Caspase-4 (Kd < 0.6 nM); Caspase-1 (Ki = 0.8 nM)

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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Selectivity of VRT-43198 against Caspases and Other Proteases. We evaluated in vitro the potency of VRT-043198 against ICE/caspase-1 and caspase-4 and its selectivity against representatives of the three subfamilies of caspases, and other proteases, including granzyme B and trypsin (serine proteases), and cathepsin B (cysteine protease). As shown in Table 1, VRT-043198 exhibited potent inhibition of ICE/caspase-1 (Ki = 0.8 nM) and caspase-4 (Ki < 0.6 nM) and at least 100-fold lower potency.

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Cytokine release [5]
Because of the evidence suggesting the involvement of cryopyrin in IL-1β production, we measured IL-1β secretion from PBMCs obtained from FCAS patients and controls. Levels of IL-1β secreted after 4- or 24-h exposure to LPS were markedly greater for PBMCs from FCAS patients than unaffected donors (Fig. 1). IL-1β released in the absence of LPS treatment was below the level of detection in PBMCs from patients and controls. Exposure to as little as 0.01 ng/ml LPS for 4 h, which produced negligible IL-1β secretion from control cells, stimulated robust secretion from PBMCs of FCAS patients. IL-1β released at the highest concentration of LPS (10 ng/ml) was 2.1- to 2.7-fold higher in PBMCs from FCAS patients. Consistent with the involvement of ICE/caspase-1 in the processing of pro-IL-1β, the caspase-1 inhibitor Belnacasan (VX765) (10 μM) markedly (>80%) inhibited release of IL-1β from both FCAS and control cells (Fig. 1).
Before its release from monocytes, IL-18, like IL-1β, undergoes proteolytic maturation by ICE/caspase-1, so levels of this cytokine in cell medium were also measured. The level of IL-18 release was ∼100-fold lower than that of IL-1β. As with IL-1β, LPS-stimulated release of IL-18 was strikingly higher in PBMCs from FCAS patients compared with control subjects and was markedly inhibited by Belnacasan (VX765) at 10 μM (Fig. 2). Also like IL-1β, there was no detectable constitutive release of IL-18 in the absence of LPS exposure, while there was significant IL-18 release from FCAS cells at low LPS concentrations that did not produce IL-18 release in normal cells.
To determine whether the abnormality in FCAS PBMCs is simply a general hyperinflammatory state or a specific defect in caspase-1 regulation, we also measured IL-6 and IL-1α in the cell medium after 24-h exposure to LPS (Fig. 3). In marked contrast to IL-1β and IL-18, secretion of IL-6 was not increased in PBMCs from FCAS compared with control subjects. Secretion of IL-1α appeared to be lower in FCAS than in controls (Fig. 3), although results were available from only two control subjects. At the highest concentration of LPS (10 ng/ml), IL-1α secretion was reduced by ∼50% in the presence of Belnacasan (VX765) (10 μM). Interestingly, IL-1α secretion is reduced in macrophages from ICE/caspase-1 knockout mice (18), suggesting that IL-1α secretion is partially dependent on ICE/caspase-1 or IL-1β levels.

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Inhibitory potency of Belnacasan (VX765) [5]
Because the mutations in FCAS appear to affect the activity state of ICE/caspase-1, we tested whether the potency of Belnacasan (VX765) to inhibit ICE/caspase-1-mediated IL-1β production is altered by the mutations. The ability of Belnacasan (VX765) to inhibit IL-1β release was determined using PBMCs from three FCAS and three control subjects, measured after 24-h exposure to 10 ng/ml LPS. Belnacasan (VX765) inhibited IL-1β release with similar potency in PBMCs from FCAS (IC50 = 0.99 ± 0.29 μM) and control (IC50 = 1.10 ± 0.61 μM) subjects (Fig. 5).

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]

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Belnacasan (VX765) 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]

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An enhanced production of IL-1beta in glia is a typical feature of epileptogenic tissue in experimental models and in human drug-refractory epilepsy. We show here that the selective inhibition of Interleukin Converting Enzyme (ICE), which cleaves the biologically active form of IL-1beta using Belnacasan (VX765), blocks kindling development in rats by preventing IL-1beta increase in forebrain astrocytes, without interfering with glia activation. The average afterdischarge duration was not altered significantly by VX-765. Up to 24 h after kindling completion and drug washout, kindled seizures could not be evoked in treated rats. VX-765 did not affect seizures or afterdischarge duration in fully kindled rats. These data indicate an antiepileptogenic effect mediated by ICE inhibition and suggest that specific anti-IL-1beta pharmacological strategies can be envisaged to interfere with epileptogenic mechanisms.[2]

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In this study, the anticonvulsant activity of Belnacasan (VX765) (a selective ICE/caspase-1 inhibitor) was examined in a mouse model of chronic epilepsy with spontaneous recurrent epileptic activity refractory to some common anticonvulsant drugs. Moreover, the effects of this drug were studied in one acute model of seizures in mice, previously shown to involve activation of ICE/caspase-1. Quantitative analysis of electroencephalogram activity was done in mice exposed to acute seizures or those developing chronic epileptic activity after status epilepticus to assess the anticonvulsant effects of systemic administration of VX-765. Histological and immunohistochemical analysis of brain tissue was carried out at the end of pharmacological experiments in epileptic mice to evaluate neuropathology, glia activation and IL-1β expression, and the effect of treatment. Repeated systemic administration of VX-765 significantly reduced chronic epileptic activity in mice in a dose-dependent fashion (12.5-200 mg/kg). This effect was observed at doses ≥ 50 mg/kg, and was reversible with discontinuation of the drug. Maximal drug effect was associated with inhibition of IL-1β synthesis in activated astrocytes. The same dose regimen of VX-765 also reduced acute seizures in mice and delayed their onset time. These results support a new target system for anticonvulsant pharmacological intervention to control epileptic activity that does not respond to some common anticonvulsant drugs [3].

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.

Before being exposed to LPS, PBMCs were pre-treated for 30 minutes with Belnacasan (VX765).

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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]

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Assay for secreted cytokines [5]
A total of 2 × 105 cells/well (100 μl cell suspension) was distributed in triplicate in flat-bottom 96-well plates. Either 50 μl of Belnacasan (VX765) (40 μM in RPMI 1640 complete medium containing 0.2% DMSO) or vehicle control was added to appropriate wells. Following a 30-min incubation at 37°C, 50 μl of LPS diluted in RPMI 1640 complete medium was added at final concentrations varying from 0.001 to 10 ng/ml. Cells were returned to a 37°C incubator. At 4 h after LPS addition, 75 μl of supernatant was removed from wells, cleared by centrifugation for 5 min at 1500 rpm, and stored at 4°C until assayed. Cells were returned to a 37°C incubator until 24 h after LPS addition, at which time 100 μl of supernatant was removed, cleared by centrifugation, and stored at 4°C. Supernatants were tested using ELISA kits for IL-1β, IL-6, IL-18, and IL-1α, according to the manufacturers’ instruction.

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Immunoblotting [5]
A total of 106 cells/well (500 μl cell suspension) was distributed in 24-well plates. Cells were treated with Belnacasan (VX765) and LPS, as described above, adjusted for 1 ml final volume. At 4 h after addition of LPS, all of the medium was transferred to 1.5-ml tubes and centrifuged at 1000 × g for 5 min at 4°C to pellet the suspended cells, and supernatants were removed. To lyse attached cells, 100 μl of 1× NuPage sample buffer plus 2-ME were added to each well, and plates were placed on an orbital shaker. The 100 μl of sample buffer in the wells was transferred to corresponding pellets and boiled for 15 min, then frozen at −20°C. Samples were boiled again before loading onto a 4–12% Bis-Tris NuPage gel and run with NuPage MES running buffer. Gels were transferred to nitrocellulose filters. Filters were blotted in TBST (0.05% Tween 20) + 5% dry milk. For IL-1β immunoblotting, blots were incubated overnight at 4°C with 1:2,000 mouse anti-IL-1βprimary Ab, then incubated with 1:10,000 HRP goat anti-mouse secondary Ab for 1 h, and developed with ECL.

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.

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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.

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Kindling development [2]
Belnacasan (VX765) (200 mg/kg) was dissolved in 20% cremophor and injected intraperitoneally (i.p.) in rats once a day for 3 consecutive days (n = 12); control rats (n = 12) received the corresponding vehicle. On the 4th day, rats received Belnacasan (VX765) or vehicle, 45 min before the beginning of the electrical stimulation. We adopted this treatment protocol since it provided significant protection from seizures induced by intrahippocamapl injection of kainic acid in rats (Ravizza et al., 2006b), and this effect was associated with inhibition of pro-IL-1β processing and of the consequent production of the biologically active form of IL-1β in the hippocampus (Ravizza et al., 2006b). During the stimulation protocol VX-765, or its vehicle, was injected 3 times every 90 min since previous experiments showed that the drug effect on kainic acid--induced seizures was maintained for 90 min slowly decreasing thereafter (Ravizza et al., 2006b). In preliminary experiments, VX-765 was also administered at 50 mg/kg (n = 5) but this dose did not affect kindling parameters (not shown).

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Fully kindled rats [2]
After the re-test session (24 h after kindling completion), rats (n = 7) were treated for 3 consecutive days with Belnacasan (VX765), as described above; on the 4th day, Belnacasan (VX765) was administered once, and 45 min later rats received 5 electrical stimulations to evoke fully kindled seizures. The same rats, after 3 days of drug washout, received vehicle using the same treatment and stimulation protocol adopted with VX-765.

Based upon the data provided in this panel, it was clear that these agents represent important new tools for caspase 1 inhibition. However, the contributing functional groups for these agents (i.e. ethyl acetals, aldehydes, nitriles and esters) are all subject to hydrolysis in various conditions. It was paramount to fully understand their stability profile to appreciate their utility as molecular probes or even clinically used agents. Therefore, we examined 1/Belnacasan (VX765) , 2b, 3, 4 and 16 within an aqueous degradation study at neutral (pH 7), acidic (pH 2), and basic (pH 8) conditions. The study was conducted by monitoring the degradation of each agent by LCMS analysis at various time points over 96 hours (Figure 3). The prodrug 1Belnacasan (VX765) / showed moderate degradation in water with over 50% of the compound decomposed after 48 hours. This degradation was amplified in both basic and acidic conditions. Conversely, the active agent 2b was very stable in both neutral and acidic conditions and its degradation at pH 8 was moderate. The potent 4 was exceedingly stable in basic conditions and its stability in neutral and acidic conditions was moderate to good (degradation of 50% in both conditions after 72 hours). The ethyl ester 3 was exceptionally stable in neutral and acidic conditions (no degradation noted), however, it was fully degraded in basic conditions after 22 hours (presumably due to saponification of the ester). Finally, the tetrazole 16 was found to be resistant to degradation in all conditions. Interestingly, this data suggests that 1/Belnacasan (VX765) may have a short half-life as an oral agent due to its instability in acidic conditions such as those found in the gastric environment (40% degradation after 3.5 hours at pH 2). In contrast, this data highly suggests that 3 and 16 will be suitable reagents for all manner of examinations (cell based and in vivo studies) and even the highly active 4 will persist beyond 24 hours.[https://pubmed.ncbi.nlm.nih.gov/20229566/]

[1]. J Pharmacol Exp Ther . 2007 May;321(2):509-16.

[2]. Neurobiol Dis . 2008 Sep;31(3):327-33.

[3]. Neurotherapeutics . 2011 Apr;8(2):304-15.

[4]. Neurobiol Dis . 2011 Dec;44(3):259-69.

[5]. J Immunol . 2005 Aug 15;175(4):2630-4.

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.

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Drug Indication
Investigated for use/treatment in inflammatory disorders (unspecified) and psoriasis and psoriatic disorders.

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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.

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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.

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(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) is an orally absorbed prodrug of (S)-3-({1-[(S)-1-((S)-2-{[1-(4-amino-3-chlorophenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidin-2yl]-methanoyl}-amino)-4-oxo-butyric acid (VRT-043198), a potent and selective inhibitor of interleukin-converting enzyme/caspase-1 subfamily caspases. VRT-043198 exhibits 100- to 10,000-fold selectivity against other caspase-3 and -6 to -9. 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]

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Interleukin (IL)-1β plays a crucial role in the mechanisms of limbic seizures in rodent models of temporal lobe epilepsy. We addressed whether activation of the IL-1β signaling occurs in rats with genetic absence epilepsy (GAERS) during the development of spike-and-wave discharges (SWDs). Moreover, we studied whether inhibition of IL-1β biosynthesis in GAERS could affect SWD activity. IL-1β expression and glia activation were studied by immunocytochemistry in the forebrain of GAERS at postnatal days (PN)14, PN20, and PN90 and in age-matched non-epileptic control Wistar rats. In PN14 GAERS, when no SWDs have developed yet, IL-1β immunostaining was undetectable, and astrocytes and microglia showed a resting phenotype similar to control Wistar rats. In 3 out of 9 PN20 GAERS, IL-1β was observed in activated astrocytes of the somatosensory cortex; the cytokine expression was associated with the occurrence of immature-type of SWDs. In all adult PN90 GAERS, when mature SWDs are established, IL-1β was observed in reactive astrocytes of the somatosensory cortex but not in adjacent cortical areas or in extra-cortical regions. An age-dependent c-fos activation was found in the somatosensory cortex of GAERS with maximal levels reached in PN90 rats; c-fos was also induced in some thalamic nuclei in PN20 and PN90 GAERS. Inhibition of IL-1β biosynthesis in PN90 GAERS by 4-day systemic administration of a specific ICE/Caspase-1 blocker, significantly reduced both SWD number and duration. These results show that IL-1β is induced in reactive astrocytes of the somatosensory cortex of GAERS at the onset of SWDs. IL-1β has pro-ictogenic properties in this model, and thus it may play a contributing role in the mechanisms underlying the occurrence of absence seizures. [4]

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Familial cold autoinflammatory syndrome (FCAS) and the related autoinflammatory disorders, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, are characterized by mutations in the CIAS1 gene that encodes cryopyrin, an adaptor protein involved in activation of IL-converting enzyme/caspase-1. Mutations in cryopyrin are hypothesized to result in abnormal secretion of caspase-1-dependent proinflammatory cytokines, IL-1beta and IL-18. In this study, we examined cytokine secretion in PBMCs from FCAS patients and found a marked hyperresponsiveness of both IL-1beta and IL-18 secretion to LPS stimulation, but no evidence of increased basal secretion of these cytokines, or alterations in basal or stimulated pro-IL-1beta levels. VX-765, an orally active IL-converting enzyme/caspase-1 inhibitor, blocked IL-1beta secretion with equal potency in LPS-stimulated cells from FCAS and control subjects. These results further link mutations in cryopyrin with abnormal caspase-1 activation, and support the clinical testing of caspase-1 inhibitors such as VX-765 in autoinflammatory disorders. [5]

C24H33CLN4O6

508.99

508.208

C, 56.63; H, 6.53; Cl, 6.97; N, 11.01; O, 18.86

273404-37-8

11398092

White to off-white solid powder

1.3±0.1 g/cm3

779.0±60.0 °C at 760 mmHg

424.9±32.9 °C

0.0±2.7 mmHg at 25°C

1.589

0.83

3

7

8

35

818

4

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]

SJDDOCKBXFJEJB-MOKWFATOSA-N

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

(2S)-1-[(2S)-2-[(4-amino-3-chlorobenzoyl)amino]-3,3-dimethylbutanoyl]-N-[(2R,3S)-2-ethoxy-5-oxooxolan-3-yl]pyrrolidine-2-carboxamide

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

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)

DMSO: ~100 mg/mL (~196.5 mM)
Ethanol: ~100 mg/mL (~196.5 mM)

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.

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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.

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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.
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 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.

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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.

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Solubility in Formulation 5: 2% DMSO+30% PEG 300+ddH2O: 5mg/mL

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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.

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