p38α (pKi = 8.1); p38β (pKi = 7.6)

Losmapimod (GW856553X, GW856553, GSK-AHAB) is a selective, potent, and orally active p38 MAPK inhibitor.

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As p38 signaling is also capable of inducing apoptosis, researchers asked whether p38 inhibition could also prevent DUX4-mediated cell death. Researchers chose to test the p38α/β inhibitor losmapimod because it is currently being investigated as a treatment for FSHD in a clinical trial (NCT04003974). p38 inhibition has been shown to prevent DUX4 expression in FSHD patient-derived myotubes, thereby preventing cell death (Oliva et al., 2019; Rojas et al., 2020); however, a role in DUX4 mediated-apoptosis independent of DUX4 expression has not been explored. As expected, losmapimod treatment did not affect doxycycline-induced DUX4 expression (Fig. S5D,E). Researchers performed a similar live-cell imaging experiment as above, treating cells with mock, 1 µM or 10 µM losmapimod at the same time as doxycycline induction. Losmapimod treatment of uninduced iDUX4 myoblasts had no effect on cell death, indicating a lack of toxicity at these concentrations (Fig. S5F). Remarkably, losmapimod treatment prevented DUX4-mediated cell death in a dose-dependent manner, as demonstrated by both the viability stain (Fig. 7C,D) and the Caspase 3/7 stain (Fig. 7E,F). Researchers conclude that p38 is activated by DUX4 expression and contributes to DUX4-dependent apoptosis, independent of its role in controlling DUX4 expression. Interestingly, simultaneous addition of 10 µM SP600125 and 1 µM losmapimod displayed decreased DUX4-mediated cell death compared to either inhibitor alone (Fig. 7G,H). This suggests that the combined inhibition of JNK and p38 signaling confers enhanced protection against DUX4 cytotoxicity.[2]

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Inhibition of p38 MAPK by Losmapimod Prevents Gefitinib-Induced Tetraploidization [3]
Researchers observed that gefitinib treatment could induce tetraploidization through p38 MAPK signaling in gefitinib-resistant cells. Thus, to further investigate whether inhibition of p38 MAPK could prevent tetraploidization, both HCC827GR and H1975 cell lines were treated with gefitinib and/or p38 MAPK inhibitors (either losmapimod or SB203580) for 24 h. Results showed that each p38 MAPK inhibitors could eliminate gefitinib-induced tetraploidy (Fig. 3A) in both resistant cell lines. In addition, Western blot analyses showed that losmapimod could inhibit p38 MAPK phosphorylation induced by gefitinib treatment. However, gefitinib-induced MKK3/6 phosphorylation was further up-regulated by losmapimod and there was no changes of YAP phosphorylation (Fig. 3B). To further examine the detailed mechanism of gefitinib-induced tetraploidization, researchers determined p-STAT3, p21 and cyclin D1 expressions after gefitinib and/or losmapimod treatment in gefitinib-resistant cells. Consistent with the results of tetraploidization and p38 MAPK inhibition, Western blotting revealed that phosphorylation of STAT3 and expressions of p21 and cyclin D1 were up-regulated with gefitinib treatment in both resistant cell lines, and that the up-regulations could be inhibited by losmapimod (Fig. 3C). All these findings suggest that gefitinib-induced tetraploidization requires p38 MAPK signaling and could be targeted by p38 MAPK inhibitors.

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Losmapimod Successfully Overcomes Gefitinib-Resistance in Lung Cancer Cells [3]
Based on the previous data, researchers determined whether losmapimod could overcome gefitinib resistance in gefitinib-resistant human lung cancer cells. First, researchers treated both HCC827GR and H1975 cells with either gefitinib or losmapimod alone and then in combination. Our results revealed that the combined treatment with gefitinib and losmapimod significantly reduced both anchorage-independent cell growth (Fig. 4A, B) and proliferation (Fig. 4C) of gefitinib-resistant cells. However, neither gefitinib nor losmapimod alone could inhibit either type of cell proliferation. In order to examine the combination effect of gefitinib and losmapimod, the IC50 value of gefitinib or losmapimod alone treatment in both cells was first determined (Supplementary Fig. 2 and Table 1) and the combination index was then calculated by using the CompuSyn program. Results showed that co-treatment of gefitinib and losmapimod synergistically inhibited cell proliferation with a combination index of 0.14 for the HCC827GR and 0.22 for the H1975 cell line (Supplementary Table 2). Overall, our data indicates that losmapimod could be a potential drug to overcome gefitinib resistance in NSCLC.

Losmapimod attenuates dyslipidemia, hypertension, cardiac remodeling, plasma renin activity (PRA), aldosterone, and interleukin-1beta (IL-1beta) after significantly enhancing survival, endothelial-dependent and -independent vascular relaxation, and indicators of renal function in spontaneously hypertensive stroke-prone rats (SHR-SP). [1]

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Losmapimod Suppresses Tumor Growth in a Gefitinib-Resistant NSCLC PDX Model [3]
To further investigate the chemotherapeutic effect of losmapimod and its clinical relevance, reserchers conducted in vivo experiments using an in-house generated gefitinib-resistant PDX model. Results showed that losmapimod alone or in combination with gefitinib markedly reduced gefitinib-resistant NSCLC PDX tumor volume and weight, whereas gefitinib alone had no effect (Fig. 6A-C). In addition, no changes in mouse body weight were observed, suggesting that toxicity was not associated with the different treatments (Fig. 6D). In addition, immunohistochemical analysis of harvested PDX tumors were conducted to evaluate the expression level of cyclin D1, p-p38 and Ki-67 (Fig. 6E, F). Our results showed that cyclin D1, p-p38 and Ki-67 were significantly reduced in both losmapimod-treated and groups treated with a combination of losmapimod and gefitinib compared with the vehicle- or gefitinib-treated group. These data provide strong evidence that losmapimod could be used as a promising candidate to overcome gefitinib-resistant lung cancers clinically.

A ligand-displacement fluorescence polarization assay is used to determine the inhibition of p38β and p38.

Live-cell imaging [2]
Cells were cultured in black-walled, 96 well plates and SP600125 or Losmapimod or DMSO (vehicle control) was added along with Incucyte Cytotox Dye and Incucyte Caspase 3/7 Dye simultaneously upon induction of DUX4. Cells were imaged every hour (Fig. 1H) or every 2 h (Fig. 7) using the IncuCyte live-cell imaging system. Images were analyzed using Incucyte software to quantify Cytotox- and Caspase3/7-positive cells.

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Flow Cytometry for Cell Cycle Analysis [3]
Cells were plated at a density of 3 × 105 cells/dish in 60-mm dishes overnight. The cells were then treated with vehicle, gefitinib, or a combination of gefitinib and a p38 MAPK inhibitor (either SB203580 or Losmapimod) for another 24 h. Cells were trypsinized, washed twice with cold PBS and then fixed with 70% ethanol overnight at -20 °C. The cells were stained with propidium Iodide and analyzed with the FACSCalibur flow cytometer. The data were then analyzed by CellQuest and Modfit LT V4.0 software

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Cell Viability Assay [3]
Cytotoxicity of Losmapimod and/or gefitinib was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Briefly, cells (3 × 103 cells/well) were plated in 96-well plates overnight for attachment. The cells were then treated with vehicle, gefitinib, a p38 MAPK inhibitor (either SB203580 or losmapimod), or a combination of gefitinib and p38 MAPK inhibitor for 72 h and MTT (0.3 mg/mL) was added to the media for 1 h at 37 °C. The reaction was terminated by the addition of 100 μL DMSO. The optical density of the MTT formazan formation was read at 490 nm on a microplate reader. Absorbance values were normalized as a percentage of untreated cells (set at 100%).

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Anchorage-Independent Cell Growth Assay [3]
Cells (8 × 103) were suspended in 1 mL RPMI-1640/10% FBS/0.33% agar with vehicle, gefitinib, a p38 MAPK inhibitor (either SB203580 or Losmapimod), or a combination of gefitinib and p38 MAPK inhibitor and plated on 3 mL solidified RPMI-1640/10% FBS/0.5% agar with the same concentration of vehicle, SB203580, losmapimod and/or gefitinib in each well of 6-well plates in triplicates and cultured for 3 weeks. Images from 5 independent fields of each well were captured by a microscope using Images-Pro Plus software. Colony numbers > 200 pixels were quantified by Image J software.

Male SHR-SPs (n=70) were divided into five groups (n=14 per group) based on body weight and randomly assigned to one of five diets: normal diet controls (ND), high salt-fat diet controls (SFD), SFD + GSK-AHAB (1.2 mg/kg/day), and SFD + GSK-AHAB (12 mg/kg/day) and SFD + MK 966 (18 mg/kg/day). All medications are taken orally by mixing with SFD. The conscious measurement of mean arterial blood pressure and heart rate is performed on a subgroup of animals from each group (n=6 per group) who have undergone anesthesia and have been surgically outfitted with radiotelemetry devices. Before the study begins, these animals are given at least 7 days to recover.

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The in vivo Gefitinib-Resistant NSCLC PDX Model [3]
Previously, an in vivo gefitinib-resistant NSCLC PDX model was generated in-house (Zhang et al., 2015). The gefitinib-resistant tumor fragments were passaged to 40 mice for an in vivo study. Mice were divided into four groups (n = 10 mice per group). The four groups were: 1) vehicle control; 2) 50 mg/kg gefitinib; 3) 12 mg/kg Losmapimod; and 4) 50 mg/kg gefitinib and 12 mg/kg Losmapimod (Willette et al., 2009). Once the tumor volumes reached approximately 25 mm3, mice were treated by oral gavage with vehicle control (dimethyl sulfoxide 5%, normal saline 50% and PEG400 50%), gefitinib and/or Losmapimod. Body weights and tumor measurements were performed twice a week and tumor volume was calculated based on the formula: length × width2 × 0.5. At the end of the experiment, mice were sacrificed prior to removal of the tumors for further analysis.

This phase 1 study characterized the safety, tolerability, pharmacokinetics, and pharmacodynamics of losmapimod and its metabolite GSK198602 following single and repeat doses of oral losmapimod in healthy Japanese volunteers. Subjects (n = 41) received single oral doses of losmapimod (2.5, 7.5, 20 mg) or matching placebo on 3 separate days (n = 20) or losmapimod 7.5 mg or matching placebo twice daily for 14 days (n = 21). Assessments included maximum observed plasma concentration (Cmax ), time to Cmax (Tmax ), apparent terminal-phase half-life (t1/ )2 , area under the curve (AUC), and change in C-reactive protein and phosphorylated heat shock protein 27 levels. No serious adverse events occurred during the study, and there were no safety concerns regarding clinical laboratory parameters, 12-lead electrocardiogram, or vital signs. The losmapimod Tmax was 3-4 hours, and the mean t1/2 was approximately 7.9-9.0 hours, with no appreciable difference in Tmax and apparent clearance following oral dosing between dosing regimens. Single and repeat oral doses of losmapimod were well tolerated in healthy Japanese volunteers. The Tmax of GSK198602 was similar to and t1/2 was slightly longer than those of losmapimod. Approximate dose-proportional increases in exposure to losmapimod and GSK198602 were observed in AUC with single-dose administration. Repeat-dose trough concentrations reached steady state within 2 days, with an observed accumulation ratio of 1.56 and 1.91 for losmapimod and GSK198602, respectively.
Reference: Clin Pharmacol Drug Dev. 2015 Jul;4(4):262-9. https://pubmed.ncbi.nlm.nih.gov/27136906/

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Aims: Evaluate safety, tolerability, pharmacokinetics (PK) and target engagement (TE) of losmapimod in blood and muscle in facioscapulohumeral dystrophy (FSHD).
Methods: This study included Part A: 10 healthy volunteers randomized to single oral doses of losmapimod (7.5 mg then 15 mg; n = 8) or placebo (both periods; n = 2); Part B: 15 FSHD subjects randomized to placebo (n = 3), or losmapimod 7.5 mg (n = 6) or 15 mg (n = 6); and Part C: FSHD subjects received open-label losmapimod 15 mg (n = 5) twice daily for 14 days. Biopsies were performed in FSHD subjects at baseline and Day 14 in magnetic resonance imaging-normal appearing (Part B) and affected muscle identified by abnormal short-tau inversion recovery sequence + (Part C). PK and TE, based on pHSP27:total HSP27, were assessed in muscle and sorbitol-stimulated blood.
Results: PK profiles were similar between healthy volunteers and FSHD subjects, with mean Cmax and AUC0-12 for 15 mg in FSHD subjects (Part B) of 85.0 ± 16.7 ng*h/mL and 410 ± 50.3 ng*h/mL, respectively. Part B and Part C PK results were similar, and 7.5 mg results were approximately dose proportional to 15 mg results. Dose-dependent concentrations in muscle (42.1 ± 10.5 ng/g [7.5 mg] to 97.2 ± 22.4 ng/g [15 mg]) were observed, with plasma-to-muscle ratio from ~0.67 to ~1 at estimated tmax of 3.5 hours postdose. TE was observed in blood and muscle. Adverse events (AEs) were mild and self-limited.
Conclusion: Losmapimod was well tolerated, with no serious AEs. Dose-dependent PK and TE were observed. This study supports advancing losmapimod into Phase 2 trials in FSHD.
Reference:Br J Clin Pharmacol. 2021 Dec;87(12):4658-4669. https://pubmed.ncbi.nlm.nih.gov/33931884/

Aims: The purpose of this study was to establish safety and tolerability of a single intravenous (IV) infusion of a p38 mitogen-activated protein kinase inhibitor, losmapimod, to obtain therapeutic levels rapidly for a potential acute coronary syndrome indication. Pharmacokinetics (PK) following IV dosing were characterized, and pharmacokinetic/pharmacodynamic (PK/PD) relationships between losmapimod and phosphorylated heat shock protein 27 (pHSP27) and high-sensitivity C-reactive protein were explored.
Methods: Healthy volunteers received 1 mg losmapimod IV over 15 min (n = 4) or 3 mg IV over 15 min followed by a washout period and then 15 mg orally (PO; n = 12). Pharmacokinetic parameters were calculated by noncompartmental methods. The PK/PD relationships were explored using modelling and simulation.
Results: There were no deaths, nonfatal serious adverse events or adverse events leading to withdrawal. Headache was the only adverse event reported more than once (n = 3 following oral dosing). Following 3 mg IV and 15 mg PO, Cmax was 59.4 and 45.9 μg l(-1) and AUC0-∞ was 171.1 and 528.0 μg h l(-1) , respectively. Absolute oral bioavailability was 0.62 [90% confidence interval (CI) 0.56, 0.68]. Following 3 mg IV and 15 mg PO, maximal reductions in pHSP27 were 44% (95% CI 38%, 50%) and 55% (95% CI 50%, 59%) occurring at 30 min and 4 h, respectively. There was a 17% decrease (95% CI 9%, 24%) in high-sensitivity C-reactive protein 24 h following oral dosing. A direct-link maximal inhibitory effect model related plasma concentrations to pHSP27 concentrations.
Conclusions: A single IV infusion of losmapimod in healthy volunteers was safe and well tolerated, and may potentially serve as an initial loading dose in acute coronary syndrome as rapid exposure is achieved.
Refrence: Br J Clin Pharmacol . 2013 Jul;76(1):99-106. https://pubmed.ncbi.nlm.nih.gov/23215699/

[1]. Differential effects of p38 mitogen-activated protein kinase and cyclooxygenase 2 inhibitors in a model of cardiovascular disease. J Pharmacol Exp Ther. 2009 Sep;330(3):964-70.

[2]. DUX4 expression activates JNK and p38 MAP kinases in myoblasts. Dis Model Mech. 2022 Nov 1;15(11):dmm049516.

[3]. Losmapimod Overcomes Gefitinib Resistance in Non-small Cell Lung Cancer by Preventing Tetraploidization. eBioMedicine. 2018 Feb 2;28:51–61.

6-[5-[(cyclopropylamino)-oxomethyl]-3-fluoro-2-methylphenyl]-N-(2,2-dimethylpropyl)-3-pyridinecarboxamide is a phenylpyridine.
Losmapimod has been investigated for the prevention of Chronic Obstructive Pulmonary Disease.
Losmapimod is an orally bioavailable inhibitor of the alpha and beta isoforms of p38 mitogen-activated protein kinase (MAPK), with potential immunomodulating and anti-inflammatory activities. Upon oral administration, losmapimod inhibits the activity of p38alpha/beta MAPK, thereby preventing p38alpha/beta MAPK-mediated signaling. This may result in the inhibition of the production of proinflammatory cytokines. p38 MAPK, a serine/threonine protein kinase, plays an important role in regulating the transcription and translation of cytokines involved in inflammation including tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-1, IL-6 and IL-8.

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The evidence is compelling for a role of inflammation in cardiovascular diseases; however, the chronic use of anti-inflammatory drugs for these indications has been disappointing. The recent study compares the effects of two anti-inflammatory agents [cyclooxygenase 2 (COX2) and p38 inhibitors] in a model of cardiovascular disease. The vascular, renal, and cardiac effects of 4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one (rofecoxib; a COX2 inhibitor) and 6-{5-[(cyclopropylamino)carbonyl]-3-fluoro-2-methylphenyl}-N-(2,2-dimethylpropyl)-3-pyridinecarboxamide [GSK-AHAB, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor], were examined in the spontaneously hypertensive stroke-prone rat (SHR-SP). In SHR-SPs receiving a salt-fat diet (SFD), chronic treatment with GSK-AHAB significantly and dose-dependently improved survival, endothelial-dependent and -independent vascular relaxation, and indices of renal function, and it attenuated dyslipidemia, hypertension, cardiac remodeling, plasma renin activity (PRA), aldosterone, and interleukin-1beta (IL-1beta). In contrast, chronic treatment with a COX2-selective dose of rofecoxib exaggerated the harmful effects of the SFD, i.e., increasing vascular and renal dysfunction, dyslipidemia, hypertension, cardiac hypertrophy, PRA, aldosterone, and IL-1beta. The protective effects of a p38 MAPK inhibitor are clearly distinct from the deleterious effects of a selective COX2 inhibitor in the SHR-SP and suggest that anti-inflammatory agents can have differential effects in cardiovascular disease. The results also suggest a method for evaluating long-term cardiovascular efficacy and safety.[1]

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Facioscapulohumeral muscular dystrophy (FSHD) is caused by misexpression of the DUX4 transcription factor in skeletal muscle that results in transcriptional alterations, abnormal phenotypes and cell death. To gain insight into the kinetics of DUX4-induced stresses, we activated DUX4 expression in myoblasts and performed longitudinal RNA sequencing paired with proteomics and phosphoproteomics. This analysis revealed changes in cellular physiology upon DUX4 activation, including DNA damage and altered mRNA splicing. Phosphoproteomic analysis uncovered rapid widespread changes in protein phosphorylation following DUX4 induction, indicating that alterations in kinase signaling might play a role in DUX4-mediated stress and cell death. Indeed, we demonstrate that two stress-responsive MAP kinase pathways, JNK and p38, are activated in response to DUX4 expression. Inhibition of each of these pathways ameliorated DUX4-mediated cell death in myoblasts. These findings uncover that the JNK pathway is involved in DUX4-mediated cell death and provide additional insights into the role of the p38 pathway, a clinical target for the treatment of FSHD.[2]

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The epidermal growth factor receptor (EGFR) is known to play a critical role in non-small cell lung cancer (NSCLC). Constitutively active EGFR mutations, including in-frame deletion in exon 19 and L858R point mutation in exon 21, contribute about 90% of all EGFR-activating mutations in NSCLC. Although oral EGFR-tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, show dramatic clinical efficacy with significantly prolonged progression-free survival in patients harboring these EGFR-activating mutations, most of these patients will eventually develop acquired resistance. Researchers have recently named genomic instability as one of the hallmarks of cancer. Genomic instability usually involves a transient phase of polyploidization, in particular tetraploidization. Tetraploid cells can undergo asymmetric cell division or chromosome loss, leading to tumor heterogeneity and multidrug resistance. Therefore, identification of signaling pathways involved in tetraploidization is crucial in overcoming drug resistance. In our present study, we found that gefitinib could activate YAP-MKK3/6-p38 MAPK-STAT3 signaling and induce tetraploidization in gefitinib-resistance cells. Using p38 MAPK inhibitors, SB203580 and losmapimod, we could eliminate gefitinib-induced tetraploidization and overcome gefitinib-resistance. In addition, shRNA approach to knockdown p38α MAPK could prevent tetraploidy formation and showed significant inhibition of cancer cell growth. Finally, in an in vivo study, losmapimod could successfully overcome gefitinib resistance using an in-house established patient-derived xenograft (PDX) mouse model. Overall, these findings suggest that losmapimod could be a potential clinical agent to overcome gefitinib resistance in NSCLC.[3]

C22H26FN3O2

383.46

383.2

C, 68.91; H, 6.83; F, 4.95; N, 10.96; O, 8.34

585543-15-3

11552706

white solid powder

1.2±0.1 g/cm3

529.4±50.0 °C at 760 mmHg

274.0±30.1 °C

0.0±1.4 mmHg at 25°C

1.582

3.3

2

4

6

28

573

0

FC1C(C([H])([H])[H])=C(C2C([H])=C([H])C(=C([H])N=2)C(N([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])=C(C=1[H])C(N([H])C1([H])C([H])([H])C1([H])[H])=O

KKYABQBFGDZVNQ-UHFFFAOYSA-N

InChI=1S/C22H26FN3O2/c1-13-17(9-15(10-18(13)23)21(28)26-16-6-7-16)19-8-5-14(11-24-19)20(27)25-12-22(2,3)4/h5,8-11,16H,6-7,12H2,1-4H3,(H,25,27)(H,26,28)

6-[5-(cyclopropylcarbamoyl)-3-fluoro-2-methylphenyl]-N-(2,2-dimethylpropyl)pyridine-3-carboxamide

GW856553; Losmapimod; GSKAHAB; GW856553X; GW-856553; GW 856553, GSK-AHAB; GSK AHAB; GW-856553X; GW 856553X; SB856553; SB-856553; SB 856553

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: 2.75 mg/mL (7.17 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 27.5 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: 2.75 mg/mL (7.17 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 27.5 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: ≥ 2.75 mg/mL (7.17 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 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.5 mg/mL (6.52 mM) in 10% EtOH + 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 EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix well.
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 5: ≥ 2.5 mg/mL (6.52 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 4% DMSO+30% PEG 300+5% Tween 80+ddH2O: 5mg/mL

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