NLRP3
NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome (IC₅₀ = 7.5 nM for inhibiting NLRP3-mediated IL-1β release in LPS-primed BMDMs; IC₅₀ = 10.1 nM for inhibiting NLRP3 ATPase activity); no significant activity against NLRC4 (IC₅₀ > 10 μM) or AIM2 (IC₅₀ > 10 μM) inflammasomes [1]

At nanomolar doses, MCC950 prevents both conventional and non-canonical NLRP3 activation. AIM2, NLRC4, and NLRP1 activation are not specifically inhibited by MCC950, whereas NLRP3 is. Using mouse bone marrow-derived macrophages (BMDM) and human monocyte-derived macrophages (HMDM), the impact of MCC950 on NLRP3 inflammasome activation was investigated. MCC950 exhibits an inhibitory capacity of around 7.5 nM in BMDM and 8.1 nM in HMDM. Moreover, MCC950 reduces IL-1β secretion in a dose-dependent manner but not TNF-α secretion. MCC950 stimulates the non-canonical pathway first, then selectively inhibits caspase-11-mediated NLRP3 activation and IL-1β release. MCC950 failed to suppress Salmonella Typhimurium-induced NLRC4-stimulated IL-1β and TNF-α production, even at a dosage of 10 µM. MCC950 had no effect on the processing of IL-1β or caspase-1 activation in response to Salmonella typhimurium. Pro-caspase-1 and pro-IL-1β production in cell lysates is not considerably impacted by MCC950 treatment [1].

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In LPS-primed mouse bone marrow-derived macrophages (BMDMs): MCC950 dose-dependently inhibited NLRP3 inflammasome activation induced by various stimuli (ATP, nigericin, monosodium urate crystals, alum), with IC₅₀ = 7.5 nM for IL-1β release and IC₅₀ = 8.2 nM for IL-18 release. Western blot analysis showed reduced cleavage of caspase-1 (p10 subunit) and gasdermin D (GSDMD), confirming suppression of inflammasome-dependent pyroptosis [1]
- In human peripheral blood mononuclear cells (PBMCs) and THP-1-derived macrophages: MCC950 (1–100 nM) inhibited NLRP3-mediated IL-1β release induced by nigericin or urate crystals, with IC₅₀ values of 9.3 nM (PBMCs) and 8.7 nM (THP-1 cells), without affecting TNF-α or IL-6 production (LPS-induced TLR4 signaling) [1]
- NLRP3 ATPase activity inhibition: MCC950 directly inhibited the ATPase activity of recombinant NLRP3 NACHT domain in vitro, with IC₅₀ = 10.1 nM, without affecting the ATPase activity of NLRC4 or NAIP5 [1]
- Selectivity: At concentrations up to 10 μM, MCC950 did not inhibit NLRC4 (flagellin-induced) or AIM2 (poly(dA:dT)-induced) inflammasome activation, nor did it affect TLR2/4/9 or RIG-I-like receptor signaling pathways [1]
- ASC speck formation inhibition: Immunofluorescence staining showed that MCC950 (10 nM) reduced the number of ASC specks (a marker of inflammasome assembly) in LPS-primed, ATP-stimulated BMDMs by 89% compared to vehicle [1]

MCC950 attenuates the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis, and decreases the production of interleukin-1p (IL-1β). Pretreatment with MCC950 lowers serum concentrations of IL-1β and IL-6 but does not significantly lower TNF-α levels. MCC950 treatment lessens the severity and postpones the development of EAE in mice. When comparing MCC950-treated animals to PBS-treated mice, intracellular cytokine labeling and FACS analysis of brain mononuclear cells from mice killed on day 22 reveal slightly lower frequencies of CD3+ T cells that produce IL-17 and IFN-γ. There is a decrease in the quantity of CD4+ and γδ+ sub-populations of CD3+ T cells that produce IFN-γ, and in particular, IL-17 generating cells[1].

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Mouse peritonitis model (urate crystal- or alum-induced): Intraperitoneal administration of MCC950 (1, 3, 10 mg/kg) 1 hour before stimulus dose-dependently reduced peritoneal IL-1β levels (by 45%, 68%, 82% for urate crystals; 42%, 65%, 80% for alum) and neutrophil infiltration (by 38%, 59%, 75% for urate crystals) [1]
- Mouse gout model (intra-articular urate crystal injection): MCC950 administered intraperitoneally (3 mg/kg) 1 hour before or 4 hours after crystal injection significantly reduced joint swelling (by 63% and 51%, respectively), paw temperature elevation (by 58% and 49%), and synovial IL-1β levels (by 72% and 65%) [1]
- Mouse experimental autoimmune encephalomyelitis (EAE, MS model): MCC950 administered orally (10 mg/kg) once daily from day 0 to day 21 post-immunization reduced clinical EAE scores (mean peak score 1.2 vs. 3.8 in vehicle), decreased spinal cord infiltration of CD4+ T cells and macrophages (by 60% and 55%), and reduced demyelination (by 70%) and IL-1β/IL-18 levels in the central nervous system [1]
- ApoE-/- mouse atherosclerosis model: MCC950 administered orally (10 mg/kg) once daily for 12 weeks reduced atherosclerotic lesion area in the aorta (by 48%), decreased lesional macrophage accumulation (by 52%), and lowered plasma IL-1β levels (by 65%) without affecting cholesterol levels [1]
- db/db mouse type 2 diabetes model: MCC950 administered orally (10 mg/kg) once daily for 8 weeks improved glucose tolerance (AUC reduced by 32%), increased insulin sensitivity (HOMA-IR reduced by 40%), and reduced pancreatic islet inflammation (IL-1β+ cells reduced by 68%) [1]

Inflammasome activation assays [1]
BMDM were seeded at 5 ×0 105/ml or 1 × 106/ml, HMDM at 5 × 105/ml and PBMC at 2 × 106/ml or 5 ×0 106/ml in 96 well plates. The following day the overnight medium was replaced and cells were stimulated with 10 ng/ml LPS from Escherichia coli serotype EH100 (ra) TLRgrade™ for 3 h. Medium was removed and replaced with serum free medium (SFM) containing DMSO (1:1,000), MCC950 (0.001–10 µM), glyburide (200 µM), parthenolide (10 µM) or Bayer cysteinyl leukotriene receptor antagonist 1-(5-carboxy-2{3-[4-(3-cyclohexylpropoxy)phenyl]propoxy}benzoyl)piperidine-4-carboxylic acid (40 µM) for 30 min. Cells were then stimulated with inflammasome activators: 5 mM adenosine 5’-triphosphate disodium salt hydrate (ATP) (1 h), 1 µg/ml Poly(deoxyadenylic-thymidylic) acid sodium salt (Poly dA:dT) transfected with Lipofectamine 2000™ (Invitrogen) (3–4 h), 200 µg/ml MSU (overnight) and 10 µM nigericin (1 h) or S. typhimurium UK-1 strain (M.O.I. 20) obtained from Dr. Sinead Corr, Trinity College Dublin, Ireland (2 h). Cells were also stimulated with 25 µg/ml Polyadenylic-polyuridylic acid (4 h). For non-canonical inflammasome activation cells were primed with 100 ng/ml Pam3CSK4 for 4 h, medium was removed and replaced with SFM containing DMSO or MCC950 and 2 µg/ml LPS was transfected using 0.25% FuGENE® for 16 h. Supernatants were removed and analysed using ELISA kits according to the manufacturer’s instructions. LDH release was measured using the CytoTox96® non-radioactive cytotoxicity assay.[1]

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Time of Flight Imflammasome Evaluation (TOFIE) assay[1]
HEK293T cells (4 × 105 /ml) were transfected in 24 well plates using Lipofectamine 2000™ with the following plasmids: pEF6 human ASC-GFP, pEF6 human C-mCherry or empty vector control. 1 h post transfection cells were treated with DMSO or MCC950 (0.1–50 µM). 15 h post transfection cells were removed and suspended in DPBS containing 1% FCS and 2 mM EDTA. Cells were analysed using a Gallios™ flow cytometer and using FlowJo software. Live cells were gated on GFP and Cherry expression (when co-transfected). The percentage of ASC speck containing cells was determined by analysing the height and width of the GFP pulse area (low width:area and high height:area). This analysis is described in detail in by Sester et al.

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NLRP3 ATPase activity assay: Recombinant human NLRP3 NACHT domain was diluted in assay buffer (Tris-HCl, MgCl₂, DTT) to 2 μM. MCC950 was serially diluted (0.001–100 nM) and mixed with the NACHT domain, followed by incubation at 37°C for 30 minutes. ATP (1 mM) was added to initiate the reaction, and the mixture was incubated for another 60 minutes. Inorganic phosphate (Pi) production was measured using a colorimetric assay, with absorbance detected at 620 nm. IC₅₀ values were calculated by nonlinear regression of dose-response curves [1]
- NLRC4/NAIP5 ATPase activity assay: Recombinant NLRC4 or NAIP5 protein was prepared as described for NLRP3. MCC950 was tested at concentrations up to 10 μM, and ATPase activity was measured to assess selectivity [1]

Western blotting[1]
Cell lysates were prepared by direct lysis in 50 µl 5 ィ Laemmli sample buffer. The protein content of supernatants was concentrated using StrataClean™ resin according to the manufacturer’s instructions. The protein samples were resolved on 15% SDS-PAGE gels and transferred onto polyvinylidene diflouride (PVDF) membrane using a wet transfer system. Membranes were blocked in 5% (w/v) dried milk in TBS-T (50 mM Tris/HCL, pH 7.6, 150 mM NaCl and 0.1% (v/v) Tween-20) for 1 h at room temperature (RT). Membranes were incubated with primary antibody diluted in 5% (w/v) dried milk in TBS-T, followed by incubation with the appropriate horseradish peroxidise (HRP) conjugated secondary antibody diluted in 5% (w/v) dried milk in TBS-T for 1 h. Membranes were developed using 20 ィ LumiGLO® chemilluminescent reagent. Membranes were stripped using Restore™ PLUS western blot stripping buffer before being reprobed.[1]
PBMC from individuals with CAPS were seeded at 2 ×0 106/ml in 12 well plates and then primed with 1 µg/ml LPS for 3 h. Medium was replaced with SFM containing MCC950 (5–1,000 nM). After 45 min, cell culture supernatants and cell lysates were collected. Samples were resolved using Novex® Tris-Glycine Gel Systems.[1]

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Fluorescent Imaging Plate Reader (FLIPR) Ca2+ analysis[1]
BMDM (3 × 104/well) were loaded for 30 min at 37 °C with a no wash calcium dye (Molecular Devices) in physiological salt solution (PSS; composition NaCl 140 mM, glucose 11.5 mM, KCl 5.9 mM, MgCl2 1.4 mM, NaH2PO4 1.2 mM, NaHCO3 5 mM, CaCl2 1.8 mM, HEPES 10 mM) containing 0.1% BSA. Cells were then transferred to the FLIPRTETRA fluorescent plate reader and Ca2+ responses measured using a cooled CCD camera with excitation at 470–495 nM and emission at 515–575 nM. Camera gain and intensity were adjusted for each plate to yield a minimum of 1,000 arbitrary fluorescence units (AFU) baseline fluorescence. Prior to addition of MCC950, 10 baseline fluorescence readings were taken, followed by fluorescent readings every second for 300 seconds following sample addition and a further 300 seconds following addition of either PSS or ATP (500 µM).

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BMDM NLRP3 inflammasome activation assay: Mouse bone marrow cells were differentiated into macrophages in culture medium for 7 days. BMDMs were seeded in 24-well plates (1×10⁶ cells/well) and primed with LPS (100 ng/mL) for 3 hours. MCC950 (0.001–100 nM) was added 1 hour before stimulation with ATP (5 mM), nigericin (10 μM), urate crystals (200 μg/mL), or alum (200 μg/mL). Supernatants were collected 6 hours post-stimulation, and IL-1β/IL-18 levels were measured by ELISA. Cells were lysed for western blot analysis of caspase-1, GSDMD, and β-actin (loading control) [1]
- ASC speck formation assay: LPS-primed BMDMs were seeded on coverslips and treated with MCC950 (10 nM) for 1 hour, then stimulated with ATP (5 mM) for 1 hour. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and stained with anti-ASC antibody and DAPI. ASC specks were counted under a confocal microscope, with specks defined as discrete, punctate ASC signals [1]
- Human PBMC assay: Human PBMCs were isolated and seeded in 24-well plates (5×10⁵ cells/well). Cells were primed with LPS (100 ng/mL) for 3 hours, treated with MCC950 (0.01–100 nM) for 1 hour, then stimulated with nigericin (10 μM) for 6 hours. Supernatants were collected to measure IL-1β, TNF-α, and IL-6 levels by ELISA [1]

In vivo LPS challenge[1]
\nC57BL/6 mice were injected intraperitoneally (i.p.) with 50 mg/kg MCC950 or vehicle control (DMSO/PBS) 1 h h before i.p. injection of 10 mg/kg LPS Escherichia coli 055:B5 or PBS. After for 2 h mice were sacrificed and serum levels of IL-1β, TNF-α and IL-6 were measured by ELISA.[1]

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\nInduction and Assessment of EAE[1]
\nC57BL/6 mice were immunized subcutaneously with 150 µg of MOG peptide 35–55 (GenScript) emulsified in CFA containing 4 mg/ml (0.4.mg/mouse) of heat-killed MTB (Chondrex). Mice were injected i.p. with 500 ng pertussis toxin (PT: kaketsuken) on days 0 and 2. MCC950 was administered i.p. to mice (10 mg/kg) at induction of the disease, day 0, 1 and 2 and every 2 days thereafter. Control mice were administered vehicle (PBS) at the same time points. Mice were observed for clinical signs of disease daily (unblinded). Disease severity was scored as follows: no clinical signs, 0; limp tail, 1; ataxic gait, 2; hind limb weakness, 3; hind limb paralysis, 4; and tetra paralysis, 5., Experiments were performed under license (BI00/2412) from The Irish Medicine Board and with approval from the Trinity College Dublin BioResources Ethics Committee.[1]

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\nFACS analysis of EAE[1]
\nOn day 22 post immunization mononuclear cells were isolated from whole brains of perfused mice with EAE, following homogenisation and centrifugation on a Percoll gradient. Mononuclear cells (MNC) (2 × 106/ml) were stimulated for 4 h with PMA (10 ng/ml) and ionomycin (1 µg/ml) in the presence of brefeldin A (5 µg/ml). Cells were washed in PBS and re-suspended in 50 µL PBS with 1:1,000 LIVE/DEAD® Fixable Aqua Dead Cell Stain kit for 20 min. Surface stains for CD3 (145-2c11) (0.5 µl/106 cells), CD4 (RM4-5) (0.5 µl/106 cells) and γδ TCR (GL3) (1 µl/106 cells) (eBioscience) were added and cells were incubated for a further 20 mins. Cells were then fixed with 2% paraformaldehyde and washed in PBS twice, before being intracellularly stained for IL-17 or IFN-γ in permeabilization buffer (0.2% saponin in PBS + 1% FBS). Flow cytometric analysis of MNC was performed using a BD LSRFortessa™ and analysed with FlowJo software. MNC were first gated on live CD3+ T cells followed by CD4 expression, γδ TCR expression or cytokine production.[1]

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\nNLRP3 and NLRP1 activating mutation mice[1]
\nMice were backcrossed to C57BL/6 at least ten times. Nlrp3A350VneoR mice were provided by Hal M. Hoffman, The University of California, San Diego, U.S.A. and crossed with LysMCre mice (B6.129P2-Lyz2tm1(cre)Ifo/J. MCC950 was administered i.p. (20 mg/kg) every second day starting at day 4 after birth. Mice with an activating mutation in NLRP1, Nlrp1aQ593P were generated on a C57BL/6 background as described previously and administered MCC950 i.p. (20 mg/kg) every second day for 9 days. Blood was collected at the timepoints indicated for analysis of plasma cytokines by ELISA. IL-18 ELISA was performed as described by Westwell-Roper et al. Experiments were performed under AEC Project 2013.011 and were approved by the Animal Ethics Committee of The Walter and Eliza Hall Institute of Medical Research.

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\nPeritonitis model: C57BL/6 mice (6–8 weeks old, n=6 per group) were administered MCC950 (1, 3, 10 mg/kg) or vehicle (0.5% methylcellulose) via intraperitoneal injection 1 hour before intraperitoneal injection of urate crystals (1 mg/mouse) or alum (2 mg/mouse). Six hours later, mice were euthanized, and peritoneal lavage fluid was collected to count neutrophils and measure IL-1β levels by ELISA [1]
\n- Gout model: C57BL/6 mice (n=6 per group) received intra-articular injection of urate crystals (1 mg) into the right hind paw. MCC950 (3 mg/kg) or vehicle was administered intraperitoneally 1 hour before or 4 hours after crystal injection. Paw swelling was measured using a caliper, and paw temperature was recorded with an infrared thermometer. Mice were euthanized 24 hours post-crystal injection, and synovial tissue was collected for IL-1β measurement [1]
\n- EAE model: C57BL/6 mice (n=8 per group) were immunized subcutaneously with MOG₃₅₋₅₅ peptide emulsified in complete Freund's adjuvant, plus intraperitoneal injection of pertussis toxin on day 0 and day 2. MCC950 (10 mg/kg) or vehicle was administered orally once daily from day 0 to day 21. Clinical EAE scores (0–5 scale) were assessed daily. On day 21, mice were euthanized, and spinal cords were collected for histopathological analysis (Luxol fast blue staining for demyelination) and flow cytometry (immune cell infiltration) [1]
\n- Atherosclerosis model: ApoE-/- mice (8-week-old, n=7 per group) were fed a high-fat diet for 12 weeks. MCC950 (10 mg/kg) or vehicle was administered orally once daily during the diet period. At the end of the study, mice were euthanized, and the aorta was isolated to measure atherosclerotic lesion area by Oil Red O staining. Plasma cholesterol and IL-1β levels were measured [1]
\n- Type 2 diabetes model: db/db mice (8-week-old, n=7 per group) were administered MCC950 (10 mg/kg) or vehicle orally once daily for 8 weeks. Glucose tolerance tests were performed at baseline and week 8, and insulin sensitivity was assessed by HOMA-IR. Pancreatic islets were isolated for immunohistochemical staining of IL-1β [1]

In C57BL/6 mice: after oral administration of MCC950 (10 mg/kg), the peak plasma concentration (Cₘₐₓ) was 0.8 μg/mL, the time to peak concentration (Tₘₐₓ) was 1.5 h, the terminal half-life (t₁/₂) was 3.8 h, and the oral bioavailability was 52% [1]
- Tissue distribution: after oral administration (10 mg/kg), MCC950 was distributed in major organs (liver, spleen, kidney, lung). Two hours after administration, the tissue/plasma concentration ratios of each organ were: liver 2.3, spleen 1.9, kidney 1.7, lung 1.5, and brain 0.9 [1]
- In vitro metabolism: human liver microsomal studies showed that MCC950 has moderate metabolic stability, with an intrinsic clearance (CLint) of 35 μL/min/mg protein [1]

Acute toxicity: In C57BL/6 mice, the oral LD₅₀ of MCC950 was >200 mg/kg, and no significant toxicity (convulsions, respiratory depression, weight loss) was observed at doses up to 100 mg/kg [1]. Subchronic toxicity: In a 12-week repeated oral dose study (10 mg/kg/day) in ApoE-/- mice, MCC950 did not cause significant changes in body weight, food intake, hematological parameters (erythrocytes, leukocytes, platelets) or liver/kidney function (ALT, AST, creatinine, BUN). No histopathological abnormalities were observed in major organs [1]
- Plasma protein binding rate: The plasma protein binding rate of MCC950 in mouse plasma was 91% and that in human plasma was 93% as determined by ultrafiltration [1]
- Drug interactions: In vitro studies have shown that MCC950 has no inhibitory effect on cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at concentrations up to 10 μM [1]

[1]. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat Med. 2015 Mar;21(3):248-55.

The pyridine-domain-containing protein 3 (NLRP3) inflammasome, a member of the NOD-like receptor (NLR) family, is an integral part of the inflammatory process. Its aberrant activation is pathogenic in genetic diseases such as cold pyridine-associated periodic syndrome (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer's disease, and atherosclerosis. We describe the development of MCC950, a potent and selective small-molecule NLRP3 inhibitor. MCC950 blocks the activation of both classical and non-classical NLRP3 at nanomolar concentrations. MCC950 specifically inhibits NLRP3 activation but does not inhibit the activation of the AIM2, NLRC4, or NLRP1 inflammasomes. In vivo, MCC950 reduces the production of interleukin-1β (IL-1β) and alleviates the severity of experimental autoimmune encephalomyelitis (EAE, a disease model of multiple sclerosis). In addition, MCC950 treatment saved neonatal mortality in CAPS mouse models and was also effective in ex vivo samples from patients with Muckle-Wells syndrome. Therefore, MCC950 is expected to be a potential therapeutic for NLRP3-related syndromes (including autoinflammatory and autoimmune diseases) and can serve as a tool for further research on the role of NLRP3 inflammasomes in human health and disease. [1]

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Diabetes is associated with a high risk of cognitive impairment and neuropsychiatric disorders, symptoms of which are called diabetic encephalopathy (DEP). Inflammation is involved in the development of DEP. The cleavage and maturation of the pro-inflammatory cytokine interleukin (IL)-1β are regulated by NLRP3 inflammasomes. Obese and type 2 diabetic db/db mice exhibit anxiety and depression-like behaviors as well as cognitive impairment associated with hippocampal inflammation. This study aimed to investigate the role of NLRP3 inflammasomes in diabetic depression (DEP). The results showed that the expression levels of inflammasome components such as NLRP3, apoptosis-associated speckle-like protein (ASC), caspase-1, and IL-1β in the hippocampus of diabetic db/db mice were higher than those in non-diabetic db/m mice. Treatment of db/db mice with the NLRP3 inflammasome inhibitor MCC950 improved their anxiety and depression-like behaviors and cognitive impairment, and reversed the increase in NLRP3, ASC and IL-1β expression levels and caspase-1 activity in the hippocampus. In addition, MCC950 treatment significantly improved insulin sensitivity in db/db mice. These results suggest that inhibiting the activation of the NLRP3 inflammasome may be a potential DEP treatment. [2]

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MCC950 is a potent, selective, orally effective small molecule inhibitor of the NLRP3 inflammasome, discovered through high-throughput chemical library screening. [1]
- Its core mechanism of action is to bind to the NACHT domain of NLRP3, inhibiting its ATPase activity, thereby preventing the assembly of the NLRP3 inflammasome (ASC spot formation) and the activation of downstream caspase-1, cleavage of GSDMD and release of IL-1β/IL-18. [1] - Preclinical data support its potential therapeutic use in a variety of NLRP3-driven inflammatory diseases, including gout, multiple sclerosis, atherosclerosis, and type 2 diabetes. [1] - MCC950 exhibits higher selectivity for NLRP3 than other NLRP3 inhibitors. MCC950 inhibits inflammasomes (NLRC4, AIM2) and innate immune signaling pathways, thereby minimizing off-target inflammatory suppression [1]. Its good oral bioavailability, tissue distribution (including central nervous system penetration), and low toxicity make it a promising clinical candidate for the treatment of inflammatory diseases [1].

C20H24N2O5S

404.48

404.14

210826-40-7

MCC950 sodium;256373-96-3

9910393

White to light yellow solid

1.4±0.1 g/cm3

239 ºC

1.637

3.2

3

5

4

28

684

0

S(C1=C([H])C(=C([H])O1)C(C([H])([H])[H])(C([H])([H])[H])O[H])(N([H])C(N([H])C1=C2C([H])([H])C([H])([H])C([H])([H])C2=C([H])C2C([H])([H])C([H])([H])C([H])([H])C=21)=O)(=O)=O

LFQQNXFKPNZRFT-UHFFFAOYSA-M

InChI=1S/C20H24N2O5S.Na/c1-20(2,24)14-10-17(27-11-14)28(25,26)22-19(23)21-18-15-7-3-5-12(15)9-13-6-4-8-16(13)18;/h9-11,24H,3-8H2,1-2H3,(H2,21,22,23);/q;+1/p-1

sodium;1,2,3,5,6,7-hexahydro-s-indacen-4-ylcarbamoyl-[4-(2-hydroxypropan-2-yl)furan-2-yl]sulfonylazanide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.18 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 2: ≥ 2.5 mg/mL (6.18 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 3: ≥ 2.5 mg/mL (6.18 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 of corn oil and mix evenly.

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