JAK2 (IC50 = 2.8 nM); JAK1 (IC50 = 3.3 nM); Tyk2 (IC50 = 19 nM); JAK3 (IC50 = 428 nM)

Ruxolitinib phosphate (INCB018424) inhibits JAK2V617F-mediated signaling and proliferation in a potent and specific manner. Ruxolitinib phosphate has an EC50 value of 186 nM, which means that it inhibits HEL cell growth. Ruxolitinib phosphate dramatically suppresses the proliferation of hematopoietic progenitor cells and raises Ba/F3-EpoR-JAK2V617F cell apoptosis in primary MPN patient samples [1].

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INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC50] = 281nM), and proliferation of JAK2V617F+ Ba/F3 cells (IC50 = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F+ polycythemia vera patients (IC50 = 67nM) versus healthy donors (IC50 > 400nM). [1]

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Furthermore, PIM1 RNA and protein levels were rapidly downregulated in a HOXA9+ patient-derived xenograft (PDX) sample with active JAK/STAT signaling after treatment with the JAK1 inhibitor ruxolitinib (Fig. 6C). This provided further evidence for a functional signaling network of PIM1 regulation downstream of JAK/STAT signaling. Exploiting this observation, we next sought to determine whether dual inhibition of PIM1 and JAK1 would be beneficial in JAK/STAT-mutant T-ALL samples. Here, we observed a synergistic response in JAK3-mutant T-ALL samples when treated with a JAK kinase inhibitor (ruxolitinib) in combination with a PIM1 inhibitor (AZD1208) within ex vivo cell culture [3].

In mice injected with JAK2V617F-expressing cells, rufolitinib phosphate (180 mg/kg, oral) decreases tumor burden without resulting in anemia or lymphopenia [1].

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In a mouse model of JAK2V617F+ MPN, oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs.[1]

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The JAK1/2 inhibitor ruxolitinib decreases WBC count and reduces splenomegaly in CSF3RT618I mice [2]
We previously demonstrated that activating CSF3R mutations lead to preferential downstream signaling via JAK kinases, and a CNL patient carrying a JAK activating CSF3RT618I mutation showed marked clinical improvement after administration of the JAK1/2 inhibitor ruxolitinib.1 To determine whether the granulocytic expansion seen in CSF3RT618I mice is dependent upon the JAK kinase pathway, we tested the effect of ruxolitinib in a second cohort of CSF3RT618I mice. Oral administration of ruxolitinib (90 mg/kg 2×/d) or vehicle was started at day 12 post transplant, at which time mice already exhibited leukocytosis. Ruxolitinib treatment resulted in a prompt reduction in WBC count and a decrease in spleen weight (Figure 2A-C). Consistent with its ability to improve constitutional symptoms such as fatigue and early satiety in myelofibrosis,12,13 ruxolitinib-treated mice had increased body weight compared with vehicle-treated mice (Figure 2D). This demonstrates that the pathologic expansion of granulocytes in the CSF3RT618I mouse model is sensitive to JAK inhibition and warrants further investigation into the therapeutic use of JAK inhibitors in patients with CNL harboring the CSF3RT618I mutation.

Biochemical assays[1] The kinase domains of human JAK1 (837-1142), JAK2 (828-1132), JAK3 (781-1124), and Tyk2 (873-1187) were cloned by PCR with N-terminal epitope tags. Recombinant proteins were expressed using Sf21 cells and baculovirus vectors and purified with affinity chromatography. JAK kinase assays used a homogeneous time-resolved fluorescence assay with the peptide substrate (-EQEDEPEGDYFEWLE). Each enzyme reaction was carried out with test compound or control, JAK enzyme, 500nM peptide, adenosine triphosphate (ATP; 1mM), and 2.0% dimethyl sulfoxide (DMSO) for 1 hour. The 50% inhibitory concentration (IC50) was calculated as the compound concentration required for inhibition of 50% of the fluorescent signal. Biochemical assays for CHK2 and c-MET enzymes were performed using standard conditions (Michaelis constant [Km] ATP) with recombinantly expressed catalytic domains from each protein and synthetic peptide substrates. An additional panel of kinase assays (Abl, Akt1, AurA, AurB, CDC2, CDK2, CDK4, CHK2, c-kit, c-Met, EGFR, EphB4, ERK1, ERK2, FLT-1, HER2, IGF1R, IKKα, IKKβ, JAK2, JAK3, JNK1, Lck, MEK1, p38α, p70S6K, PKA, PKCα, Src, and ZAP70) was performed using standard conditions (CEREP; www.cerep.com) using 200nM INCB018424. Significant inhibition was defined as more than or equal to 30% (average of duplicate assays) compared with control values.

Cell proliferation assay[1]
Cells were seeded at 2000/well of white bottom 96-well plates, treated with compounds from DMSO stocks (0.2% final DMSO concentration), and incubated for 48 hours at 37°C with 5% CO2. Viability was measured by cellular ATP determination using the Cell-Titer Glo luciferase reagent or viable cell counting. Values were transformed to percent inhibition relative to vehicle control, and IC50 curves were fitted according to nonlinear regression analysis of the data using PRISM GraphPad.

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Apoptosis[1]
Annexin V staining. Cells were treated for 20 to 24 hours and stained with annexin V and propidium iodide for analysis of early apoptotic and dead cells, respectively. Analysis was performed using a FACSCaliber flow cytometer. Mitochondrial membrane potential. Cells were treated for 24 hours and then incubated with 2μM of the dye JC-1. Analysis was performed by flow cytometry using 488-nm excitation and 530-nm and 585-nm emission filters. JC-1 exhibits potential-dependent accumulation in the mitochondria where its emission is in the red spectrum (590nM). A fluorescence shift from red (590nM) to green (530nM) indicates redistribution of the dye to the cytoplasm resulting from loss of mitochondrial membrane potential, an early marker for apoptosis.

In vivo treatment with INCB018424 in a myeloproliferative neoplasm mouse model [1]
Mice were fed standard rodent chow and provided with water ad libitum. Ba/F3-JAK2V617F cells (105 per mouse) were inoculated intravenously into 6- to 8-week-old female BALB/c mice. Survival was monitored daily, and moribund mice were humanely killed and considered deceased at time of death. Treatment with vehicle (5% dimethyl acetamide, 0.5% methocellulose) or INCB018424 began within 24 hours of cell inoculation, twice daily by oral gavage. Hematologic parameters were measured using a Bayer Advia120 analyzed, and statistical significance was determined using Dunnett testing.

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Histology and morphometric analysis [1]
Tissue samples of spleen were fixed in 10% neutral buffered formalin and processed through graded alcohols and a clearing agent, infiltrated and embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. To quantify the effects of INCB018424 on white pulp, a simple morphometric method using point-counts was devised. Images of spleen at 2 times magnification were overlaid with a standardized grid. Point counts were by made tabulating the grid intersects that overlaid total spleen and white pulp. Point counts for white pulps were summed for each group, that is, naive (N = 3), vehicle-treated (N = 6), and INCB018424-treated (N = 6), and mean values calculated. An approximate mean mass of white pulp was calculated using the mean weights of spleens for each group and the relative mean point counts for total spleen and white pulp. Photographic images were acquired with a Nikon Eclipse E800 microscope equipped with a Nikon 20×/0.75 Plan Apo objective, and a Nikon DXM1200 digital camera. Images were processed on a Dell computer with Nikon ACT/1 software and Adobe Photoshop 7.0.

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In Vivo and Ex Vivo Treatment of PDX Samples [3]
PDX samples were transplanted in 8-week-old NSG mice through tail-vein injection. Human leukemic cell expansion was monitored through human CD45 staining on blood samples. Single cells were isolated from the spleen, which at time of sacrifice contained >85% human CD45+ cells. Spleen cells were seeded in 96-well plate (5 × 105 cells/well) and incubated with vehicle (DMSO) or inhibitor. Cell viability was assessed at 48 hours using ATP-Lite. CompuSyn was used to calculate the combination index. For in vivo treatment studies, the XC65 was transduced overnight with lentivirus pCH-SFFV-eGFP-P2A-fLuc. The GFP-positive cells were then sorted using the S3 Sorter and retransplanted back into recipient NSG mice. Upon confirmation that XC65 was greater than >95% GFP positive, leukemic cells were isolated from the spleen and reinjected into a larger cohort of NSG mice for acute 7-day in vivo treatment. Ruxolitinib was dissolved in 0.5% methylcellulose, AZD1208 was dissolved in 50% PEG400/0.5% methylcellulose, and both were administered by oral gavage.

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Dissolved in 5% dimethyl acetamide, 0.5% methocellulose; 180 mg/kg/day; Oral gavage

JAK2V617F-driven mouse model

Absorption, Distribution and Excretion
Following oral administration, ruxotinib is rapidly absorbed, reaching peak plasma concentrations within 1 hour of administration. The mean maximum plasma concentration (Cmax) increases proportionally with a single dose ranging from 5 mg to 200 mg. Cmax ranges from 205 nM to 7100 nM, and AUC ranges from 862 nM·h to 30700 nM·h. The time to peak concentration (Tmax) after oral administration is 1 to 2 hours. Oral bioavailability is at least 95%. Following a single oral dose of radiolabeled ruxotinib, the drug is primarily eliminated through metabolism. Approximately 74% of the total dose is excreted in the urine and 22% in the feces, mainly as ruxotinib's hydroxyl and oxytocin metabolites. Unmetabolized parent drug accounts for less than 1% of the total radioactive excretion.
In patients with myelofibrosis, the mean volume of distribution at steady state (coefficient of variation %) was 72 L (29%), and in patients with polycythemia vera, it was 75 L (23%). It is unclear whether ruxotinib crosses the blood-brain barrier.
In female patients with myelofibrosis, the clearance of ruxotinib (coefficient of variation %) was 17.7 L/h, and in male patients, it was 22.1 L/h. In patients with polycythemia vera, the clearance was 12.7 L/h (42%), and in patients with acute graft-versus-host disease, it was 11.9 L/h (43%).
After oral administration, ruxotinib is absorbed at approximately 95%, and the mean systemic bioavailability is estimated to be approximately 80%. Peak plasma concentrations are reached within 1–2 hours after oral administration of ruxotinib. …In healthy subjects, after a single oral administration of radiolabeled ruxotinib, the drug is primarily eliminated through metabolism, with 74% and 22% of the radioactive material excreted in urine and feces, respectively. Less than 1% of the total excreted radioactivity is unmetabolized.
Metabolisms/Metabolites
Over 99% of orally administered ruxotinib is metabolized via CYP3A4, with less metabolism via CYP2C9. The major circulating metabolites in human plasma are M18, generated by 2-hydroxylation, and M16 and M27 (stereoisomers), generated by 3-hydroxylation. Other identified metabolites include M9 and M49, formed by hydroxylation and ketone bodies, respectively. The structures of not all metabolites have been fully characterized; it is presumed that many metabolites exist in stereoisomer form. Ruxotinib metabolites exhibit lower inhibitory activity against JAK1 and JAK2 than their parent drug. Cytochrome P-450 (CYP) isoenzyme 3A4 is the major enzyme responsible for ruxotinib metabolism. Two major active metabolites were identified in the plasma of healthy individuals; all active metabolites contribute 18% of the overall pharmacodynamic activity of ruxotinib. Ruxotinib is primarily metabolized by cytochrome P-450 (CYP) isoenzyme 3A4.
Biological Half-Life
The mean elimination half-life of ruxotinib is approximately 3 hours, and the mean half-life of its metabolites is approximately 5.8 hours. Following a single oral dose of ruxotinib, its mean half-life is approximately 3 hours, and the mean half-life of ruxotinib and its metabolites is approximately 5.8 hours.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Currently, there is no information on the clinical use of ruxotinib during lactation. Because ruxotinib binds to plasma proteins at a rate as high as 97%, its concentration in breast milk may be very low. The manufacturer recommends discontinuing breastfeeding during ruxotinib treatment, and for two weeks after the last dose of oral tablets or four weeks after the last dose of topical cream.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.

[1]. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood, 2010, 115(15), 3109-3117.

[2]. The CSF3R T618I mutation causes a lethal neutrophilic neoplasia in mice that is responsive to therapeutic JAK inhibition. Blood. 2013 Nov 21;122(22):3628-31.

[3]. HOXA9 Cooperates with Activated JAK/STAT Signaling to Drive Leukemia Development. Cancer Discov. 2018 May;8(5):616-631.

Ruxotinib phosphate is a phosphate form of ruxotinib prepared by reacting ruxotinib with an equivalent amount of phosphoric acid. It is used to treat intermediate- or high-risk myelofibrosis patients, including primary myelofibrosis, post-polycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis. It is an anti-tumor drug and also an EC 2.7.10.2 (non-specific protein tyrosine kinase) inhibitor. Its main component is ruxotinib. Ruxotinib phosphate is the phosphate form of ruxotinib, a highly bioavailable oral Janus kinase (JAK) inhibitor with potential anti-tumor and immunomodulatory activities. Ruxotinib specifically binds to and inhibits JAK1 and JAK2 protein tyrosine kinases, potentially reducing inflammation and inhibiting cell proliferation. The JAK-STAT (signal transduction and transcription activator) pathway plays a crucial role in the signaling of many cytokines and growth factors and is involved in cell proliferation, growth, hematopoiesis, and immune responses; JAK kinases may be upregulated in inflammatory diseases, myeloproliferative disorders, and various malignancies. See also: Ruxotinib (containing the active ingredient).

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Drug Indications
Opzelura is indicated for the treatment of non-segmental vitiligo affecting the face in adults and adolescents aged 12 years and older.
Myelofibrosis (MF): Jakavi is indicated for the treatment of disease-related splenomegaly or symptoms in adult patients with primary myelofibrosis (also known as chronic idiopathic myelofibrosis), post-polycythemia vera myelofibrosis, or post-essential thrombocythemia vera myelofibrosis. Polycythemia vera (PV): Jakavi is indicated for the treatment of adult patients with polycythemia vera who are resistant to or intolerant of hydroxyurea. Graft-versus-host disease (GvHD): Jakavi is indicated for the treatment of acute or chronic graft-versus-host disease in patients aged 12 years and older who have an inadequate response to corticosteroids or other systemic therapies (see Section 5.1).
Treatment of atopic dermatitis
Treatment of chronic graft-versus-host disease (cGvHD)
Treatment of acute graft-versus-host disease (aGvHD)
Treatment of vitiligo.

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The JAK2V617F mutation leads to constitutive activation of JAK2 in hematopoietic cells, reproducing the myeloproliferative neoplasm (MPN) phenotype in mice, suggesting that JAK2 inhibition is a potential therapeutic strategy. Although most patients with polycythemia vera carry the JAK2V617F mutation, about half of patients with essential thrombocythemia or essential myelofibrosis do not carry this mutation, suggesting that there may be other mechanisms of constitutive activation of the JAK-STAT signaling pathway in MPN. Most patients with essential myelofibrosis have elevated levels of JAK-dependent pro-inflammatory cytokines (such as interleukin-6), consistent with our observed JAK1 overactivation. Therefore, we evaluated the efficacy of selective JAK1/2 inhibitors in MPN-related experimental models and reported the effect of INCB018424, the first potent and selective orally administered JAK1/JAK2 inhibitor to enter clinical trials. INCB018424 inhibited the interleukin-6 signaling pathway (IC50 = 281 nM) and the proliferation of JAK2V617F(+) Ba/F3 cells (IC50 = 127 nM). In primary culture, INCB018424 preferentially inhibited colony formation of erythroid progenitor cells from JAK2V617F(+) polycythemia vera patients (IC50 = 67 nM), while the IC50 in healthy donors was > 400 nM. In the JAK2V617F(+) MPN mouse model, oral administration of INCB018424 significantly reduced splenomegaly and decreased levels of circulating inflammatory cytokines, preferentially clearing tumor cells and thus significantly prolonging mouse survival without myelosuppression or immunosuppression. Preliminary clinical results support these preclinical data and confirm INCB018424 as a promising oral treatment for MPN. [1]

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We recently identified a targetable mutation of CSF3R (GCSFR) in 60% of patients with chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL negative) chronic myeloid leukemia (aCML). This paper demonstrates that the most common activating mutation, CSF3R T618I, is sufficient to induce fatal myeloproliferative disease in a mouse bone marrow transplantation model. Mice transplanted with hematopoietic cells expressing CSF3R T618I developed myeloproliferative disease characterized by excessive granulocyte proliferation and granulocyte infiltration in the spleen and liver, ultimately leading to death. Treatment with the JAK1/2 inhibitor ruxotinib reduced white blood cell count and spleen weight. This suggests that activating mutations in CSF3R are sufficient to drive a myeloproliferative disorder similar to aCML and CNL, and that the disease is sensitive to JAK inhibitors. This mouse model is an excellent tool for further research into neutrophilic myeloproliferative neoplasms and suggests that JAK inhibitors may be used to treat this disease. [2]

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Leukemia is caused by the accumulation of multiple genomic damages in hematopoietic progenitor cells. However, how these events synergistically interact in the process of oncogenic transformation remains poorly understood. We investigated the synergistic effect between activated JAK3/STAT5 signaling pathways and HOXA9 overexpression, two events that were found to coexist significantly in T-cell acute lymphoblastic leukemia. Expression of mutant JAK3 and HOXA9 led to rapid progression of leukemia originating from pluripotent or lymphotropic progenitor cells, with a significantly shorter disease latency compared to expression of JAK3 or HOXA9 alone. Integrated RNA sequencing, chromatin immunoprecipitation sequencing, and transposase-accessible chromatin sequencing (ATAC-seq) revealed that STAT5 and HOXA9 co-localize in the genome, leading to enhanced STAT5 transcriptional activity and ectopic activation of FOS/JUN (AP1). Our data suggest that oncogenic transcription factors such as HOXA9 provide favorable conditions for the activation of specific signaling pathways, explaining why JAK/STAT pathway mutations accumulate in cells expressing HOXA9. Significance: The mechanism of synergistic effects of oncogenes in cancer development remains unclear. This study constructed a model of the synergistic effect of JAK/STAT signaling pathway activation and ectopic expression of HOXA9 during the development of T-cell leukemia. We found that STAT5 and HOXA9 have a direct synergistic effect at the transcriptional level and identified PIM1 kinase as a potential drug target in JAK/STAT/HOXA9 mutation-positive leukemia cases. [3]

C₁₇H₂₁N₆O₄P

404.36

404.136

1092939-17-7

Ruxolitinib;941678-49-5;Ruxolitinib (S enantiomer);941685-37-6;Ruxolitinib sulfate;1092939-16-6

25127112

Typically exists as white to gray solids at room temperature

2.537

4

8

4

28

503

1

N#CC[C@H](C1CCCC1)N2N=CC(C3=C4C=CNC4=NC=N3)=C2.O=P(O)(O)O

JFMWPOCYMYGEDM-XFULWGLBSA-N

InChI=1S/C17H18N6.H3O4P/c18-7-5-15(12-3-1-2-4-12)23-10-13(9-22-23)16-14-6-8-19-17(14)21-11-20-16;1-5(2,3)4/h6,8-12,15H,1-5H2,(H,19,20,21);(H3,1,2,3,4)/t15-;/m1./s1

(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propanenitrile;phosphoric acid

INCB-018424 phosphate, INCB 018424, INCB018424; INC424, INC424, INC-424; INCB18424, INCB 18424, 1092939-17-7; (R)-3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate; OPZELURA; Ruxolitinib (phosphate); ruxolitinib monophosphate; INCB-18424; Jakafi and Jakavi (trade name)

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: ≥ 2.75 mg/mL (6.80 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (6.80 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.08 mg/mL (5.14 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 20.8 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 4: ≥ 2.08 mg/mL (5.14 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 20.8 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 5: ≥ 2.08 mg/mL (5.14 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 2% DMSO+30% PEG 300+ddH2O:5mg/mL

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Solubility in Formulation 7: 10 mg/mL (24.73 mM) in 0.5% MC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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