mSmo ( IC50 = 1.3 nM ); hSmo ( IC50 = 2.5 nM )

Sonidegib (NVP-LDE225) has an IC50 value of more than 10 μM for the main human CYP450 drug-metabolizing enzymes[1]. When administered alone or in conjunction with nilotinib, sonidegib (LDE225), a small molecule SMO inhibitor under clinical investigation, inhibits the Hh pathway in CD34+ chronic phase (CP)-chronic myeloid leukemia (CML) cells, thereby decreasing the quantity and potential for self-renewal of CML leukaemia stem cells (LSC). Similar to cyclopamine, sonidegib directly interacts with SMO to decrease the expression of downstream Hh signaling targets. Serum-free medium (SFM)±Sonidegib is used to cultivate primary CD34+ CP-CML cells during 6, 24, and 72 hours (h). After being exposed to Sonidegib at 10 nM; 0.78-fold and 100 nM; 0.73-fold, respectively (p<0.01), GLI1 is considerably downregulated at 72 hours, however there is diversity throughout the biological samples[2].

Sonidegib (NVP-LDE225) has a pKa of 4.2, making it a weak base with comparatively low solubility in water. Sonidegib dose-related antitumor activity in the subcutaneous Ptch+/-p53-/- medulloblastoma allograft mouse model was observed 10 days after oral administration of a suspension of the diphosphate salt. Sonidegib exhibits a significant tumor growth inhibition at a dose of 5 mg/kg/day qd, with a corresponding T/C value of 33% (p<0.05) in comparison to vehicle controls. Sonidegib provides 51 and 83% regression when administered at doses of 10 and 20 mg/kg/day qd, respectively[1]. Secondary recipient mice are transplanted with bone marrow and spleen cells from a subset of treated mice. Comparing bone marrow (BM) or spleen cells transplanted from mice treated with Sonidegib (LDE225)+Nilotinib to Sonidegib or Nilotinib alone, the latter reduces the development of leukemia in secondary recipients and lowers the white cell count (WCC)[2].

Fluorescence binding assays using BODIPY-cyclopamine [1]
Fluorescence binding assays using BODIPY FL or BODIPY® 558/568 labeled binding assays were conducted as described. Briefly, binding assays were conducted in 384-well plates using fixed CHO cells stably expressing mouse or human Smo. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed, covered in PBS buffer containing 0.5% fetal bovine serum, and incubated with fluorescence labeled BODIPY-cyclopamine (20 nM) and the test compounds [e.g. Sonidegib (Erismodegib; LDE225; NVP-LDE225)] for 4 h at 37 °C. The treated cells then were washed with PBS, stained with Hoechst 33258, and analyzed by ImageXpress® Ultra imaging system.

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TM3-Gli-Luc reporter gene assay [1]
Test compounds [e.g. Sonidegib (Erismodegib; LDE225; NVP-LDE225)] were prepared for assay by serial dilution in DMSO and then added to empty assay plates. TM3Hh12 cells (TM3 cells containing Hh-responsive reporter gene construct pTA8xGli-Luc) were cultured in F12 Ham’s/DMEM (1:1) containing 5% horse serum, 2.5% fetal bovine serum (FBS), and 15 mM HEPES, pH 7.3. Cells were harvested by trypsin treatment, resuspended in F12 Ham’s/DMEM (1:1) containing 5% horse serum and 15 mM HEPES, pH 7.3, added to assay plates, and incubated with test compounds for approximately 30 min at 37 °C in 5% CO2. Then 1 or 25 nM Ag1.5 was added to assay plates and incubated at 37 °C in the presence of 5% CO2. After 48 h, either Bright-Glo (Promega E2650) or MTS reagent was added to the assay plates and luminescence or absorbance at 492 nm was determined. IC50 values, defined as the inflection point of the logistic curve, were determined by nonlinear regression of the Gli-driven luciferase luminescence or absorbance signal from MTS assay vs log10 (concentration) of test compounds using the R statistical software package. [1]
LLDE225 blocks the TM3 luciferized cell line with 0.6 nM and 8 nM of Hh agonist Ag1.5 present, respectively.

Proliferation/apoptosis/cell cycle analysis[2]
CD34+ CP-CML cells were seeded in SFM alone ± Sonidegib (Erismodegib; LDE225; NVP-LDE225) ± nilotinib and cultured for 24–72 h prior to assessment. Proliferation was measured using colorimetric assessment of BrDU incorporation. Proportion of viable cells versus those in early and late apoptosis was assessed by flow cytometry using annexin V–FITC and 7-amino-actinomycin D (7-AAD, Via-Probe solution) according to the manufacturer’s instructions. Cell cycle status was assessed as previously described using Ki67 (FITC) expression and 7-AAD incorporation55.

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CFC assay/re-plating assay[2]
CD34+ CP-CML cells were seeded in SFM ± Sonidegib (Erismodegib; LDE225; NVP-LDE225) ± nilotinib and cultured for 72 h then washed three times, inoculated at a concentration of 4 × 103/ml into methylcellulose supplemented with growth factors and cultured in duplicate for 14d prior to colony assessment. Following assessment, at least 20 colonies (granulocyte-erythroid-megakaryocyte-macrophage [GEMM] or granulocyte macrophage [GM]) colonies were plucked from each experimental arm and serially re-dispersed in Methocult with secondary and tertiary colony formation assessed after 7d intervals.

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LTC-IC assay[2]
Primary CD34+ normal and CP-CML cells were cultured in SFM ±Sonidegib (Erismodegib; LDE225; NVP-LDE225) ± nilotinib for 72 h. Following this, they were thoroughly washed and inoculated into pre-prepared long term cultures comprising a stromal feeder layer (a 1:1 mix of irradiated (80 Gy) SL/SL and M210B4 murine fibroblasts) in long term myeloid culture medium (MyeloCult supplemented with hydrocortisone) as previously described35. These cultures were maintained for 5 weeks with 50% media changes performed weekly. Following this, the contents of the wells were harvested and cells counted prior to seeding into Methocult to perform CFC assays as described above.

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Long term stromal co-culture[2]
CD34+ CP-CML cells were inoculated directly into pre-prepared stromal co-cultures, as described above, in the presence of Sonidegib (Erismodegib; LDE225; NVP-LDE225) ± nilotinib. Cultures were maintained for 5 weeks with 80% media changes and addition of fresh drug weekly. Co-cultures were examined weekly by microscopy to ensure that the stromal layer remained morphologically normal and adherent. After 5 weeks, CFC assays were performed as described.

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Prior to assessment, CD34⁺ CP-CML cells are cultured for 24-72 hours in SFM alone±Sonidegib±Nilotinib. BrDU incorporation colorimetric assessment is used to quantify proliferation. Utilizing annexin V-FITC and 7-amino-actinomycin D (7-AAD, Via-Probe solution), flow cytometry is used to determine the ratio of viable cells to those in early and late apoptosis. Ki67 (FITC) expression and 7-AAD incorporation are used to determine the cell cycle status.

Subcutaneous Ptch+/-p53-/- medulloblastoma allograft model. [1]
Mouse Ptch+/-p53-/- medulloblastoma cells ((1.0-5.0) × 106 ), dissociated directly from tumor fragments, were inoculated subcutaneously into the right flank of Harlan nu/nu mice. Treatment was initiated approximately 7 days after implantation. Animals were randomized into treatment groups with similar mean tumor volumes of 271 mm3 with individual tumor sizes ranging from approximately 200 to 340 mm3 . Tumor volumes (mm3 ) and body weights (g) were recorded two or three times per week from all groups for analysis. Dose was body weight adjusted at time of dosing. Comparisons between treatment groups was performed using ANOVA rank sum test.

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Orthotopic Ptch+/-p53-/- medulloblastoma allograft model. [1]
Twenty four athymic nude mice (age 6 week, body weight 21.31 ± 1.52 g) were implanted with 100,000 tumor cells 17 days prior to the intiation of dosing. Tumor cells were stereotactically implanted subcortically at a depth of 3 mm and at 1.5 mm posterior to and 2.5 mm right of bregma. MRI was performed on day 4 prior to initiation of treatment for randomization into treatment group (baseline measurement). Nine animals were excluded from the study based on tumor size. The remaining 16 mice were sorted into a vehicle-treated group and a 5m-treated group so that the mean and SEM were similar. One animal in the5m -treatment group was subsequently excluded from the analysis because the tumor volume did not change over the observation period, and the finding was confirmed by histological evaluation. The mean (± SEM) tumor volume of the 5m-treated group was 3.39 ± 0.26 mm3 , and the mean (± SEM) tumor volume of the vehicle-treated group was 3.19 ± 0.39 mm3 . Treatment (vehicle or 5m at 40 mg/kg/day p.o. b.i.d) was initiated on day 0 (17 days following tumor implantation). Doses are provided as free base equivalents started on day 0. MRI scans were performed on days -4, 0 and +4 In reference to initiation of dosing) Mice were euthanized when they exhibited signs of morbidity.

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Demonstration of an intact blood-brain barrier in the orthotopic Ptch+/-p53-/- medulloblastoma allograft model. [1]
Animals (8 total; 4 groups of 2 each) were implanted with 50,000 or 100,000 tumor cells, and treated with either with 40 mg/kg/day po bid 5m or vehicle. MRI was performed at day 9 post implantation. Images were acquired before and after intraperitoneal administration of 0.4 ml/kg of the contrast agent gadopentetate dimeglumine (Gd-DTPA). In 7 out of 8 animals, the brain was unenhanced after contrast injection, while surrounding cranial muscles indicating the integrity of the blood-brain barrier (Figure 1). No difference was observed between the treatment groups. The remaining animal was in the vehicle-treated group implanted with 100,000 cells. In this case, the tumor grew along the great cerebral vein of Galen, and disrupting the blood-brain barrier, resulting in a hyperintense tumor.

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Imaging of orthotopic Ptch+/-p53-/- medulloblastoma allograft model. [1]
MRI was performed in a Bruker BioSpec 7.0 T scanner, using a 35 mm innerdiameter birdcage resonator for transmission and reception. The mice were anaesthetized with 1.2% – 1.5% isoflurane in oxygen. The head of animal was fixed by a tooth bar and a facemask to minimize motion. Respiration rate and body temperature were monitored continuously and temperature maintained between 32 – 35°C by heated airThe T2-weighted anatomical images were acquired in the coronal view to image the whole mouse brain with a multislice multi-spinecho sequence. The following parameters including: repetition time of 3000 ms, echo train length of 8, echo spacing of 11.5 ms, effective echo time of 51.75 ms, 160×128 matrix, field of view of 20×20 mm2 , spatial resolution of 0.125×0.156 mm2 /pixel, bandwidth of 50000 Hz, 2×2 oversampling, 2 averages, 30 slices, slice thickness 0.5 mm, and a total scan time of 25 min 36 sec were used. These images were segmented to quantify tumor volume using ITK-SNAP [Yushkevich, P. A., Piven, J., Hazlett, H. C., Smith, R. G., Ho, S., Gee, J. C. and Gerig, G. Neuroimage 2006, 31, 1116-1128.] For assessment of blood-brain-barrier integrity, T1- weighted images were acquired with a gradient-echo sequence using the following parameters: repetition time of 200 ms, echo time of 2.7 ms, 128×128 matrix, field of view of 20×20 mm2, spatial resolution of 0.156×0.156 mm2/pixel, 2×1 oversampling, flip angle of 90°, 8 averages, bandwidth of 50505.1 Hz, echo position at 40%, 30 slices, slice thickness 0.5 mm, and a total scan time of 3 min 25 sec.

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Dissolved in 0.5% sodium carboxymethyl cellulose, and diluted in saline; 40 mg/kg; Oral administration

Orthotopic Ptch+/-p53-/- medulloblastoma allograft model in athymic nude mice

Absorption, Distribution and Excretion
Sonidegib is rapidly absorbed in the fasted state with peak concentrations occurring 2-4 hours after administration. (2) However, the total absorption of Sonidegib is low (roughly 6-7%). (1)
Around 70% of Sonidegib is eliminated in the feces, while 30% is eliminated in the urine. (2)
Estimated volume of distribution = 9166 L (2)

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Metabolism / Metabolites
Sonidegib is primarily metabolized via oxidation and amide hydrolysis. (1) The enzyme responsible for the majority of metabolism is the cytochrome P450 (CYP) 3A4 enzyme. (2)

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Biological Half-Life
Half-life ~ 28 days (2)

Hepatotoxicity
Most clinical trials of sonidegib included few patients and rates of liver tests abnormalities were often not reported. In isolated trials, serum ALT elevations were reported in 15% to 27% of patients and to rise above 5 times the upper limit of normal (ULN) in 1% to 6%. Rates of serum enzyme elevations were greater with higher doses, and all were apparently transient and resolved either spontaneously or with dose reductions or discontinuation. In these trials, there were no cases of clinically apparent liver injury, hepatitis with jaundice or death from liver failure. The product label for sonidegib mentions serum enzyme elevations as a possible adverse event, but does not mention liver injury with jaundice or hepatic failure. Since its approval and more widespread use, there have been no published cases of hepatotoxicity attributed to sonidegib, but it is an uncommonly used antineoplastic agent. Serum enzyme elevations were also rare with the initial hedgehog inhibitor, vismodegib, which has been implicated in causing at least one case of acute, self-limited cholestatic hepatitis (Case 1 in Vismodegib).
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of sonidegib during breastfeeding. Because sonidegib is 97% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 28 days and it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during sonidegib therapy.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Sonidegib is over 97% bound to plasma proteins, and binding is independent of concentration. (2)

[1]. Discovery of NVP-LDE225, a Potent and Selective Smoothened Antagonist. ACS Med Chem Lett. 2010 Mar 16;1(3):130-4.

[2]. Deregulated hedgehog pathway signaling is inhibited by the smoothened antagonist LDE225 (Sonidegib) in chronic phase chronic myeloid leukaemia. Sci Rep. 2016 May 9;6:25476.

Sonidegib phosphate is a phosphate salt obtained by reaction sonidegib with two equivalent of phosphoric acid. Used for treatment of locally advanced basal cell carcinoma. It has a role as an antineoplastic agent, a SMO receptor antagonist and a Hedgehog signaling pathway inhibitor. It contains a sonidegib.
See also: Sonidegib (has active moiety).

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Drug Indication
Odomzo is indicated for the treatment of adult patients with locally advanced basal cell carcinoma (BCC) who are not amenable to curative surgery or radiation therapy.

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Sonidegib is a member of the classo of biphenyls that is the amide obtained by formal condensation of the carboxy group of 2-methyl-4'-(trifluoromethoxy)[1,1'-biphenyl]-3-carboxylic acid with the amino group of 6-(2,6-dimethylmorpholin-4-yl)pyridin-3-amine. Used (as its phosphate salt) for treatment of locally advanced basal cell carcinoma. It has a role as an antineoplastic agent, a SMO receptor antagonist and a Hedgehog signaling pathway inhibitor. It is a member of morpholines, an aminopyridine, a member of biphenyls, a member of benzamides, an aromatic ether, an organofluorine compound and a tertiary amino compound.
Sonidegib is a Hedgehog signaling pathway inhibitor (via smoothened antagonism) developed as an anticancer agent by Novartis. It was FDA approved in 2015 for the treatment of basal cell carcinoma.
Sonidegib is a Hedgehog Pathway Inhibitor. The mechanism of action of sonidegib is as a Smoothened Receptor Antagonist.
Sonidegib is a small molecule kinase inhibitor that blocks signaling in the hedgehog pathway and is used in the therapy of unresectable or metastatic basal cell carcinoma. Sonidegib therapy is associated with a low rate or transient serum aminotransferase elevations during therapy, but has not been linked to instances of clinically apparent acute liver injury.
Sonidegib is an orally bioavailable small-molecule smoothened (Smo) antagonist with potential antineoplastic activity. Sonidegib selectively binds to the hedgehog (Hh)-ligand cell surface receptor Smo, which may result in the suppression of the Hh signaling pathway and, so, the inhibition of tumor cells in which this pathway is abnormally activated. The Hh signaling pathway plays an important role in cellular growth, differentiation and repair. Inappropriate activation of Hh pathway signaling and uncontrolled cellular proliferation, as is observed in a variety of cancers, may be associated with mutations in the Hh-ligand cell surface receptor Smo.
See also: Sonidegib Phosphate (active moiety of).

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Drug Indication
Sonidegib is approved for use in the US and EU for treatment of adults with locally advanced basal cell carcinoma (BCC) that has recurred post surgery or radiation therapy. It is also approved for adult patients with BCC who are not eligible for surgery or radiation therapy. (2)
FDA Label
Odomzo is indicated for the treatment of adult patients with locally advanced basal cell carcinoma (BCC) who are not amenable to curative surgery or radiation therapy.
Treatment of medulloblastoma

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Mechanism of Action
The hedgehog pathway is involved in many human cancers. Sonidegib effectively inhibits the regulator called smoothened (Smo), preventing the hedgehog pathway from functioning. As a result, tumours that depend on the hedgehog pathway are unable to grow.

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The blockade of aberrant hedgehog (Hh) signaling has shown promise for therapeutic intervention in cancer. A cell-based phenotypic high-throughput screen was performed, and the lead structure (1) was identified as an inhibitor of the Hh pathway via antagonism of the Smoothened receptor (Smo). Structure-activity relationship studies led to the discovery of a potent and specific Smoothened antagonist N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4'-(trifluoromethoxy)biphenyl-3-carboxamide (5m, NVP-LDE225), which is currently in clinical development.[1]

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Targeting the Hedgehog (Hh) pathway represents a potential leukaemia stem cell (LSC)-directed therapy which may compliment tyrosine kinase inhibitors (TKIs) to eradicate LSC in chronic phase (CP) chronic myeloid leukaemia (CML). We set out to elucidate the role of Hh signaling in CP-CML and determine if inhibition of Hh signaling, through inhibition of smoothened (SMO), was an effective strategy to target CP-CML LSC. Assessment of Hh pathway gene and protein expression demonstrated that the Hh pathway is activated in CD34(+) CP-CML stem/progenitor cells. LDE225 (Sonidegib), a small molecule, clinically investigated SMO inhibitor, used alone and in combination with nilotinib, inhibited the Hh pathway in CD34(+) CP-CML cells, reducing the number and self-renewal capacity of CML LSC in vitro. The combination had no effect on normal haemopoietic stem cells. When combined, LDE225 + nilotinib reduced CD34(+) CP-CML cell engraftment in NSG mice and, upon administration to EGFP(+) /SCLtTA/TRE-BCR-ABL mice, the combination enhanced survival with reduced leukaemia development in secondary transplant recipients. In conclusion, the Hh pathway is deregulated in CML stem and progenitor cells. We identify Hh pathway inhibition, in combination with nilotinib, as a potentially effective therapeutic strategy to improve responses in CP-CML by targeting both stem and progenitor cells.[2]

C26H32F3N3O11P2

681.49

681.146

C, 45.82; H, 4.73; F, 8.36; N, 6.17; O, 25.82; P, 9.09

1218778-77-8

Sonidegib;956697-53-3; 1218778-77-8 (phosphate)

45138699

White to off-white solid powder

4.413

7

16

5

45

741

2

C[C@@H]1CN(C[C@@H](O1)C)C2=NC=C(C=C2)NC(=O)C3=CC=CC(=C3C)C4=CC=C(C=C4)OC(F)(F)F.OP(=O)(O)O.OP(=O)(O)O

RWIVSVMMGFFZIJ-VWDRLOGHSA-N

InChI=1S/C26H26F3N3O3.2H3O4P/c1-16-14-32(15-17(2)34-16)24-12-9-20(13-30-24)31-25(33)23-6-4-5-22(18(23)3)19-7-10-21(11-8-19)35-26(27,28)29;2*1-5(2,3)4/h4-13,16-17H,14-15H2,1-3H3,(H,31,33);2*(H3,1,2,3,4)/t16-,17+;;

N-[6-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-3-yl]-2-methyl-3-[4-(trifluoromethoxy)phenyl]benzamide;phosphoric acid

Sonidegib phosphate; Sonidegib diphosphate; LDE 225 phosphate; LDE 225 diphosphate;LDE-225 phosphate; LDE225 phosphate; LDE-225 diphosphate; LDE225 diphosphate; NVP-LDE225 diphosphate; NVP LDE-225 diphosphate; NVP-LDE225 phosphate; NVP LDE-225 phosphate; NVP LDE225 phosphate; Erismodegib phosphate; Erismodegib diphosphate; trade name of Odomzo

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (3.67 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 (3.67 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 (3.67 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: 0.2% Tween80 + and 0.5% methyl cellulose

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Solubility in Formulation 5: 5 mg/mL (7.34 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication (<60°C).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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