Caspase; caspase-3
Emricasan, also known as IDN-6556 or PF-03491390, is a sub- to nanomolar active inhibitor of activated caspases. While Emricasan exhibits neuroprotective activity for hNPCs, ZIKV replication is not prevented[2].

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Emricasan, a pan-caspase inhibitor, was identified as the most potent anti-death compound with IC50 values of 0.13 – 0.9 μM in both caspase activity and cell viability assays for SNB-19 cells against three ZIKV strains: MR766 (1947 Ugandan strain), FSS13025 (2010 Cambodian strain), and PRVABC59 (2015 Puerto Rican strain) (Fig. 1a). It was also effective for all three cell types tested (Supplementary Fig. 1f and Supplementary Table 5). In addition, Emricasan reduced the number of active (cleaved) caspase-3-expressing forebrain-specific hNPCs infected by FSS13025 in both monolayer and 3D organoid cultures (Fig. 1b–c). Emricasan treatment of ZIKV-exposed brain organoids did not appear to affect hNPC proliferation compared to the mock treatment, as evaluated by phospho-Histone3 (PH3) expression (106 ± 10%; n = 8; P = 0.7; One-way ANOVA). Notably, ZIKV antigen persisted in both 2D and 3D cultures after Emricasan treatment (Fig. 1b–c). Therefore, Emricasan displays neuroprotective activity for hNPCs, but does not suppress ZIKV replication.[2]

Emricasan, also known as IDN-6556 or PF-03491390, is a sub- to nanomolar active inhibitor of activated caspases. While Emricasan exhibits neuroprotective activity for hNPCs, ZIKV replication is not prevented[2].

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Emricasan, a pan-caspase inhibitor, was identified as the most potent anti-death compound with IC50 values of 0.13 – 0.9 μM in both caspase activity and cell viability assays for SNB-19 cells against three ZIKV strains: MR766 (1947 Ugandan strain), FSS13025 (2010 Cambodian strain), and PRVABC59 (2015 Puerto Rican strain) (Fig. 1a). It was also effective for all three cell types tested (Supplementary Fig. 1f and Supplementary Table 5). In addition, Emricasan reduced the number of active (cleaved) caspase-3-expressing forebrain-specific hNPCs infected by FSS13025 in both monolayer and 3D organoid cultures (Fig. 1b–c). Emricasan treatment of ZIKV-exposed brain organoids did not appear to affect hNPC proliferation compared to the mock treatment, as evaluated by phospho-Histone3 (PH3) expression (106 ± 10%; n = 8; P = 0.7; One-way ANOVA). Notably, ZIKV antigen persisted in both 2D and 3D cultures after Emricasan treatment (Fig. 1b–c). Therefore, Emricasan displays neuroprotective activity for hNPCs, but does not suppress ZIKV replication.[2]

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Emricasan inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors (hNPCs) in both monolayer and 3-dimensional organoid cultures. [2]
In SNB-19 glioblastoma cells infected with three ZIKV strains (MR766, FSS13025, PRVABC59), Emricasan inhibited caspase activity with IC50 values of 0.13 µM (MR766), 0.17 µM (FSS13025), and 0.19 µM (PRVABC59), and improved cell viability with IC50 values of 1.06 µM (MR766), 0.84 µM (FSS13025), and 0.45 µM (PRVABC59). [2]
Emricasan reduced the number of active (cleaved) caspase-3-expressing forebrain-specific hNPCs infected by ZIKV strain FSS13025 in both monolayer and 3D organoid cultures. [2]
Emricasan treatment did not suppress ZIKV replication, as ZIKV antigen persisted in both 2D and 3D cultures after treatment. [2]
Emricasan treatment of ZIKV-exposed brain organoids did not significantly affect hNPC proliferation compared to mock treatment, as evaluated by phospho-Histone3 (PH3) expression. [2]
The two-drug combination of Emricasan and the antiviral compound PHA-690509 exhibited an additive effect in inhibiting caspase-3 activity in SNB-19 cells and in preserving astrocyte viability after ZIKV infection. Emricasan did not interfere with PHA-690509's ability to inhibit ZIKV infection in the combination treatment. [2]
Sequential treatment of PRVABC59-infected hNPCs with Emricasan for 72 hours followed by Niclosamide (an antiviral compound) for 48 hours led to the recovery of ZIKV-negative hNPCs. [2]

Emricasan reduces NASH-related liver damage but not metabolic disturbance. It lessens inflammation as well. The pan-caspase inhibitor Emricasan reduces hepatic fibrogenesis and stellate cell activation in the murine NASH model[1]. Phase 2 clinical trials are currently being conducted on emricasan to determine whether it can lessen the hepatic damage and liver fibrosis brought on by chronic HCV infection[2].

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Mice fed a HFD diet demonstrate a five-fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5-fold and 1.3-fold increase in caspase-3 and-8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD-Em). Likewise, liver injury and inflammation were reduced in mice fed HFD-Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1-β, TNF-α, monocyte chemoattractant protein (MCP-1) and C-X-C chemokine ligand-2 (CXCL2) quantitative reverse-transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR. Conclusion: In conclusion, these data demonstrate that in a murine model of NASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy in NASH.[2]

A quantitative high-throughput screening (qHTS), in which each compound was assayed in four concentrations (0.37, 1.84, 9.2, and 46 μM), was performed in singlet for the primary compound screen. While a single compound concentration (in singlet) has been traditionally used for HTS of large compound collections (such as 1 to 3 million compounds), the qHTS format with multiple compound concentrations has recently been used for medium or small compound collections such as approved drug library. Specifically, SNB-19 cells and hNPCs were seeded onto PDL coated 1536-well assay plates at 250 cells per 3 μl/well and incubated at 37°C in 5% CO2 for 16 hours. Test compounds dissolved in DMSO were transferred to assay plates at a volume of 23 nl/well by an automated pintool workstation. Compounds were incubated with cells for 30 minutes at 37°C in 5% CO2, immediately followed by the addition of 2 μl/well of ZIKV (2 FFU/cell). Incubation time of compound-treated cells with ZIKV varied based on assay format. Experiments measuring virus-induced caspase-3/7 activity required a 6-hour incubation of ZIKV in the presence compounds at 37°C in 5% CO2. Following this incubation, 3.5 μl/well of caspase-3/7 reagent mixture was added to assay plates. The plates were incubated for 30 minutes at room temperature, and the resultant luminescence signal was measured using a ViewLux plate reader. Experiments measuring virus-induced cell death required a 72-hour incubation of ZIKV in the presence of compounds at 37°C in 5% CO2. Following this incubation, 3.5 μl/well of ATP content detection reagent was added to assay plates. The plates were incubated for 30 minutes at room temperature, and the resultant luminescence signal was measured in a ViewLux plate reader. Step-by-step assay protocols are listed in Supplementary Tables 1 and 2.[2]

Astrocytes are mock-infected, treated with DMSO, or treated with 2 M niclosamide, 92 M PHA-690509, 9 μM emricasan, or a combination of 92 μM PHA-690509 and 9 M emricasan for 1 h prior to infection with PRVABC59 (MOI = 0.5). Following infection for 24 hours, cells are fixed and stained for ZIKVE and nuclei.

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ATP content assay for cell viability and compound cytotoxicity[2]
The ATPlite luminescence assay system assay kit was used to determine cell viability. The reagent was reconstituted and prepared as described by the manufacturer. In order to measure the cell death caused by ZIKV infection, cells were cultured for 16 hours at 37°C with 5% CO2 in assay plates, followed by addition of ZIKV solution and incubation at 37°C with 5% CO2 for 72 hours. ATPlite, the ATP monitoring reagent, was then added to the assay plates and they were incubated for 15 minutes. The resulting luminescence was measured using the ViewLux plate reader. Data were normalized using wells without cells as a control for 100% cell killing, and cell-containing wells without ZIKV infection as full cell viability (0% cell killing). For analysis of potential toxicity of select compounds, cells were seeded in 96-well plates. One day later, cells were treated with indicated compounds and concentrations for 24–48 hours prior to the addition of Cell Titer-Glo substrate and measured according to manufacturer instructions.

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Caspase-3/7 activity was measured using a luminescence-based assay kit. Cells were seeded in assay plates and cultured. ZIKV solution was added to cells, followed by incubation. Caspase-Glo-3/7 reagent was then added to each well and incubated at room temperature. The luminescence intensity was measured using a plate reader. Data were normalized using uninfected cells as a negative control and ZIKV-infected cells as a positive control. [2]
Cell viability was determined using a luminescence-based ATP content assay kit. Cells were cultured in assay plates, followed by addition of ZIKV solution and incubation for 72 hours. ATP monitoring reagent was then added to the assay plates and incubated. The resulting luminescence was measured. Data were normalized using wells without cells as a control for 100% cell killing, and uninfected cell-containing wells as full cell viability. [2]
For immunocytochemistry, cells were fixed with paraformaldehyde. Samples were permeabilized and blocked, then incubated with primary antibodies (e.g., anti-cleaved caspase-3) overnight at 4°C, followed by incubation with secondary antibodies. Samples were mounted with mounting medium containing DAPI. Images were taken by confocal microscopes. Quantitative analyses were conducted on randomly selected fields. [2]
For organoid studies, forebrain-specific organoids were exposed to ZIKV. After fixation and immunostaining (e.g., for cleaved caspase-3 and DAPI), quantitative analyses were conducted on randomly selected cortical structures captured by confocal microscope in a blind fashion. Cell death was quantified by counting activated Caspase-3-positive nuclei over total DAPI-positive nuclei in the ventricular structures. [2]

Reagents and formulation [1]
Emricasan (formerly named IDN-6556 or PF-03491390) was suspended in vehicle [2% (v/v) DMSO in 0.5% (w/v) methylcellulose] and administered to mice per os daily. The high fat diet (HFD) used was used, which contains 47% of calories from fat (mostly from Milk fat, 50% saturated fat) with 2% of cholesterol, 35% from carbohydrate (78% of carbohydrate from Sucrose) and 18% of calories from protein, and was designed to approximate the typical human diet from patients with NASH.

Animals[1]
Studies were performed in male C57BL/6J mice. All animals were maintained in a temperature (24°C) and light controlled (12:12 h light:dark) facility, and had free access to food and water. Animals were age-matched and used at approximately 12–16 weeks of age. Four groups were studied (n = 60) with 15 mice per group. Groups 1 and 3 received regular chow. Groups 2 and 4 received HFD and 50 g/L (Sucrose) was added to drinking water for 20 weeks. Groups 3 and 4 received Emricasan 0.3 mg/kg/day per os, and Group 1 and 2 received the vehicle. The dosing was based on previous data 21 that demonstrates that oral administration of Emricasan at doses of 0.3 mg/kg corresponded to the ED90 value to prevent liver injury in the model of α-Fas-induced liver injury. Total body weight was measured at 0, 5, 10, 15 and 20 weeks.

Emricasan Pharmacokinetics [https://pmc.ncbi.nlm.nih.gov/articles/PMC6175779/]
Table 1 summarizes the key pharmacokinetic data. On days 1 and 4, the geometric means of Emricasan AUC0–8, Cmax, and AUC0–last increased approximately dose-proportionally in both the 5 mg and 50 mg dose groups. No plasma accumulation was observed in any treatment group on day 4 compared to day 1. Overall, inter-subject variability of all pharmacokinetic parameters was lower in the low-dose treatment groups compared to the 50 mg group, with coefficients of variation (CV) ranging from 28% to 48% for the IDN-6556 5 mg and 25 mg groups, and from 99% to 258% for the IDN-6556 50 mg group. Pharmacokinetic analysis in rats showed rapid drug clearance after intravenous, intraperitoneal, and subcutaneous injection, with terminal half-life (t_1/2) ranging from 46 to 51 minutes. While the absolute bioavailability after oral administration was lower (2.7–4%), the portal vein concentration was three times higher than the systemic concentration, and the terminal half-life was prolonged by 3.7 times, indicating a significant first-pass effect. Hepatic drug concentrations remained stable for at least 4 hours after oral administration, reaching a C_max of 2558 ng/g liver at 120 minutes. Finally, 51 ± 20% and 4.9 ± 3.4% of IDN-6556 were excreted intact via bile after intravenous and oral administration, respectively. This assessment demonstrates that IDN-6556 has significant therapeutic efficacy in a liver disease model after oral administration, making it an ideal candidate drug for treating liver diseases characterized by excessive apoptosis. [https://pubmed.ncbi.nlm.nih.gov/14742742/]

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It has been reported that after oral administration (twice daily for 4 consecutive days), the total concentration and maximum concentration of Emricasan in human blood were 1.90 µg/ml (3.35 µM) and 2.36 µg/ml (4.15 µM), respectively. [2]
The reported human plasma concentration of Emricasan was approximately 10 times higher than the IC50 value for inhibiting the increase in caspase-3 activity and cell death induced by Zika virus infection in vitro. [2]

Safety [https://pmc.ncbi.nlm.nih.gov/articles/PMC6175779/]
As shown in Figure 2, a total of 10 deaths occurred across all treatment groups. Of these, 5 occurred during the study period, 2 occurred during the one-month follow-up period after completion of the full course of treatment, and 5 were reported as serious adverse events (SAEs) after the study ended or after discontinuation of treatment. All of these deaths were attributed to progressive liver disease. Adverse events (AEs) were reported in 17 of the 21 patients, of which 13 were SAEs. All SAEs were determined to be treatment-independent. The only AE considered to be treatment-related was nausea and vomiting reported by one patient in the placebo group. Adverse events (AEs) and serious adverse events (SAEs) are listed in Table 5.

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Emricasan was well tolerated in human trials (for the treatment of chronic hepatitis C virus infection) with no significant adverse events. [2]

[1]. Liver Int . 2015 Mar;35(3):953-66.

[2]. Nat Med . 2016 Oct;22(10):1101-1107.

Emricasan is the first caspase inhibitor to undergo human trials and receive FDA orphan drug designation. Developed by Pfizer, its mechanism of action involves protecting hepatocytes from excessive apoptosis.

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Drug Indications
It is being investigated for the treatment of hepatitis (viral, hepatitis C), liver disease, and liver transplantation (organ or tissue transplantation).
Treatment of Nonalcoholic Steatohepatitis (NASH)Mechanism of Action
IDN-6556 significantly improves liver damage markers in patients infected with hepatitis C virus (HCV). HCV infection affects up to 170 million patients worldwide. IDN-6556 represents a new class of drugs that protect the liver from inflammation and cellular damage caused by viral infection and other factors. Multiple studies have shown that this drug significantly reduces aminotransferase activity in patients with hepatitis C virus (HCV) and is well-tolerated.

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Background and Objectives: Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis (NASH), leading to liver damage and fibrosis. Although both intrinsic and extrinsic apoptosis pathways are involved in the pathogenesis of NASH, the final common step in apoptosis is performed by a class of cysteine proteases called caspases. Therefore, our aim was to determine whether administration of the pan-cysteine inhibitor Emricasan could improve liver injury and fibrosis in a mouse model of NASH. Methods: C57/BL6J mice were fed either a normal diet or a high-fat diet (HFD) for 20 weeks. All mice received either a vector or Emricasan treatment. Results: TUNEL assays in the high-fat diet (HFD) group showed a 5-fold increase in hepatocyte apoptosis, with caspase-3 and caspase-8 activities increasing by 1.5-fold and 1.3-fold, respectively; while apoptosis was significantly reduced in the Emricasan-treated group (HFD-Em). Furthermore, compared to the HFD group, the HFD-Em group mice showed reduced liver injury and inflammation in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, NAS histological scores, and quantitative reverse transcription polymerase chain reaction (qPCR) results for IL-1β, TNF-α, monocyte chemoattractant protein-1 (MCP-1), and CXC chemokine ligand 2 (CXCL2). Since the liver triglyceride content was similar in both HFD groups, these differences were not due to differences in hepatic steatosis. In the HFD animal model, Emricasan alleviated liver fibrosis by reducing αSMA (a marker of hepatic stellate cell activation), fibrosis score, Sirius red staining, liver hydroxyproline content, and the levels of pro-fibrotic cytokines detected by qPCR. Conclusion: In summary, these data indicate that inhibiting hepatocyte apoptosis can suppress liver injury and fibrosis in a NASH mouse model, suggesting that Emricasan may be a promising anti-fibrotic therapy for NASH. [1]

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In light of the Zika virus (ZIKV) outbreak and its association with microcephaly and other neurological disorders, we conducted a drug repurposing screening of approximately 6,000 compounds, including approved drugs, clinical trial candidates, and pharmacologically active compounds; we screened for compounds that could inhibit ZIKV infection or inhibit infection-induced caspase-3 activity in different neural cells. The pan-caspase inhibitor emricasan inhibited Zika virus (ZIKV)-induced increases in caspase-3 activity and protected human cortical neural progenitor cells in monolayer and three-dimensional organoid cultures. Ten structurally unrelated cyclin-dependent kinase inhibitors inhibited ZIKV replication. Nicoloxamide (an FDA-approved Category B anthelmintic) also inhibited ZIKV replication. Finally, combination therapy using compounds from both neuroprotective and antiviral classes further enhanced the protection of human neural progenitor cells and astrocytes from ZIKV-induced cell death. Our results demonstrate the effectiveness of this screening strategy and identify lead compounds for the development of anti-ZIKV drugs. [2]

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Background: Cirrhosis and acute exacerbation of chronic liver failure (ACLF) are associated with systemic inflammation and caspase-mediated hepatocyte death. Emricasan is a novel pan-caspasesin inhibitor. This study aimed to evaluate the pharmacokinetics, pharmacodynamics, safety, and clinical efficacy of emricasan in patients with acute decompensated cirrhosis (AD).
Methods: This was a phase II, multicenter, double-blind, randomized controlled trial. The primary objective was to evaluate the pharmacokinetics, pharmacodynamics, and safety of emricasan in patients with cirrhosis and organ failure. AD was defined as an acute decompensated event lasting ≤6 weeks. Patients were pro rataly randomized to emricasan 5 mg bid, emricasan 25 mg bid, emricasan 50 mg bid, or placebo. Treatment continued for up to 28 days, or patients voluntarily discontinued treatment. Results: 23 subjects were randomized, with 21 receiving the drug (placebo n=4; 5 mg n=5; 25 mg n=7; 50 mg n=5). Pharmacokinetic data showed lower plasma drug concentrations (<50 ng/ml) in the 5 mg group, while the 25 mg and 50 mg groups had similar pharmacokinetic profiles. Therefore, for secondary endpoint analysis, the placebo and 5 mg groups were grouped as the "placebo/low-dose" group, and the 25 mg and 50 mg groups were grouped as the "high-dose" group. Five of the 21 patients died, all due to liver disease progression (2 in the placebo/low-dose group and 3 in the high-dose group). On day 7, there were no statistically significant changes in MELD or CLIF-C ACLF scores from baseline in either the placebo/low-dose or high-dose groups (MELD -1 vs -1, CLIF-C ACLF 0.7 vs 0.8). The levels of cleaved keratin M30 fragments were initially reduced in both the placebo/low-dose and high-dose groups (relative percentage change: Day 2: -11.6 vs -42.6, P = 0.017; Day 4: -3.5 vs -38.9, P = 0.017), but this reduction did not persist into Day 7 (-3.1 vs -20.8, P = 0.342). Conclusion: This study demonstrates that emricasan is safe and well-tolerated in patients with advanced liver disease. However, this study failed to provide proof-of-concept support for the use of caspase inhibitors as a treatment strategy for acute liver failure (ACLF). Emricasan (also known as IDN-6556 or PF-03491390) is an activated caspase inhibitor. [2] Emricasan is currently undergoing a phase II clinical trial to evaluate its efficacy in reducing liver injury and liver fibrosis caused by chronic hepatitis C virus (HCV) infection. [2] Emricasan has neuroprotective effects on human neural progenitor cells (hNPCs) and resists Zika virus (ZIKV)-induced cell death, but does not inhibit ZIKV replication. [2] Future animal studies are crucial for evaluating the efficacy of emricasan against ZIKV in vivo. The safety of using emricasan to treat Zika virus infection during pregnancy needs to be evaluated through preclinical toxicology studies and clinical trials. [2]

C26H27F4N3O7

569.5021

569.178

C, 54.83; H, 4.78; F, 13.34; N, 7.38; O, 19.66

254750-02-2

254750-02-2;

12000240

white solid powder

1.4±0.1 g/cm3

1.550

4.63

4

11

11

40

934

2

FC1C(=C([H])C(=C(C=1OC([H])([H])C([C@]([H])(C([H])([H])C(=O)O[H])N([H])C([C@]([H])(C([H])([H])[H])N([H])C(C(N([H])C1=C([H])C([H])=C([H])C([H])=C1C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)=O)=O)=O)F)F)F

SCVHJVCATBPIHN-SJCJKPOMSA-N

InChI=1S/C26H27F4N3O7/c1-12(31-24(38)25(39)32-16-8-6-5-7-13(16)26(2,3)4)23(37)33-17(10-19(35)36)18(34)11-40-22-20(29)14(27)9-15(28)21(22)30/h5-9,12,17H,10-11H2,1-4H3,(H,31,38)(H,32,39)(H,33,37)(H,35,36)/t12-,17-/m0/s1

(3S)-3-[[(2S)-2-[[2-(2-tert-butylanilino)-2-oxoacetyl]amino]propanoyl]amino]-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid

Emricasan; PF 03491390; PF-03491390; IDN-6556; (S)-3-((S)-2-(2-(2-TERT-BUTYLPHENYLAMINO)-2-OXOACETAMIDO)PROPANAMIDO)-4-OXO-5-(2,3,5,6-TETRAFLUOROPHENOXY)PENTANOIC ACID; (S)-3-((S)-2-(2-((2-(tert-Butyl)phenyl)amino)-2-oxoacetamido)propanamido)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid; C26H27F4N3O7; PF03491390; IDN-6556; IDN6556; IDN 6556

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 (~175.6 mM)
Ethanol: ~25 mg/mL (~43.9 mM)

Solubility in Formulation 1: 2.5 mg/mL (4.39 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (4.39 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (4.39 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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Solubility in Formulation 4: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 5mg/ml

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