chamber of a 96-well transwell plate (pore size 5 um). CCL2 (10-50 ng/mL) is placed in the lower chamber as a chemoattractant. ECL1i TFA (0.1-100 uM) is added to both chambers. After 2-4 h incubation at 37degC, cells that migrated to the lower chamber are quantified by cell counting (hemocytometer) or by fluorescence (if pre-labeled with Calcein-AM). IC₅0 values for inhibition of chemotaxis are calculated. For calcium flux assays, THP-1 cells are loaded with Fluo-4 AM, and changes in fluorescence (λex 494 nm, λem 516 nm) upon CCL2 stimulation (with/without ECL1i TFA) are measured using a fluorescence plate reader.
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| Cell Assay |
For cell-based assays, primary human monocytes are isolated from peripheral blood mononuclear cells (PBMCs) by CD14+ magnetic selection. Cells (1×10⁵ cells/well) are seeded in 96-well plates in RPMI-1640 with 10% FBS. ECL1i TFA (1-100 uM) is added 30 min prior to CCL2 stimulation (10-50 ng/mL). After 2-4 h, cell migration is assessed as described above. For mechanistic studies, THP-1 cells are treated with ECL1i TFA for 30 min, then stimulated with CCL2. Cells are lysed, and ERK1/2 phosphorylation is assessed by Western blotting using p-ERK (Thr202/Tyr204)-specific antibodies. Total ERK is used as a loading control. Cytotoxicity is assessed by LDH release or MTT assay to ensure that observed reductions in migration are not due to cell death.
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| Animal Protocol |
Experimental autoimmune encephalomyelitis (EAE) mouse model: Female C57BL/6 mice (6-8 weeks old, n=10-15/group) are immunized subcutaneously with myelin oligodendrocyte glycoprotein (MOG35-55) peptide (200 ug) emulsified in complete Freund's adjuvant (CFA). Pertussis toxin (200 ng) is injected intraperitoneally on days 0 and 2. ECL1i TFA is formulated in 5% DMSO + 5% Tween 80 + 90% saline (or 0.5% methylcellulose) and administered intraperitoneally at doses of 10-30 mg/kg once daily from day 0 to day 21. Control groups receive vehicle alone. Clinical signs of EAE are scored daily (0 = no clinical signs; 0.5 = partial tail limpness; 1 = complete tail limpness; 2 = hind limb weakness; 3 = complete hind limb paralysis; 4 = tetraplegia; 5 = moribund or death). At the study endpoint (day 21), spinal cords are harvested for histology (H&E staining and luxol fast blue staining to assess demyelination) and for flow cytometry to quantify infiltrating CCR2+ monocytes. ECL1i TFA treatment significantly reduces clinical scores and CNS inflammation.
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| ADME/Pharmacokinetics |
No specific PK data for ECL1i TFA is publicly available. As a cyclic peptide (MW 780.97), the TFA salt form is used to enhance water solubility. Following intraperitoneal (IP) administration in rodents, ECL1i TFA is absorbed into the systemic circulation. Peptide drugs generally have a short plasma half-life (usually 30 min - 2 h) due to rapid proteolytic degradation and renal clearance. The compound is likely metabolized by proteases in the blood, liver, and kidneys, with excretion primarily in urine. For research use, the compound is typically formulated in 5% DMSO + 5% Tween 80 + 90% saline or 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline for IP administration. The in vivo formulation should be prepared fresh before each dosing session.
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| Toxicity/Toxicokinetics |
For ECL1i TFA, GHS hazard statements: H315 (Causes skin irritation), H319 (Causes serious eye irritation), H335 (May cause respiratory irritation). Signal word: Warning. Precautionary statements: P261 (Avoid breathing dust/fume/gas/mist/vapors/spray), P280 (Wear protective gloves/protective clothing/eye protection/face protection), P305+P351+P338 (IF IN EYES: Rinse cautiously with water for several minutes; remove contact lenses if present and easy to do; continue rinsing). Storage: powder at -20degC for 3 years, 4degC for 2 years; in solvent (e.g., DMSO) at -80degC for 6 months, -20degC for 1 month. Store in a sealed container, protected from light and moisture, and under inert gas (e.g., nitrogen). Not for human consumption.
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| References | |
| Additional Infomation |
ECL1i TFA (CAS# for parent ECL1i: 1417312-35-6) is a research-grade cyclic peptide CCR2 allosteric antagonist (sequence: d(LGTFLKC)). It is used in studies of inflammation (multiple sclerosis), cancer (tumor-associated macrophages, MDSCs), and atherosclerosis. ECL1i TFA is not an FDA-approved drug. For research use only (RUO), not for diagnostic or therapeutic applications.
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| Related CAS # |
ECL1i
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|---|---|
| Sequence |
d(Leu-Gly-Thr-Phe-Leu-Lys-Cys)d(LGTFLKC)
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| Appearance |
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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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.) |
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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