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, ruxolitinib undergoes rapid absorption and the peak concentrations are reached within one hour after administration. Over a single-dose range of 5 mg to 200 mg, the mean maximal plasma concentration (Cmax) increases proportionally. Cmax ranged from 205 nM to 7100 nM and AUC ranged from 862 nM x hr to 30700 nM x hr. Tmax ranges from one to two hours following oral administration. Oral bioavailability is at least 95%.
Following oral administration of a single radiolabeled dose of ruxolitinib, the drug was mainly eliminated through metabolism. About 74% of the total dose was excreted in urine and 22% was excreted in feces, mostly in the form of hydroxyl and oxo metabolites of ruxolitinib. The unchanged parent drug accounted for less than 1% of the excreted total radioactivity.
The mean volume of distribution (%coefficient of variation) at steady-state is 72 L (29%) in patients with myelofibrosis and 75 L (23%) in patients with polycythemia vera. It is not known whether ruxolitinib crosses the blood-brain barrier.
Ruxolitinib clearance (% coefficient of variation) is 17.7 L/h in women and 22.1 L/h in men with myelofibrosis. Drug clearance was 12.7 L/h (42%) in patients with polycythemia vera and 11.9 L/h (43%) in patients with acute graft-versus-host disease.
Following oral administration, absorption of ruxolitinib is approximately 95%, and mean systemic bioavailability is estimated to be about 80%. Following oral administration of ruxolitinib, peak plasma concentrations are achieved within 1-2 hours. ... Following administration of a single oral dose of radiolabeled ruxolitinib in healthy individuals, elimination was predominantly through metabolism with 74 and 22% of radioactivity excreted in urine and feces, respectively. Unchanged drug accounted for less than 1% of the excreted total radioactivity.

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Metabolism / Metabolites
More than 99% of orally-administered ruxolitinib undergoes metabolism mediated by CYP3A4 and, to a lesser extent, CYP2C9. The major circulating metabolites in human plasma were M18 formed by 2-hydroxylation, and M16 and M27 (stereoisomers) formed by 3-hydroxylation. Other identified metabolites include M9 and M49, which are formed by hydroxylation and ketone formation. Not all metabolite structures are fully characterized and it is speculated that many metabolites exist in stereoisomers. Metabolites of ruxolitinib retain inhibitory activity against JAK1 and JAk2 to a lesser degree than the parent drug.
Cytochrome P-450 (CYP) isoenzyme 3A4 is the major enzyme responsible for metabolism of ruxolitinib. Two major active metabolites were identified in the plasma of healthy individuals; all active metabolites contribute 18% of the overall pharmacodynamic activity of ruxolitinib.
Ruxolitinib is metabolized mainly by cytochrome P-450 (CYP) isoenzyme 3A4.

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Biological Half-Life
The mean elimination half-life of ruxolitinib is approximately 3 hours and the mean half-life of its metabolites is approximately 5.8 hours.
The mean half-life of ruxolitinib following a single oral dose is approximately 3 hours, and the mean half-life of ruxolitinib and its metabolites is approximately 5.8 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of ruxolitinib during breastfeeding. Because ruxolitinib is 97% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during ruxolitinib therapy and for 2 weeks after the last dose for the oral tablets and for 4 weeks after the last dose for the topical cream.
◉ 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.

[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.

Ruxolitinib phosphate is a phosphate salt obtained by reaction ruxolitinib with one equivalent of phosphoric acid. Used for the treatment of patients with intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis. It has a role as an antineoplastic agent and an EC 2.7.10.2 (non-specific protein-tyrosine kinase) inhibitor. It contains a ruxolitinib.
Ruxolitinib Phosphate is the phosphate salt form of ruxolitinib, an orally bioavailable Janus-associated kinase (JAK) inhibitor with potential antineoplastic and immunomodulating activities. Ruxolitinib specifically binds to and inhibits protein tyrosine kinases JAK 1 and 2, which may lead to a reduction in inflammation and an inhibition of cellular proliferation. The JAK-STAT (signal transducer and activator of transcription) pathway plays a key role in the signaling of many cytokines and growth factors and is involved in cellular proliferation, growth, hematopoiesis, and the immune response; JAK kinases may be upregulated in inflammatory diseases, myeloproliferative disorders, and various malignancies.
See also: Ruxolitinib (has active moiety).

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Drug Indication
Opzelura is indicated for the treatment of non-segmental vitiligo with facial involvement in adults and adolescents from 12 years of age.
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 polycythaemia vera myelofibrosis or post essential thrombocythaemia myelofibrosis. Polycythaemia vera (PV)Jakavi is indicated for the treatment of adult patients with polycythaemia vera who are resistant to or intolerant of hydroxyurea. Graft versus host disease (GvHD)Jakavi is indicated for the treatment of patients aged 12 years and older with acute graft versus host disease or chronic graft versus host disease who have 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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Constitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice, establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation, half of those with essential thrombocythemia or primary myelofibrosis do not, suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg, interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly, we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424, the first potent, selective, oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC(50)] = 281nM), and proliferation of JAK2V617F(+) Ba/F3 cells (IC(50) = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F(+) polycythemia vera patients (IC(50) = 67nM) versus healthy donors (IC(50) > 400nM). 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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We have recently identified targetable mutations in CSF3R (GCSFR) in 60% of chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL-negative) chronic myeloid leukemia (aCML) patients. Here we demonstrate that the most prevalent, activating mutation, CSF3R T618I, is sufficient to drive a lethal myeloproliferative disorder in a murine bone marrow transplantation model. Mice transplanted with CSF3R T618I-expressing hematopoietic cells developed a myeloproliferative disorder characterized by overproduction of granulocytes and granulocytic infiltration of the spleen and liver, which was uniformly fatal. Treatment with the JAK1/2 inhibitor ruxolitinib lowered the white blood count and reduced spleen weight. This demonstrates that activating mutations in CSF3R are sufficient to drive a myeloproliferative disorder resembling aCML and CNL that is sensitive to pharmacologic JAK inhibition. This murine model is an excellent tool for the further study of neutrophilic myeloproliferative neoplasms and implicates the clinical use of JAK inhibitors for this disease. [2]

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Leukemia is caused by the accumulation of multiple genomic lesions in hematopoietic precursor cells. However, how these events cooperate during oncogenic transformation remains poorly understood. We studied the cooperation between activated JAK3/STAT5 signaling and HOXA9 overexpression, two events identified as significantly co-occurring in T-cell acute lymphoblastic leukemia. Expression of mutant JAK3 and HOXA9 led to a rapid development of leukemia originating from multipotent or lymphoid-committed progenitors, with a significant decrease in disease latency compared with JAK3 or HOXA9 alone. Integrated RNA sequencing, chromatin immunoprecipitation sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that STAT5 and HOXA9 have co-occupancy across the genome, resulting in enhanced STAT5 transcriptional activity and ectopic activation of FOS/JUN (AP1). Our data suggest that oncogenic transcription factors such as HOXA9 provide a fertile ground for specific signaling pathways to thrive, explaining why JAK/STAT pathway mutations accumulate in HOXA9-expressing cells.Significance: The mechanism of oncogene cooperation in cancer development remains poorly characterized. In this study, we model the cooperation between activated JAK/STAT signaling and ectopic HOXA9 expression during T-cell leukemia development. We identify a direct cooperation between STAT5 and HOXA9 at the transcriptional level and identify PIM1 kinase as a possible drug target in mutant JAK/STAT/HOXA9-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.)