NLRP3

CY-09 demonstrates responsiveness to monosodium urate (MSU), MSU, ATP-induced caspase-1 activation, and IL-1β walking in LPS-challenged bone marrow-derived macrophages (BMDM) at concentrations of 1 to 10 μM. Death-dependent inhibition. Cytosolic LPS-induced non-canonical NLRP3 activation in BMDM can also be swollen by CY-09 administration. CY-09 buffer activates the NLRP3 inflammasome and has no effect on the LPS-induced priming effect. CY-09 treated major inhibitory factors and found that CY-09 treated the interaction of Flag-NLRP3 and mCherry-NLRP3 in HEK-293T cells, indicating that CY-09 prevented NLRP3 oligomerization [1].

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CY-09 specifically blocks NLRP3 activation in macrophages.CY-09 blocks NLRP3 inflammasome activation. CY-09 inhibits NLRP3 oligomerization and inflammasome assembly. CY-09 directly binds to NLRP3 and inhibits its ATPase activity. CY-09 binds to the ATP-binding site of NLRP3 NACHT domain. CY-09 inhibits NLRP3 ATPase activity[1].

The monosodium urate (MSU) injection-induced IL-1β generation and neutrophil influx were significantly suppressed in chambers treated with CY-09, suggesting that CY-09 can prevent MSU-induced NLRP3 inflammasome activation within the chamber. Even when therapy was terminated on day 25, CY-09 treatment raised the even rate of NLRP3 mutations to 30 to 48 days. CY-09 also inhibits the even processing of caspase-1 (caspase-1) channels, which is shown in adipose tissue on a high-fat diet (HFD) [1].

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CY-09 inhibits NLRP3 activation in vivo and prevents neonatal lethality in a mouse model of CAPS. CY-09 inhibits NLRP3 activation in MSU-induced peritonitis and a mouse model of MWS. CY-09 reverses metabolic disorders in diabetic mice by inhibition of NLRP3-dependent inflammation. Treatment of metabolic disorders in HFD-induced diabetic mice with CY-09. CY-09 suppresses NLRP3-dependent metainflammation in diabetic mice. CY-09 is active ex vivo for cells from healthy human or gouty patients. CY-09 is active for cells from healthy humans or patients with gout [1].

MST assay [1]
The KD value was measured using the Monolith NT.115 instrument. A range of concentrations of CY-09 (from 0.025 mM to 1.2 nM) were incubated with 200 nM of purified His-GFP-NLRP3 protein for 40 min in assay buffer (50 mM Hepes, 10 mM MgCl2, 100 mM NaCl, pH 7.5, and 0.05% Tween 20). The samples were loaded into the NanoTemper glass capillaries, and MST was performed using 100% LED power and 80% MST power. The KD value was calculated using the mass action equation via the NanoTemper software from duplicate reads of an experiment.

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Microsomal stability [1]
Microsomal stability was determined in a CRO company. CY-09 or control compound DMSO stock solution (10 mM) diluted with 50% methanol to a concentration of 100 µM. 6 µl of 100 µM compound solution was combined with 534 µl of liver microsome solution (0.7 mg protein/ml potassium phosphate buffer) to produce the compound working solution. A 90-µl aliquot of this working solution was incubated at 37°C for 10 min before the addition of NADPH regenerating system (10 µl) to start the reaction. Reactions were stopped at 0, 10, 30, and 60 min by addition of 300 µl cold acetonitrile (containing tolbutamide as internal standard at 500 nM) and centrifuged at 4,000 rpm for 20 min. The supernatant (100 µl) was added to water (300 µl) and analyzed by liquid chromatography/tandem mass spectrometry. The ratio of peak area of test compound remaining/internal standard was used to determine reduction in concentration of test compound over time. The t1/2 and hepatic clearance (CLhep) were then calculated.

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NLRP3 ATPase activity and ATP binding assay [1]
For ATPase activity assay, purified recombinant human proteins (1.4 ng/µl) were incubated at 37°C with indicated concentrations of CY-09 for 15 min in the reaction buffer. ATP (25 µm, Ultra-Pure ATP) was then added, and the mixture was further incubated at 37°C for another 40 min. The amount of ATP converted into adenosine diphosphate (ADP) was determined by luminescent ADP detection with ADP-Glo Kinase Assay kit (Promega, Madison, MI, USA) according to the manufacturer’s protocol. The results were expressed as percentage of residual enzyme activity to the vehicle-treated enzyme. For ATP binding assay, purified NLRP3 protein (0.1 ng/µl) were incubated with ATP binding agarose for 1 h and then different concentrations of CY-09 was added and incubated for 2 h with motion at 4°C. Beads were washed and boiled in loading buffer. Samples were subjected to immunoblotting analysis.

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ASC oligomerization assay [1]
BMDMs were seeded at 1 × 106/ml in 6-well plates. The following day, the medium was replaced, and cells were primed with 50 ng/ml LPS for 3 h. The cells were treated with CY-09 for 30 min and then stimulated with nigericin for 30 min. The supernatants were removed, cells were rinsed in ice-cold PBS, and then cells were lysed by NP-40 for 30 min. Lysates were centrifuged at 330 g for 10 min at 4°C. The pellets were washed twice in 1 ml ice-cold PBS and resuspended in 500 µl PBS. 2 mM disuccinimydyl suberate was added to the resuspended pellets, which were incubated at room temperature for 30 min with rotation. Samples were then centrifuged at 330 g for 10 min at 4°C. The cross-linked pellets were resuspended in 30 µl sample buffer and then boiled and analyzed by immunoblotting.

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Cytochrome P450 inhibition [1]
Cytochrome P450 inhibitory effects of CY-09 against five major CYP isozymes (1A2, 2C9, 2C19, 2D6, and 3A4) were determined at Wuxi AppTech. A working stock solution of CY-09 (10 mM in DMSO) was diluted using phosphate buffer to a concentration of 100 µM. Five inhibitor stock solutions were prepared at a concentration of 3 mM in DMSO: α-naphthoflavone, sulfaphenazole, N-3-benzylnirvanol, quinidine, and ketoconazole. The inhibitor stocks were diluted using phosphate buffer to a concentration of 30 µM. A cocktail of substrates (phenacetin, diclofenac, S-mephenytoin, dextromethorphan, and midazolam) for five major CYP isozymes (1A2, 2C9, 2C19, 2D6, and 3A4) was prepared by dilution of methanol stock solutions using phosphate buffer. CY-09, known inhibitor, or blank solution (20 µl of working stock solution) and substrate cocktail solution (20 µl) were incubated at 37°C with human liver microsomes (158 µl 0.253 mg/ml in phosphate buffer) for 10 min before addition of the cofactor NADPH (20 µl 10 mM solution in 33 mM MgCl2). Incubation was continued, and aliquots were removed at 0, 5, 10, 20, 30, and 60 min and then mixed with cold acetonitrile (containing tolbutamide as internal MS standard at 200 ng/ml) and centrifuged at 4,000 rpm for 20 min. The supernatant was analyzed by liquid chromatography/tandem mass spectrometry to determine the peak area of metabolite and internal standard. Peak areas determined in the presence and absence of test compound were used to determine percentage inhibition. SigmaPlot v.11 was used to plot percentage control activity versus the test compound concentrations and for nonlinear regression analysis of the data. IC50 values were determined using a three-parameter logistic equation. IC50 values were reported as “>50 µM” when percentage inhibition at the highest concentration (50 µM) was <50%.

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hERG channel assay [1]
The manual patch-clamp method (QPatchHTX) was used at a CRO company to evaluate the effects of CY-09 on the hERG potassium channel. CHO cells that stably express hERG potassium channels from Aviva Biosciences were used. The inhibition of CY-09 and amitriptyline as positive control on whole-cell hERG currents were determined.

To induce NLRP3 inflammasome activation, 5 × 105/ml BMDMs and 6 × 106/ml PBMCs were plated in 12-well plates. The following morning, the medium was replaced, and cells were stimulated with 50 ng/ml LPS or 400 ng/ml Pam3CSK4 (for noncanonical inflammasome activation) for 3 h. After that, CY-09 or other inhibitors were added into the culture for another 30 min, and then the cells were stimulated for 4 h with MSU (150 µg/ml), Salmonella typhimurium (multiplicity of infection) or for 30 min with ATP (2.5 mM) or nigericin (10 µM). Cells were transfected with poly(dA:dT) (0.5 µg/ml) for 4 h or LPS (500 ng/ml) overnight using Lipofectamine 2000. Cell extracts and precipitated supernatants were analyzed by immunoblot.

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Confocal microscopy [1]
Confocal analysis was performed as previously described (Yan et al., 2015). In brief, 2 × 105/ml BMDMs were plated on coverslips. On the following day, the medium was replaced with Opti-MEM (1% FBS) containing LPS (50 ng/ml) for 3 h, and then the indicated doses of CY-09 were added for another 30 min. BMDMs were then used for stimulation and staining with MitoTracker Red (50 nM) or MitoSOX (5 µM). After three times washing with ice-cold PBS, the cells were fixed with 4% PFA in PBS for 15 min at room temperature and then washed with PBS with Tween 20 three times. Confocal microscopic analyses were performed using a Zeiss LSM700.

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Intracellular potassium or chloride detection [1]
For accurate measurement of intracellular potassium, BMDMs were plated overnight in 6-well plates and then primed with 50 ng/ml LPS for 3 h. After that, cells were treated with CY-09 for 30 min and then stimulated with nigericin for 30 min. Culture medium was thoroughly aspirated and lysed with 65% ultrapure HNO3. Intracellular K+ measurements were performed by inductively coupled plasma optical emission spectrometry with a PerkinElmer Optima 2000 DV spectrometer using yttrium as the internal standard.
For accurate measurement of the intracellular chloride, BMDMs were plated overnight in 12-well plates and then primed with 50 ng/ml LPS for 3 h. After that, cells were treated with CY-09 or MCC950 for 30 min and then stimulated with nigericin for 15 min. The supernatants of 12-well plates were removed, ddH2O was added (200 µl/well), and the supernatants were kept 15 min at 37°C. The lysates were transferred to 1.5-ml EP tube, and centrifuged at 10,000 g for 5 min. 160-µl supernatants were then transferred to a new 1.5-ml EP tube and mixed with 40 µl MQAE (10 µM). Absorbance was tested using BioTek Multi-Mode Microplate Readers (Synergy2). A control was settled in every experiment to determine the extracellular amount of chloride remaining after aspiration, and this value was subtracted.

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Immunoprecipitation (IP) and pulldown assay [1]
For the endogenous IP assay, BMDMs were stimulated and lysed with NP-40 lysis buffer with complete protease inhibitor. The cell lysates were incubated overnight at 4°C with the primary antibodies and Protein G Mag Sepharose (GE Healthcare). The proteins bound by antibody were precipitated by protein G beads and subjected to immunoblotting analysis. For the exogenous IP assay, HEK-293T cells (3 × 105/ml) were transfected with plasmids in 6-well plates via polyethylenimine. After 24 h, cells were collected and lysed with NP-40 lysis buffer. Protein extracts were immunoprecipitated with anti-Flag antibody–coated beads and then assessed by immunoblot analysis.
For pull-down assay, BMDMs or 293T lysates were collected and centrifuged at 8,000 rpm. The supernatant was transferred to another tube and the cell debris thoroughly discarded. Prewashed streptavidin beads were added into the supernatant, allowing 2 h preincubation with motion at 4°C and centrifuging at 8,000 rpm. The supernatant was transferred to another tube, and the streptavidin beads were discarded to remove unspecific binding proteins. The pretreated supernatant and purified human recombinant NLRP3 proteins (dissolving in the lysis buffer) were incubated with indicated doses of free CY-09, followed by incubation with indicated doses of biotin–CY-09 for 1 h. After that, the samples were then incubated with prewashed streptavidin beads overnight. Beads were respectively washed twice with 0.1% Tween 20 in PBS and 1% NP-40 in PBS to remove unspecific binding proteins and boiled in SDS buffer.

With the formulation of DMA:EL:HP-β-CD (10%, wt/vol) = 5:5:90 (vol/vol/v), CY-09 achieved a concentration of 1 mg/ml at pH 7.4. Using the formulation of DMSO:Solutol HS 15:saline = 10%:10%:80% (vol/vol/v), CY-09 reached a concentration of 5 mg/ml at pH 9.0. For the in vivo experiments, CY-09 was formulated in a vehicle containing 10% DMSO, 10% Solutol HS 15, and 80% saline.[1]

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MSU-induced peritonitis [1]
C57BL/6J mice were injected i.p. with 40 mg/kg CY-09 or vehicle 30 min before i.p. injection of MSU (1 mg MSU crystals dissolved in 0.5 ml sterile PBS). After 6 h, mice were killed, and peritoneal cavities underwent lavage with 10 ml ice-cold PBS. Peritoneal lavage fluid was assessed by flow cytometry with the neutrophil markers Ly6G and CD11b for analysis of the recruitment of polymorph nuclear neutrophils. IL-1β production in serum or peritoneal lavage fluid was determined using ELISA.

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Determination of the pharmacokinetic properties of CY-09 in mice [1]
The pharmacokinetics of CY-09 were determined after single i.v. and oral administration in C57BL/6J mice (n = 3 at each time point) at doses of 5 and 10 mg/kg, respectively. Blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, 10, and 24 h (i.v.) and 0.25, 0.5, 1, 2, 4, 8, 10, and 24 h (oral) after administration. Then the samples were quantified by liquid chromatography/tandem mass spectrometry, and data analysis was conducted using WinNonlin v.6.3.

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MWS mouse model [1]
Nlrp3A350VneoR mice were crossed with LysM-Cre mice (B6.129P2-Lyz2tm1(cre)Ifo/J). CY-09 (20 mg/kg) or MCC950 (20 mg/kg) were administered orally every day starting at day 4 after birth. The weight and survival of mice were monitored every day.

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HFD and CY-09 treatment [1]
WT or Nlrp3−/− mice at the age of 6 wk, with similar plasma glucose levels and body weights were randomized into different groups. For generation of HFD-induced diabetic mice, mice were fed with HFD for 14 wk. The diabetic mice were treated with CY-09 (i.p.) at a dose of 2.5 mg/kg once a day for 6 wk. The mice were maintained with HFD when used for CY-09 treatment and the subsequent experiments.

Next, we investigated the pharmacokinetic characteristics of the compound and then evaluated the therapeutic potential of CY-09 in vivo. First, the metabolic stability of CY-09 was assessed using human and mouse liver microsomes, showing good stability with half-lives greater than 145 minutes in both (Table S1). We tested the inhibitory activity of CY-09 against five major cytochrome P450 enzymes: 1A2, 2C9, 2C19, 2D6, and 3A4, with half-maximal inhibitory concentrations (IC50) of 18.9, 8.18, >50, >50, and 26.0 µM, respectively (Table S2), indicating a low risk of drug interactions. To assess its potential cardiotoxicity, we used automated patch-clamp technique (QPatchHTX) to detect the effect of CY-09 on the human ether-a-go-go (hERG) potassium channel, showing that CY-09 had no activity against the hERG channel at a concentration of 10 µM (Table S3). Subsequently, we further evaluated the pharmacokinetic characteristics of CY-09 in C57BL/6J mice, which were administered via single intravenous or oral injection. CY-09 exhibited favorable pharmacokinetic characteristics with a half-life of 2.4 h, an area under the curve of 8,232 (h·ng)/ml, and a bioavailability of 72% (Table S4). Based on these data, we further evaluated the in vivo efficacy of CY-09. [1]

[1]. Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders. J Exp Med. 2017 Nov 6;214(11):3219-3238.

The NLRP3 inflammasome is closely related to the pathogenesis of various human diseases. Although some compounds have been developed to inhibit the activation of the NLRP3 inflammasome, there are currently no compounds that directly and specifically target NLRP3, so it is unclear whether NLRP3 itself can serve as a target for disease prevention or treatment. This study demonstrates that the compound CY-09 can specifically block the activation of the NLRP3 inflammasome. CY-09 directly binds to the ATP-binding motif of the NLRP3 NACHT domain, inhibiting the ATPase activity of NLRP3, thereby inhibiting the assembly and activation of the NLRP3 inflammasome. Importantly, CY-09 treatment showed significant therapeutic effects in mouse models of cold pyridine-associated autoinflammatory syndrome (CAPS) and type 2 diabetes. Furthermore, CY-09 also showed activity in vitro against monocytes from healthy individuals or synovial cells from gout patients. Therefore, our results provide a highly selective and direct-acting small-molecule NLRP3 inhibitor and suggest that NLRP3 can serve as an in vivo target for combating NLRP3-driven diseases. [1]

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In this study, we describe a highly potent, selective, and direct-acting NLRP3 inhibitor that exhibits significant NLRP3 inflammasome inhibitory activity in both mice and human cells. CY-09 will serve as a versatile tool for pharmacological studies of NLRP3 biology and its role in inflammatory diseases.
Several compounds have shown potent inhibitory activity against NLRP3 inflammasomes and have been tested in animal models, but their nonspecific effects limit their potential for clinical application. The inhibitory effect of sulforaphane on AIM2 or NLRC4 inflammasomes suggests that it may weaken the role of these inflammasomes in host defense (Greaney et al., 2016). The broad anti-inflammatory activities of sulforaphane, isoliquiritigenin, β-hydroxybutyric acid (BHB), parthenolide, BAY 11-7082, and INF39 (Heiss et al., 2001; Yip et al., 2004; Honda et al., 2012; Strickson et al., 2013; Fu et al., 2015; Cocco et al., 2017) suggest that these compounds may cause immunosuppressive side effects and increase the risk of infection. The effects of flufenamic acid and mefenamic acid on chloride efflux and the effect of BHB on potassium efflux indicate that these compounds target the upstream signaling pathway of NLRP3 and possess other unavoidable biological activities (Youm et al., 2015; Daniels et al., 2016). MCC950 has shown potent inhibitory activity and beneficial effects in various mouse models of NLRP3-related diseases (Coll et al., 2015; Dempsey et al., 2017), but its mechanism remains unclear. This study demonstrates that MCC950 can block NLRP3 agonist-induced chloride efflux, which is considered an upstream signaling event for NLRP3 activation (Daniels et al., 2016). This suggests that MCC950 may target volume-regulating anion channels or other chloride channels to inhibit NLRP3 inflammasome activation and may have a non-specific effect. This study describes CY-09, a specific NLRP3 inflammasome inhibitor that directly targets NLRP3 itself. We found that CY-09 has significant preventive or therapeutic effects in mouse models of CAPS, type 2 diabetes, and gout. Therefore, our study describes a direct and specific NLRP3 inhibitor with the potential to treat NLRP3-related diseases. Our results indicate that pharmacological inhibition of NLRP3 ATPase activity can effectively treat NLRP3-related diseases. Previous studies have reported that MNS, paternoside, BAY 11-7082, and INF39 can inhibit NLRP3 ATPase activity and have shown inhibitory activity against the NLRP3 inflammasome in vitro. However, these compounds are not specific NLRP3 inhibitors and possess a variety of biological activities, such as inhibitory activity against tyrosine kinases or the NF-κB signaling pathway (Yip et al., 2004; Wang et al., 2006; Strickson et al., 2013; Cocco et al., 2017). Furthermore, MNS, patenosides, and BAY 11-7082 have not been tested in vivo in animal models of NLRP3-driven disease. CY-09 binds directly to the NACHT domain of NLRP3 and inhibits its ATPase activity, which is crucial for NLRP3 oligomerization and inflammasome assembly (Duncan et al., 2007). Moreover, mutations in the Walker A motif in the NACHT domain (which is essential for ATP binding to NLRP3) (MacDonald et al., 2013) weaken the binding ability of CY-09 to NLRP3. Furthermore, our results clearly demonstrate that CY-09 competitively binds to NLRP3 with ATP, inhibiting its ATPase activity and subsequent NLRP3 oligomerization and inflammasome assembly. Importantly, our findings indicate that the inhibition of NLRP3 ATPase activity by CY-09 significantly reduced NLRP3 inflammasome activation and symptoms in type 2 diabetes (T2D) and CAPS mouse models. Therefore, our results suggest that ATPase activity could serve as a target for screening candidate drugs for NLRP3-driven diseases. Currently, clinical treatments for NLRP3-related diseases use drugs targeting IL-1β, but directly targeting NLRP3 itself with highly specific small molecule inhibitors (such as CY-09) may have certain advantages. In the CAPS mouse model, CY-09 effectively prevented death, but blocking IL-1β alone did not achieve the same effect (Brydges et al., 2009). A possible reason is that IL-18 production or pyroptosis induced by inflammasome activation may also be involved in the pathological process. Furthermore, IL-1β can also be produced by other inflammasomes or in a non-inflammasome-dependent manner (Davis et al., 2011; Netea et al., 2015), therefore, inhibiting NLRP3 itself may have fewer immunosuppressive side effects than blocking IL-1β. In fact, our results show that CY-09 has no effect on the AIM2 or NLRC4 inflammasomes, suggesting that CY-09 may not impair the role of these inflammasomes in host defense. Moreover, small molecule compounds are generally more cost-effective than biologics (Fautrel, 2012). Type 2 diabetes is characterized by insulin resistance and hyperglycemia, and can cause a variety of complications, including neurological and renal damage. However, currently available drugs cannot effectively correct the underlying cause of insulin resistance, and most patients require lifelong medication (Nathan et al., 2009; Qaseem et al., 2012). Our study shows that CY-09 inhibition of NLRP3-dependent metabolic inflammation can effectively reverse metabolic disorders in diabetic mice. CY-09 treatment significantly improved metabolic inflammation, hyperglycemia, and insulin resistance in diabetic mice. Therefore, this study suggests that correcting NLRP3-dependent metabolic inflammation may be an effective treatment for type 2 diabetes. Considering the role of NLRP3-dependent inflammation in the progression of gout, Alzheimer's disease, and atherosclerosis, CY-09 or its derivatives could be used to develop novel NLRP3-targeted therapies for these diseases. [1]

C19H12F3NO3S2

423.43

423.021

C, 53.90; H, 2.86; F, 13.46; N, 3.31; O, 11.34; S, 15.14

1073612-91-5

44561595

Light yellow to yellow solid powder

4.9

1

8

4

28

672

0

S1C(N(C(/C/1=C\C1C=CC(C(=O)O)=CC=1)=O)CC1C=CC=C(C(F)(F)F)C=1)=S

DJTINRHPPGAPLD-DHDCSXOGSA-N

InChI=1S/C19H12F3NO3S2/c20-19(21,22)14-3-1-2-12(8-14)10-23-16(24)15(28-18(23)27)9-11-4-6-13(7-5-11)17(25)26/h1-9H,10H2,(H,25,26)/b15-9-

4-[[4-Oxo-2-thioxo-3-[[3-(trifluoromethyl)phenyl]methyl]-5-thiazolidinylidene]methyl]benzoic acid

CY 09; CY-09; 1073612-91-5; CY 09; 4-[[4-Oxo-2-thioxo-3-[3-(trifluoromethyl)benzyl]thiazolidin-5-ylidene]methyl]benzoic Acid; 4-[[4-oxo-2-sulfanylidene-3-[[3-(trifluoromethyl)phenyl]methyl]-1,3-thiazolidin-5-ylidene]methyl]benzoic acid; MFCD31619349; CY09; DJTINRHPPGAPLD-UHFFFAOYSA-N; (Z)-4-((4-Oxo-2-thioxo-3-(3- (trifluoromethyl)benzyl)thiazolidin-5- ylidene)methyl)benzoic acid; CY09

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 : ≥ 150 mg/mL (~354.25 mM)
H2O : ~1.1 mg/mL (~2.60 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.90 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 (5.90 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.)