Belnacasan  (Synonyms: VX-765)

Enzyme inhibition is assayed by tracking of the rate of hydrolysis of an appropriate substrate labeled with either p-nitroaniline or aminomethyl coumarin (AMC) as follows: ICE/caspase-1, suc-YVAD-p-nitroanilide; caspase-4, Ac-WEHD-AMC; caspase-6, Ac-VEID-AMC; caspase-3, -7, -8, and -9, Ac-DEVD-AMC; and granzyme B, Ac-IEPD-AMC. Enzymes and substrates are incubated in a reaction buffer [10 mM Tris, pH 7.5, 0.1% (w/v) CHAPS, 1 mM dithiothreitol, and 5% (v/v) DMSO] for 10 min at 37°C. Glycerol is added to the buffer at 8% (v/v) for caspase-3, -6, and -9 and granzyme B to improve stability of enzymes. The rate of substrate hydrolysis is monitored using a fluorometer. Assays for cathepsin B and trypsin are performed^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

A total of 2×10⁵ cells/well (100 μL cell suspension) is distributed in triplicate in flat-bottom 96-well plates. Either 50 μL of Belnacasan (40 μM in RPMI 1640 complete medium containing 0.2% DMSO) or vehicle control is added to appropriate wells. Following a 30-min incubation at 37°C, 50 μL of LPS diluted in RPMI 1640 complete medium is added at final concentrations varying from 0.001 to 10 ng/mL. Cells are returned to a 37°C incubator. At 4 h after LPS addition, 75 μL of supernatant is removed from wells, cleared by centrifugation for 5 min at 1500 rpm, and stored at 4°C until assayed. Cells are returned to a 37°C incubator until 24 h after LPS addition, at which time 100 μL of supernatant is removed, cleared by centrifugation, and stored at 4°C. Supernatants are tested using ELISA kits for IL-1β, IL-6, IL-18, and IL-1α^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[2]
Single doses of Belnacasan (10, 21, 43, and 84 mg/kg) in vehicle (25% Cremophor EL in water) are administered via oral gavage. Blood samples (approximately 0.25-0.3 mL) are collected before dose administration and 0.167, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 h after dosing via the retroorbital sinus and processed for plasma. A high-performance liquid chromatography/mass spectrometry methodology is used to determine the concentration of Belnacasan and VRT-043198 in plasma samples. Noncompartmental analysis is carried out using WinNonlin Pro, version 4.0.1.
Rats^[3]
Male Sprague-Dawley rats (250-280 g) are used. Belnacasan (25, 50, 200 mg/kg) is dissolved in 20% Cremophor and injected ip in rats once a day for 3 consecutive days. On the fourth day, rats receive Belnacasan, 45 min and 10 min before intrahippocampal injection of kainic acid. Respective controls are similarly injected with vehicle before kainic acid.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Stack JH, et al. IL-converting enzyme/caspase-1 inhibitor VX-765 blocks the hypersensitive response to an inflammatory stimulus in monocytes from familial cold autoinflammatory syndrome patients. J Immunol. 2005 Aug 15;175(4):2630-4.  [Content Brief]

  [2]. Wannamaker W, et al. (S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765), an orally available selective interleukin (IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1beta and IL-18. J Pharmacol Exp Ther. 2007 May;321(2):509-16.  [Content Brief]

  [3]. Ravizza T, et al. Inactivation of caspase-1 in rodent brain: a novel anticonvulsive strategy. Epilepsia. 2006 Jul;47(7):1160-8.  [Content Brief]