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Purity: ≥98%
Belnacasan (also known as VX-765) is an orally bioactive, novel, potent, and selective prodrug of VRT-043198, a powerful and specific inhibitor of caspase-1 with Ki values of 0.8 nM and less than 0.6 nM for caspase-1 and caspase-4, respectively. Belnacasan blocks the release of IL-1beta and IL-18, exhibiting strong anti-inflammatory effects. The caspase-1 subfamily of caspases includes caspase-1 and the IL-converting enzyme (ICE). Normal metabolism of VX-765 results in the active molecular VRT-043198. While VRT-043198 did not affect the secretion of other cytokines like IL-α, TNFα, IL-6, or IL-8 in cultures of peripheral blood mononuclear cells stimulated with bacterial products, it did inhibit the release of IL-1β nd IL-18. In other models that demonstrate how Caspase-1 works, this product is also utilized.
| Targets |
Caspase-4 (Kd < 0.6 nM); Caspase-1 (Ki = 0.8 nM)
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| ln Vitro |
VRT-043198, which exhibits potent inhibition against ICE/caspase-1 and caspase-4 with Ki of 0.8 nM and less than 0.6 nM, respectively, is an orally absorbed prodrug of VX-765. Additionally, VRT-043198 blocks the release of IL-1β from PBMCs and whole blood with IC50 values of 0.67 μM and 1.9 μM, respectively.[1]
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| ln Vivo |
In the collagen-induced arthritis mouse model, VX-765 (200 mg/kg) inhibits LPS-induced IL-1β production by about 60%, leading to a dose-dependent, statistically significant decrease in the inflammation scores and efficient joint protection.[1]
Without significantly affecting the length of the afterdischarge, VX-765 blocks kindling epileptogenesis in rats in vivo by preventing the growth of IL-1β in the forebrain astrocytes.[2] In the mouse model of acute seizures, VX-765 (50 mg/kg-200 mg/kg) causes the anticonvulsant effect by delaying the time until the first seizure begins and reducing the number of seizures as well as their total duration by an average of 50% and 64%.[3] After the third injection, VX-765 significantly decreases the cumulative duration and quantity of spike-and-wave discharges (SWDs) in adult rats with genetic absence epilepsy (GAERS) by blocking IL-1 biosynthesis with a specificity that results in a reduction of 55% on average.[4] |
| Enzyme Assay |
The rate of hydrolysis of a suitable substrate labeled with either p-nitroaniline or aminomethyl coumarin (AMC) is monitored to determine whether an enzyme is inhibited: Granzyme B, Ac-IEPD-AMC; caspase-3, -7, -8, and -9; caspase-4, Ac-WEHD-AMC; caspase-6, Ac-VEID-AMC; and ICE/caspase-1, suc-YVAD-p-nitroanilide. The reaction buffer, which contains 10 mM Tris, pH 7.5, 0.1% (w/v) CHAPS, 1 mM dithiothreitol, and 5% (v/v) dimethyl sulfoxide, is incubated with the enzymes and substrates for 10 minutes at 37 °C. To increase the stability of caspase-3, -6, and -9 and granzyme B, glycerol is added to the buffer at a concentration of 8% (v/v). Using a fluorometer, the rate of substrate hydrolysis is measured.
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| Cell Assay |
Before being exposed to LPS, PBMCs were pre-treated for 30 minutes with VX-765.
The therapeutic potential of VX-765 was assessed by determining the effects of VRT-043198 on cytokine release by monocytes in vitro and of orally administered VX-765 in several animal models in vivo. In cultures of peripheral blood mononuclear cells and whole blood from healthy subjects stimulated with bacterial products, VRT-043198 inhibited the release of interleukin (IL)-1beta and IL-18, but it had little effect on the release of several other cytokines, including IL-1alpha, tumor necrosis factor-alpha, IL-6 and IL-8. In contrast, VRT-043198 had little or no demonstrable activity in cellular models of apoptosis, and it did not affect the proliferation of activated primary T cells or T-cell lines. VX-765 was efficiently converted to VRT-043198 when administered orally to mice, and it inhibited lipopolysaccharide-induced cytokine secretion. In addition, VX-765 reduced disease severity and the expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation. These data suggest that VX-765 is a novel cytokine inhibitor useful for treatment of inflammatory diseases.[1] |
| Animal Protocol |
Mice: Belnacasan is injected intravenously as single doses (10, 21, 43, and 84 mg/kg) in a vehicle (25% Cremophor EL in water). Through the retroorbital sinus, blood samples (roughly 0.25-0.3 mL) are taken before the dose is administered as well as 0.167, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h later. These samples are processed for plasma. The concentration of Belnacasan and VRT-043198 is measured in plasma samples using a high-performance liquid chromatography/mass spectrometry methodology. Using WinNonlin Pro, version 4.0.1, noncompartmental analysis is performed.
Rats: Male Sprague-Dawley rats weighing 250–280 g are employed. Belnacasan (25, 50, or 200 mg/kg) is dissolved in 20% Cremophor and injected intraperitoneally (i.p.) into rats once daily for three straight days. Rats are given Belnacasan on the fourth day, 45 minutes and 10 minutes before intrahippocampal injections of kainic acid. Prior to the injection of kainic acid, respective controls receive a similar vehicle injection. |
| References | |
| Additional Infomation |
Belnacasan is a dipeptide.
VX-765 is the orally available prodrug of a potent and selective competitive inhibitor of ICE/caspase-1 (VRT-043198). VX-765 is currently under clinical development for the treatment of inflammatory and autoimmune conditions, as it blocks the hypersensitive response to an inflammatory stimulus. Drug Indication Investigated for use/treatment in inflammatory disorders (unspecified) and psoriasis and psoriatic disorders. Mechanism of Action VX-765 is a potent and selective inhibitor of ICE/caspase-1 sub-family caspases. In preclinical trials, VX-765 was efficiently converted to VRT-043198 when administered orally to mice and inhibited LPS-induced cytokine secretion. The result was a blocking of IL-1beta and IL-18 secretion, with out much effect on the release of several other cytokines, including IL-1{alpha}, tumor necrosis factor-{alpha}, IL-6 and IL-8. There was also no demonstrable activity in cellular models of apoptosis and it did not affect the proliferation of activated primary T-cells or T-cell lines. Pharmacodynamics VX-765 is an orally-absorbed pro-drug of VRT-043198, a potent and selective inhibitor of ICE/caspase-1 sub-family caspases. It has been shown to reduce disease severity and the expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation, suggesting that it may be useful for treatment of inflammatory diseases. |
| Molecular Formula |
C24H33CLN4O6
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| Molecular Weight |
508.99
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| Exact Mass |
508.208
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| Elemental Analysis |
C, 56.63; H, 6.53; Cl, 6.97; N, 11.01; O, 18.86
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| CAS # |
273404-37-8
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| Related CAS # |
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| PubChem CID |
11398092
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
779.0±60.0 °C at 760 mmHg
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| Flash Point |
424.9±32.9 °C
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| Vapour Pressure |
0.0±2.7 mmHg at 25°C
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| Index of Refraction |
1.589
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| LogP |
0.83
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
35
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| Complexity |
818
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| Defined Atom Stereocenter Count |
4
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| SMILES |
ClC1=C(C([H])=C([H])C(=C1[H])C(N([H])[C@]([H])(C(N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(N([H])[C@@]1([H])C([H])([H])C(=O)O[C@@]1([H])OC([H])([H])C([H])([H])[H])=O)=O)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)N([H])[H]
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| InChi Key |
SJDDOCKBXFJEJB-MOKWFATOSA-N
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| InChi Code |
InChI=1S/C24H33ClN4O6/c1-5-34-23-16(12-18(30)35-23)27-21(32)17-7-6-10-29(17)22(33)19(24(2,3)4)28-20(31)13-8-9-15(26)14(25)11-13/h8-9,11,16-17,19,23H,5-7,10,12,26H2,1-4H3,(H,27,32)(H,28,31)/t16-,17-,19+,23+/m0/s1
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| Chemical Name |
(2S)-1-[(2S)-2-[(4-amino-3-chlorobenzoyl)amino]-3,3-dimethylbutanoyl]-N-[(2R,3S)-2-ethoxy-5-oxooxolan-3-yl]pyrrolidine-2-carboxamide
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| Synonyms |
Belnacasan; VX 765; VX765; Belnacasan (VX-765); Belnacasan (VX765); Belnacasan [USAN]; (S)-1-((S)-2-(4-amino-3-chlorobenzamido)-3,3-dimethylbutanoyl)-N-((2R,3S)-2-ethoxy-5-oxotetrahydrofuran-3-yl)pyrrolidine-2-carboxamide; VX-765
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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) |
DMSO: ~100 mg/mL (~196.5 mM)
Ethanol: ~100 mg/mL (~196.5 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 3.33 mg/mL (6.54 mM) in 15% Cremophor EL + 85% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.91 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (4.91 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 2.5 mg/mL (4.91 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly. Solubility in Formulation 5: 2% DMSO+30% PEG 300+ddH2O: 5mg/mL Solubility in Formulation 6: 5 mg/mL (9.82 mM) in 50% PEG300 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9647 mL | 9.8234 mL | 19.6468 mL | |
| 5 mM | 0.3929 mL | 1.9647 mL | 3.9294 mL | |
| 10 mM | 0.1965 mL | 0.9823 mL | 1.9647 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT05164120 | Completed | Drug: Placebo Drug: Belnacasan |
COVID-19 | MedStar Health | December 14, 2021 | Phase 2 |
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