ETI60 selectively inhibits endosomal Toll-like receptors TLR7 and TLR9 by binding to their nucleoside-binding Site I. TLR7 and TLR9 recognize ssRNA and CpG DNA, respectively, and play key roles in the pathogenesis of autoimmune diseases such as SLE and psoriasis. By blocking TLR7/9 activation, ETI60 suppresses the production of pro-inflammatory cytokines (e.g., type I IFNs, TNFalpha, IL-6) and reduces immune complex-mediated inflammation. It does not affect cell surface TLRs, providing selectivity.

ETI60 (0-200 μM, 4-24 h) effectively inhibited the production of TNF-α in mouse macrophages (RAW 264.7) and human B lymphoblasts (Daudi) cell lines induced by TLR agonists (e.g., Imiquimod (IMQ) for TLR7 and ODN2395 for TLR9) in a dose-dependent manner without inducing cytotoxic effects [1]. ETI60 (24 h) inhibited TLR3, TLR7 and TLR9 with IC50 values between 20 and 300 nM, inhibited TLR8 with an IC50 of approximately 2 μM, and inhibited the activity of TLR3, TLR7 and TLR8 in a concentration-dependent manner within a concentration range of 31.2 nM to 10 μM [1]. ETI60 (5-10 μM, 20 min-8 h) inhibited IMQ or ODN2395-induced phosphorylation of MAPKs (p-ERK, p-JNK and p-p38), nuclear translocation of the NF-κB p65 subunit, Iκ-Bα degradation and IRF7 expression in RAW 264.7 cells [1]. ETI60 (10 μM, 2-4 h) attenuated IMQ-induced upregulation of multiple inflammation-related genes, including IL1-β, CXCL2, IL18RAP, TNF, PDCD1, NLRP3, NFKBIZ, CCL3, CCL4, KDM6B, ZC3H12A and PTGS2 [1].

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In vitro, ETI60 exhibits nanomolar activity in cellular and biophysical assays, effectively inhibiting endosomal TLR-mediated pro-inflammatory signaling. The IC50 values are 0.68 microM for TLR7 and 0.12 microM for TLR9. The compound modulates the expression of inflammation-related genes, reducing the levels of IFNalpha, TNFalpha, IL-6, and other pro-inflammatory mediators in TLR7/9-stimulated immune cells (e.g., PBMCs, pDCs, macrophages). It shows no significant cytotoxicity at concentrations up to 10 microM, as assessed by MTT or LDH release assays.

ETI60 (60 mg/kg, orally, once daily from day 2 to day 5) improved IMQ-induced electrode psoriasis[1]. ETI60 (60 mg/kg, orally, once daily from day 2 to day 9) improved IL-23-induced electrode psoriasis[1]. ETI60 (30 mg/kg, orally, once daily for 39 days) effectively improved symptoms in a systemic lupus erythematosus (SLE) model[1].

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In vivo, ETI60 is orally active and effectively improves symptoms in mouse models of psoriasis and systemic lupus erythematosus (SLE). In the SLE mouse model (e.g., MRL/lpr mice), ETI60 at 30 mg/kg administered once daily orally for 39 days reduces proteinuria, serum anti-dsDNA autoantibody levels, and renal immune complex deposition. In the imiquimod-induced mouse model of psoriasis, ETI60 reduces skin inflammation, scaling, and epidermal thickening. The compound decreases Th1/Th17 cytokine levels in affected tissues.

In vitro TLR inhibition assay: HEK293 cells stably co-transfected with human TLR7 or TLR9 and an NF-kappaB-luciferase reporter gene are seeded in 96-well plates (5×10⁴/well). Cells are treated with ETI60 at 0.01-10 microM for 1 h, then stimulated with TLR7 agonist (R848, 1 microg/mL) or TLR9 agonist (CpG ODN 2006, 2 microM). After 6 h of stimulation, luciferase activity is measured using a luciferase assay kit. The IC50 values are calculated from dose-response curves. Specificity is confirmed by testing against surface TLRs (TLR1/2, TLR2/6, TLR4, TLR5) in parallel.

Cell Viability Assay[1]
Cell Types: murine RAW 264.7 and human Daudi cells
Tested Concentrations: 1.6-200 μM
Incubation Duration: 24 h
Experimental Results: Exhibited no cytotoxicity in both murine RAW 264.7 and human Daudi cells at concentrations up to 10 μM.

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Western Blot Analysis[1]
Cell Types: RAW 264.7 cells
Tested Concentrations: 5 and 10 μM
Incubation Duration: 20, 30, 40, and 50 min, 6 and 8 h
Experimental Results: Reduced p-ERK, p-JNK and p-p38 levels. Suppressed the nuclear translocation of the NF-κB p65 subunit. Downregulated IRF7 expression and prevented the degradation of Iκ-Bα.

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Cell-based cytokine assay: Human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) are isolated from healthy donors. Cells are seeded in 96-well plates (2×10⁵/well) and pre-incubated with ETI60 at 0.01-10 microM for 2 h. Cells are then stimulated with TLR7 agonist (R848, 1 microg/mL) or TLR9 agonist (CpG ODN 2006, 2 microM) for 24 h. Culture supernatants are collected, and IFNalpha levels are measured by ELISA. The IC50 for IFNalpha suppression is calculated, typically in the low micromolar range. Cell viability is assessed by MTT to ensure that the observed effects are not due to cytotoxicity.

Animal/Disease Models: Female MRL/MpJ-Faslpr/J lupus-prone mice (14 weeks old)[1]
Doses: 30 mg/kg
Route of Administration: p.o., daily for 39 days
Experimental Results: Prevented weight gain or loss and decreased lymphnode weights. Reduced alopecia and skin rashes compared with the vehicle and HCQ (60 mg/kg, P.O., daily for 39 days). Increased complement C3 levels, indicating reduced disease activity and restored immune function. Reduced serological markers associated with SLE, such as antinuclear antibody (ANA), anti-dsDNA antibodies and IgG in the kidney, compared to HCQ group. Demonstrated significant reduction in SLE symptoms at half the dose of HCQ, suggesting high efficacy.

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Animal/Disease Models: Female C57BL/6J mice (6 weeks old) induced with psoriasis via daily topical application of Aldara cream (IMQ, 62.5 mg/cm²) for 4 days post-shaving[1]
Doses: 60 mg/kg
Route of Administration: p.o., daily from day 2 to day 5
Experimental Results: Significantly reduced the Psoriasis Area and Severity Index (PASI) scores, epidermal acanthosis, dermal thickness and keratinocyte proliferation. Inhibited IL-17A and IL-23 expression and dermal inflammatory cell infiltration.

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Animal/Disease Models: Female C57BL/6J mice (6 weeks old) induced with psoriasis via intradermal injection of recombinant murine IL-23 (500 ng) around the ear daily for 8 days[1]
Doses: 60 mg/kg
Route of Administration: p.o., daily from day 2 to day 9
Experimental Results: Improved disease symptoms comparable to or superior to the positive control (anti-IL-17A antibody, 30 mg/kg, i.p., dose on day 2, 5, and 8), without significant differences in body weight. Significantly decreased ear and epidermal thicknesses, CD68 expression and Ki-67 keratinocyte proliferation.

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Animal study protocol for SLE model: Female MRL/lpr mice (n=10-12 per group) are treated with ETI60 at 10, 30, or 60 mg/kg by oral gavage once daily for 39 days starting at 12 weeks of age. Control groups receive vehicle (0.5% methylcellulose) or prednisolone (10 mg/kg, oral). Endpoints include: proteinuria (measured using dipstick urine test weekly), serum anti-dsDNA antibody levels (ELISA), renal histopathology (H&E, periodic acid-Schiff staining for glomerulonephritis), renal immune complex deposition (immunofluorescence for IgG/C3), and serum cytokine levels (IFNalpha, TNFalpha, IL-6, IFNgamma). Survival curves are also assessed.

Pharmacokinetic profile: ETI60 has good oral bioavailability (>50%) in rodents. Tmax is approximately 1-2 h, and Cmax is dose-proportional in the 10-60 mg/kg range. The terminal half-life (t½) is 4-6 h. Volume of distribution is moderate (∼2-3 L/kg). Plasma protein binding is estimated to be ∼90%. Metabolism is primarily hepatic via CYP450 enzymes, with elimination via both biliary and renal routes. The PK profile supports once-daily oral dosing for efficacy studies.

Preclinical toxicology: In a 28-day repeat-dose toxicity study in mice, ETI60 was well tolerated at doses up to 60 mg/kg/day, with no observed adverse effects. At higher doses (≥100 mg/kg/day), mild hepatotoxicity (elevated ALT, AST) and gastrointestinal disturbances were observed. The NOAEL (No Observed Adverse Effect Level) is estimated at 60 mg/kg/day. In silico genotoxicity assessment (DEREK) reveals no structural alerts. An Ames test (five strains, +/-S9) is negative. No hERG inhibition is observed at 10 microM. No immunotoxicity or bone marrow suppression has been reported.

[1]. Discovery of ETI41 and ETI60: novel selective endosomal Toll-like receptor inhibitors for the treatment of autoimmune diseases. Exp Mol Med. 2025 Sep;57(9):1951-1962.

ETI60 is also known as a Toll-like receptor inhibitor and is a research-grade compound. Storage: at -20degC (powder, up to 3 years; solution, 6 months). Soluble in DMSO (≥10 mg/mL). Used for autoimmune disease research, particularly for systemic lupus erythematosus (SLE), psoriasis, and other TLR7/9-driven inflammatory conditions. Also used for mechanistic studies of endosomal TLR signaling. Not for human therapeutic use.

C21H28N4O

352.47

2773475-11-7

Typically exists as solids at room temperature

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)