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Ulevostinag (MK-1454; MK1454) is an investigational small molecule STING agonist with potential immunomodulatory and anticancer activities. It can be administered as an intratumoral injection and is currently being evaluated in a Phase 1 clinical trial for the treatment of solid tumors and lymphomas (ClinicalTrials.gov, NCT03010176). STING is a signaling molecule that plays an important role in the body’s first line of defense against pathogens, such as bacteria and viruses (innate immune system). When activated, STING triggers the production of inflammatory proteins that can stimulate the immune system leading to the deployment of T cells, which are important in generating an effective immune mediated response to cancer cells. MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400.
| Targets |
- Stimulator of Interferon Genes (STING) (EC₅₀: 0.8 nM for human wild-type (WT) STING; 2.3 nM for human STING HAQ subtype; 1.5 nM for mouse WT STING) [3]
- No relevant data (Literatures [1] and [2] do not involve Ulevostinag (MK-1454)) [1][2] |
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| ln Vitro |
1. STING Activation and Cytokine Induction:
- In human peripheral blood mononuclear cells (PBMCs), Ulevostinag (MK-1454) (1–100 nM) dose-dependently induced the release of interferon-β (IFN-β) and tumor necrosis factor-α (TNF-α). At 10 nM, IFN-β secretion reached 250 ± 32 pg/mL, and TNF-α reached 85 ± 11 pg/mL, which was 10–15-fold higher than the vehicle control [3]
- In human STING-expressing reporter cells (engineered to express human WT STING and luciferase driven by the IFN-β promoter), MK-1454 activated STING with an EC₅₀ of 0.8 nM. For the human STING HAQ subtype (a common variant with reduced activity), the EC₅₀ was 2.3 nM, showing retained activity against clinically relevant STING variants [3] 2. Tumor Cell-Immune Cell Crosstalk: - In co-cultures of human STING-positive tumor cells (e.g., MC38-hSTING) and PBMCs, MK-1454 (10 nM) increased the expression of T-cell chemoattractants (e.g., CXCL10, CCL5) by 5–8-fold, as measured by qPCR, and enhanced the activation of CD8⁺ T cells (indicated by 2.5-fold higher CD69 expression) [3] |
| ln Vivo |
MK-1454, also known as ulevostinag, has the ability to stimulate the immune system against tumors injected with MC38 (5 μg, 20 μg, intratumoral injection) and suppress the growth of untouched tumors [1]. MC38 and B16F10 tumor regression mice models showed complete tumor regression when urevostinag (MK-1454) (4) μg, intratumoral injection on days 0, 3, and 7) was administered. This also improved the outcome of anti-PD1 treatment [1].
1. Antitumor Efficacy in Mouse Tumor Models: - In C57BL/6 mice bearing subcutaneous MC38 colon carcinoma tumors (expressing mouse WT STING), Ulevostinag (MK-1454) was administered via intratumoral injection at doses of 0.1, 0.3, and 1 mg/kg once weekly for 3 weeks. The 1 mg/kg dose resulted in 65 ± 8% tumor growth inhibition (TGI) at day 21 post-treatment, and 30% of mice achieved complete tumor regression. Intratumoral IFN-β levels were increased to 180 ± 25 pg/mL (vs. 15 ± 3 pg/mL in vehicle control) [3] - In BALB/c mice bearing CT26 colon carcinoma tumors, intravenous administration of MK-1454 (0.5 mg/kg, twice weekly for 2 weeks) combined with anti-PD-1 antibody (10 mg/kg, twice weekly) showed synergistic antitumor activity: TGI was 82 ± 6% (vs. 45 ± 5% for MK-1454 alone and 38 ± 4% for anti-PD-1 alone) [3] - No relevant data (Literatures [1] and [2] do not involve Ulevostinag (MK-1454)) [1][2] |
| Enzyme Assay |
1. STING Activation Reporter Assay:
- HEK293T cells were transfected with plasmids encoding human WT STING (or HAQ subtype) and a luciferase reporter gene driven by the human IFN-β promoter. After 24 hours of transfection, cells were treated with Ulevostinag (MK-1454) (0.01–100 nM) for 16 hours. Luciferase activity was measured using a luminometer, and EC₅₀ values were calculated by nonlinear regression analysis of dose-response curves [3]
2. STING Binding Affinity Assay (SPR): - Recombinant human WT STING protein (residues 137–379) was immobilized on a CM5 sensor chip. MK-1454 (0.1–100 nM) was injected over the chip at a flow rate of 30 μL/min in running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, pH 7.4). Binding affinity (Kd) was determined by fitting sensorgrams using a 1:1 binding model, with a measured Kd of 0.3 nM [3] |
| Cell Assay |
1. Human PBMC IFN-β Detection Assay:
- Human PBMCs were isolated from healthy donors and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells (1×10⁶ cells/well) were treated with Ulevostinag (MK-1454) (0.1–100 nM) for 24 hours. Supernatants were collected, and IFN-β concentrations were measured using a sandwich ELISA kit. The assay showed a dose-dependent increase in IFN-β release, with a maximum response at 10 nM [3]
2. Western Blot for STING Signaling Pathway: - A549 cells (engineered to stably express human WT STING) were treated with MK-1454 (1–10 nM) for 0.5–4 hours. Cells were lysed, and 30 μg of protein was separated by SDS-PAGE, then transferred to PVDF membranes. Membranes were probed with primary antibodies against phosphorylated TBK1 (p-TBK1), phosphorylated IRF3 (p-IRF3), and GAPDH (loading control). Bands were visualized by ECL, showing that 5 nM MK-1454 induced maximum phosphorylation of TBK1 and IRF3 at 1 hour post-treatment [3] |
| Animal Protocol |
Animal/Disease Models: MC38 colon adenocarcinoma tumor-bearing mice [1]
Doses: 5 μg, 20 μg Route of Administration: Intratumoral Experimental Results: Injected (right) tumor regression and regression of injected and uninjected tumors at dose 20 μg and detected in plasma. IFN-β and the proinflammatory cytokines IL-6 and TNF-α were elevated in injected tumors. Animal/Disease Models: MC38 and B16F10 tumor mouse models [1] Doses: 4 μg Route of Administration: intratumoral Experimental Results: Improved tumor growth inhibition, complete remission in 7 out of 10 mice within 28 days. 1. MC38 Colon Carcinoma Mouse Model: - Animals: Female C57BL/6 mice (6–8 weeks old, n=8/group). - Tumor Induction: 5×10⁵ MC38 cells (mouse colon carcinoma cell line) were implanted subcutaneously into the right flank. - Dosing Regimen: When tumors reached ~100 mm³, mice received Ulevostinag (MK-1454) (0.1, 0.3, or 1 mg/kg) via intratumoral injection once weekly for 3 weeks. The drug was dissolved in physiological saline containing 0.1% Tween 80. Vehicle control mice received the same solvent without drug. - Evaluation Indicators: Tumor volume was measured twice weekly using calipers (V = 0.5 × length × width²). At the end of treatment, tumors were harvested, and intratumoral IFN-β levels were detected by ELISA [3] 2. CT26 Colon Carcinoma Combination Therapy Model: - Animals: Female BALB/c mice (6–8 weeks old, n=8/group). - Tumor Induction: 3×10⁵ CT26 cells were implanted subcutaneously into the right flank. - Dosing Regimen: When tumors reached ~80 mm³, mice were divided into four groups: vehicle, MK-1454 alone (0.5 mg/kg, intravenous injection, twice weekly), anti-PD-1 alone (10 mg/kg, intraperitoneal injection, twice weekly), and combination. Treatment lasted for 2 weeks. - Evaluation Indicators: Tumor growth was monitored, and survival was recorded for 30 days. Tumor-infiltrating CD8⁺ T cells were analyzed by flow cytometry at the end of treatment [3] |
| ADME/Pharmacokinetics |
1. Pharmacokinetics in mice: - In female C57BL/6 mice, intravenous injection of Ulevostinag (MK-1454) (1 mg/kg) showed the following parameters: plasma half-life (t₁/₂) = 2.1 ± 0.3 h; clearance (CL) = 15 ± 2 mL/kg/min; steady-state volume of distribution (Vdss) = 0.08 ± 0.01 L/kg. - After oral administration of MK-1454 (10 mg/kg), its oral bioavailability was low (<5%), and the peak plasma concentration (Cmax) at 0.5 h after administration was 8 ± 1 nM [3]
2. Metabolic profile: - In mouse liver microsomes, MK-1454 was metabolized very little (turnover ≤10% after 2 hours of incubation), and no major metabolites were detected, indicating that it has high metabolic stability [3] |
| Toxicity/Toxicokinetics |
1. Acute toxicity in mice: - Female C57BL/6 mice were given a single intravenous injection of Ulevostinag (MK-1454) (10 mg/kg, 5 times the therapeutic dose). No significant weight loss (<5% change) or clinical discomfort (e.g., lethargy, diarrhea) was observed within 7 days. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were within the normal range (ALT: 45 ± 5 U/L; AST: 80 ± 8 U/L), indicating no acute hepatotoxicity [3] 2. Chronic toxicity in mice: - Mice were given intravenous injections of MK-1454 (1 mg/kg) twice a week for 4 weeks. Compared with the vector control group, no significant changes were observed in hematological parameters (red blood cells, white blood cells, platelets) or organ weight (liver, kidney, spleen) [3]
3. Plasma protein binding: - In human plasma, MK-1454 showed a high plasma protein binding rate (>95%), as determined by balanced dialysis. A similar binding rate (>94%) was also observed in mouse plasma [3] |
| References | |
| Additional Infomation |
1. Mechanism of action: - Ulevostinag (MK-1454) is a cyclic dinucleotide STING agonist that binds to STING, inducing its dimerization and activation. Activated STING recruits and phosphorylates TBK1, which in turn phosphorylates IRF3; phosphorylated IRF3 translocates to the nucleus, driving the transcription of type I interferons (e.g., IFN-β) and pro-inflammatory cytokines (e.g., TNF-α), thereby activating the innate immune system and enhancing anti-tumor immunity [3] 2. Clinical development background: - MK-1454 was developed for the treatment of cancer, with a focus on enhancing anti-tumor immunity by activating STING. Preclinical data showed that MK-1454 was effective as a monotherapy or in combination with immune checkpoint inhibitors (such as anti-PD-1 antibodies) in various mouse tumor models [3]. 3. Selectivity: - MK-1454 is highly selective for STING, superior to other immune-related targets (such as TLR4, TLR9, RIG-I), and these targets did not show significant activation at concentrations up to 1000 nM [3].
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| Molecular Formula |
C20H22F2N10O9P2S2
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| Molecular Weight |
710.52
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| Exact Mass |
710.0456
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| Elemental Analysis |
C, 33.81; H, 3.12; F, 5.35; N, 19.71; O, 20.27; P, 8.72; S, 9.02
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| CAS # |
2082743-96-0
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| Related CAS # |
Ulevostinag (isomer 1);2231258-61-8
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| PubChem CID |
149419198
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| Appearance |
White to off-white solid powder
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| LogP |
-1.2
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
19
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
45
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| Complexity |
1320
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| Defined Atom Stereocenter Count |
8
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| SMILES |
O=C1NC(N)=NC2N([C@H]3[C@]4(C([C@@]([H])(CO[P@@](=O)(S)O[C@@]5([C@]([H])(O[C@H]([C@H]5F)N5C=NC6C(N)=NC=NC5=6)CO[P@@](S)(O4)=O)[H])O3)(F)[H])[H])C=NC1=2
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| InChi Key |
YSUIQYOGTINQIN-UZFYAQMZSA-N
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| InChi Code |
InChI=1S/C20H22F2N10O9P2S2/c21-8-6-1-36-42(34,44)40-12-7(39-18(9(12)22)31-4-27-10-14(23)25-3-26-15(10)31)2-37-43(35,45)41-13(8)19(38-6)32-5-28-11-16(32)29-20(24)30-17(11)33/h3-9,12-13,18-19H,1-2H2,(H,34,44)(H,35,45)(H2,23,25,26)(H3,24,29,30,33)/t6-,7-,8-,9+,12-,13-,18-,19-,42?,43?/m1/s1
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| Chemical Name |
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| Synonyms |
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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 |
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| 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) |
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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 | 1.4074 mL | 7.0371 mL | 14.0742 mL | |
| 5 mM | 0.2815 mL | 1.4074 mL | 2.8148 mL | |
| 10 mM | 0.1407 mL | 0.7037 mL | 1.4074 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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