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Purity: ≥98%
Vesatolimod (formerly known as GS9620; GS-9620) is a novel potent, selective and orally bioactive small molecule agonist of Toll-like receptor 7 (TLR-7) with antiviral activity. It activates TLR-7 with an EC50 of 291 nM. Vesatolimod has the potential for the treatment of chronic hepatitis B viral (HBV) infection. GS-9620 selectively induces IFN-α, cytokines and chemokines. The minimum effective concentrations for IFN-α induction were similar in pDCs and in PBMCs from HCV-positive donors. GS-9620 demonstrates an EC50 of 291 nM for human TLR7, which is 30-fold selectivity over TLR8 with EC50 of 9 μM.
| Targets |
Vesatolimod (GS-9620) targets Toll-Like Receptor 7 (TLR7) with an EC50 of 0.13 μM in human TLR7-transfected HEK293 cells [1]
Vesatolimod (GS-9620) specifically activates human TLR7 with an EC50 of 0.08 μM, rhesus monkey TLR7 with EC50 0.11 μM, and mouse TLR7 with EC50 0.32 μM (selective over TLR3, TLR8, TLR9 with EC50 >10 μM) [3] |
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| ln Vitro |
Vesatolimod (GS-9620) enters cells quickly, localizes to endo-lysosomal compartments preferentially, and communicates from these areas. The kinetics of the compound's cellular absorption in Daudi cells using tritiated Vesatolimod (3H-GS-9620) is studied in order to test this notion. The buildup of 3H-GS-9620 proceeds quickly; it takes about thirty minutes to attain a concentration-dependent steady-state equilibrium. The concentration of 3H-Vesatolimod measured within cells is five times more than the concentration of 3H-GS-9620 used outside cells for treatment. Elevations in 3H-GS-9620 concentrations are approximately correlated with increases in intracellular 3H-Vesatolimod concentrations[1].
Vesatolimod (GS-9620) induced dose-dependent activation of TLR7 in HEK293 cells transfected with human TLR7, resulting in increased luciferase reporter activity (EC50=0.13 μM) and upregulated mRNA expression of IFN-α, IL-6, and TNF-α [1] Vesatolimod (GS-9620) stimulated human peripheral blood mononuclear cells (PBMCs) to secrete IFN-α (EC50=0.27 μM), IL-12p40 (EC50=0.31 μM), and TNF-α (EC50=0.45 μM), with no significant activation of TLR3, TLR8, or TLR9 even at 10 μM [3] Vesatolimod (GS-9620) enhanced the production of antiviral cytokines (IFN-α, IFN-γ) in primary human hepatocytes co-cultured with immune cells, and inhibited HBV replication in vitro (IC50=0.38 μM in HepG2.2.15 cells) [2] |
| ln Vivo |
In uninfected chimpanzees, single oral doses of Vesatolimod (GS-9620) at 0.3 and 1 mg/kg show a dose- and exposure-related elevation of serum IFN-α, some cytokines/chemokines, and IFN-stimulated genes (ISG) in the liver and peripheral circulation. The Cmax values of Vesatolimod (GS-9620) after oral administration at 0.3 (n=3) and 1 mg/kg (n=3 and n=4) are 3.6±3.5, 36.8±34.5, and 55.4± 81.0 nM, respectively. After 8 hours, serum IFN responses reach their peak. At dosages of 0.3 and 1 mg/kg, the mean peak values of induced serum IFN-α are 479 and 66 pg/mL, respectively. Treatment with vesatolimod (GS-9620) produces ISG transcripts in peripheral blood mononuclear cells (PBMC) at 0.3 mg/kg and in the liver at 1 mg/kg. These transcripts include ISG15, OAS-1, MX1, IP-10 (CXCL10), and I-TAC (CXCL11)[2].
In chronically HBV-infected chimpanzees, oral administration of Vesatolimod (GS-9620) (1 mg/kg once weekly for 12 weeks) reduced serum HBV DNA by 1.8–2.5 log10 copies/mL, with suppression maintained for 16–24 weeks post-treatment; serum HBsAg levels decreased by 0.3–0.8 log10 IU/mL, and intrahepatic cccDNA levels were reduced by 30–40% [2] In C57BL/6 mice, oral Vesatolimod (GS-9620) (0.3–3 mg/kg) induced dose-dependent increases in serum IFN-α (peak at 6–12 h) and IL-6 (peak at 24 h), with IFN-α mRNA upregulated in spleen (8-fold) and liver (5-fold) at 3 mg/kg [3] |
| Enzyme Assay |
Construct expression vectors containing full-length human TLR7, TLR3, TLR8, or TLR9, and transfect them into HEK293 cells along with a NF-κB-responsive luciferase reporter plasmid. After 24 h of transfection, treat cells with serial dilutions of Vesatolimod (GS-9620) (0.001–10 μM) and incubate for 18 h. Lyse cells and measure luciferase activity to determine EC50 for each TLR subtype [1]
Perform a TLR ligand competition assay using biotin-labeled TLR7 agonist. Incubate recombinant human TLR7 protein with Vesatolimod (GS-9620) (0.01–10 μM) for 1 h, then add biotin-labeled ligand and incubate for another 2 h. Detect bound biotin-ligand using streptavidin-HRP and measure absorbance to assess competitive binding affinity [3] |
| Cell Assay |
Isolate human PBMCs from healthy donors and resuspend in RPMI 1640 medium supplemented with 10% FBS. Seed cells into 96-well plates (2×105 cells/well) and treat with Vesatolimod (GS-9620) at concentrations of 0.01–10 μM. After 24 h of incubation, collect supernatants to measure IFN-α, IL-12p40, and TNF-α levels by ELISA. For mRNA analysis, lyse cells at 6 h post-treatment, extract total RNA, and perform qPCR to quantify IFN-α, IL-6, and TLR7 mRNA expression [1]
Culture HepG2.2.15 cells (HBV-replicating hepatocytes) in DMEM medium with 10% FBS. Treat cells with Vesatolimod (GS-9620) (0.05–5 μM) for 72 h. Collect culture supernatants to measure HBV DNA levels by real-time PCR, and intracellular HBV core antigen (HBcAg) by immunofluorescence staining [2] |
| Animal Protocol |
1 and 2 mg/kg; oral
Chimpanzees Chimpanzees chronically infected with HBV (serum HBV DNA >104 copies/mL, HBsAg positive for >6 months) were used. Vesatolimod (GS-9620) was formulated as an oral suspension in 0.5% methylcellulose. Animals were dosed orally at 1 mg/kg once weekly for 12 consecutive weeks. Serum samples were collected weekly for HBV DNA, HBsAg, and cytokine (IFN-α, IL-6) analysis. Liver biopsies were performed before and after treatment to measure intrahepatic cccDNA and HBV RNA levels [2] C57BL/6 mice (6–8 weeks old) were divided into 4 groups (n=5/group). Vesatolimod (GS-9620) was administered via oral gavage at doses of 0.3 mg/kg, 1 mg/kg, or 3 mg/kg. A control group received vehicle (0.5% methylcellulose). Blood samples were collected at 2 h, 6 h, 12 h, 24 h, and 48 h post-dosing to measure serum cytokines and drug concentrations. Spleen and liver tissues were harvested at 6 h post-dosing for mRNA analysis [3] |
| ADME/Pharmacokinetics |
In rhesus monkeys, after oral administration of vesatolimod (GS-9620) (1 mg/kg), the peak plasma concentration (Cmax) was 89 ng/mL 2 hours after administration, the oral bioavailability was 42%, the terminal half-life (t1/2) was 18.3 hours, the volume of distribution (Vd) was 1.2 L/kg, and the total clearance (CL) was 0.04 L/h/kg [3]. In chimpanzees, after oral administration of vesatolimod (GS-9620) (1 mg/kg), the peak plasma concentration (Cmax) was 102 ng/mL 3 hours after administration, the t1/2 was 21.5 hours, and the steady-state plasma concentration remained above 10 ng/mL for 7 days after administration [2].
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| Toxicity/Toxicokinetics |
In a 4-week subchronic toxicity study in rhesus monkeys, no significant adverse reactions were observed with oral doses up to 5 mg/kg/week of Vesatolimod (GS-9620). No changes were observed in body weight, hematological parameters (white blood cells, red blood cells, platelets) or clinical chemistry indicators (ALT, AST, creatinine, BUN). Histopathological examination of the liver, kidneys, spleen and lymph nodes revealed no drug-related lesions [3]. No acute or chronic toxicity was observed in chimpanzees treated with 1 mg/kg/week for 12 weeks. Serum liver enzymes (ALT, AST) and renal function indicators (creatinine, BUN) remained within the normal range throughout the study period [2].
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| References | |
| Additional Infomation |
Vesatolimod is an investigational drug being studied for the treatment of HIV infection. Vesatolimod belongs to a class of HIV drugs called immunomodulators. Immunomodulators (also known as immunomodulators) are substances that can activate, enhance or restore normal immune function. The potential role of Vesatolimod as a latency reversal agent has also been evaluated in preclinical and clinical trials; however, the results to date have been mixed. Vesatolimod has been used in clinical trials to study the treatment of hepatitis B and chronic hepatitis B. Vesatolimod (GS-9620) activates TLR7 by binding to the intracellular domain of the receptor, triggering downstream NF-κB and IRF signaling pathways, and inducing type I interferon and pro-inflammatory cytokines, thereby exerting an anti-HBV effect [1]. Vesatolimod (GS-9620) exhibits high selectivity for TLR7 and, at concentrations up to 10 μM, shows no significant activation of other TLR family members (TLR1-TLR6, TLR8-TLR13) [3]. In chronically HBV-infected chimpanzees, HBV suppression induced by Vesatolimod (GS-9620) was associated with elevated intrahepatic HBV levels. This resulted in the production of IFN-γ-producing T cells and a reduction in HBV-specific regulatory T cells [2].
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| Molecular Formula |
C22H30N6O2
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|---|---|---|
| Molecular Weight |
410.52
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| Exact Mass |
410.243
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| CAS # |
1228585-88-3
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| Related CAS # |
1228585-88-3;1454806-18-8 (HCl);
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| PubChem CID |
46241268
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| Appearance |
White to yellow solid powder
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| Density |
1.2±0.1 g/cm3
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| Index of Refraction |
1.622
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| LogP |
3.1
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
30
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| Complexity |
558
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
VFOKSTCIRGDTBR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H30N6O2/c1-2-3-11-30-22-25-20(23)19-21(26-22)28(15-18(29)24-19)14-17-8-6-7-16(12-17)13-27-9-4-5-10-27/h6-8,12H,2-5,9-11,13-15H2,1H3,(H,24,29)(H2,23,25,26)
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| Chemical Name |
4-amino-2-butoxy-8-[[3-(pyrrolidin-1-ylmethyl)phenyl]methyl]-5,7-dihydropteridin-6-one
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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) |
Solubility in Formulation 1: ≥ 1.67 mg/mL (4.07 mM) (saturation unknown) in 10% DMSO + 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 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: 1.67 mg/mL (4.07 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 1.67 mg/mL (4.07 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4359 mL | 12.1797 mL | 24.3593 mL | |
| 5 mM | 0.4872 mL | 2.4359 mL | 4.8719 mL | |
| 10 mM | 0.2436 mL | 1.2180 mL | 2.4359 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.
 GS-9620 rapidly distributes to and signals through the endo-lysosomal compartments. Fig 3. Amino acid changes due to described single nucleotide polymorphisms (SNPs) do not impact GS-9620-dependent TLR7 activation.PLoS One.2016 Jan 19;11(1):e0146835. |
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 Structure-based mutational analysis identifies residues in TLR7 that are essential for GS-9620 in vitro activity.. From: Molecular Determinants of GS-9620-Dependent TLR7 Activation.PLoS One.2016 Jan 19;11(1):e0146835. |
 GS-9620 induces phosphorylation of NF-κB and Akt in pDCs.
TLR7 dimers exist independent of GS-9620 binding.PLoS One.2016 Jan 19;11(1):e0146835. |
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