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

Ruxolitinib produces a dose-dependent increase in apoptosis, a doubling of cells with depolarized mitochondria in Ba/F3 cells, and a powerful and specific inhibition of JAK2V617F-mediated signaling and proliferation. Ruxolitinib reduced the proliferation of erythroid progenitor cells from normal donors and polycythemia vera patients with IC50 values of 407 nM and 223 nM, respectively, and demonstrated substantial anti-erythroid colony formation with an IC50 of 67 nM [1].

In JAK2V617F-driven mouse models, rufolitinib (180 mg/kg, PO, twice daily) did not cause myelosuppression or immunosuppression, but it did significantly prolong survival by reducing splenomegaly and circulating levels of inflammatory cytokines and preferentially eliminating tumor cells. At day 22, survival rates were above 90% [1]. In the myelofibrosis double-blind trial, 41.9% of patients in the ruxolitinib group and 0.7% of patients in the placebo group achieved the primary endpoint. Ruxolitinib improves overall symptom scores by 50% or more while maintaining spleen volume reduction [2].

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.

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Colony-forming assay[1]
Mononuclear cells were isolated from peripheral blood from patients with PV or normal control persons by centrifugation through Ficoll. A total of 2 × 105 cells from control or patients with PV were plated onto methocult H88434 supplemented with recombinant cytokines (50 ng/mL stem cell factor, 10 ng/mL granulocyte-macrophage colony-stimulating factor, 10 ng/mL granulocyte colony-stimulating factor, 10 ng/mL IL-3, and 3 U/mL erythropoietin) and with indicated concentrations of INCB018424 or DMSO vehicle. For evaluation of endogenous erythroid colony growth, 3 to 4 × 105 cells from PV patients were plated onto minimal methocult medium with INCB018424 or vehicle. Each condition was performed in triplicate. Colonies derived from erythroid (burst-forming units [BFU] and colony-forming units [CFU]-E) and myeloid (CFU-granulocyte macrophage) progenitor cells were counted after 14 days.

JAK2V617F-driven mouse model
In vivo treatment with INCB018424 in a myeloproliferative neoplasm mouse model
All of the procedures were conducted in accordance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. 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[1].

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.

Toxicity Summary
IDENTIFICATION AND USE: Ruxolitinib phosphate is used for the treatment of intermediate- or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis. Ruxolitinib is designated an orphan drug by the US Food and Drug Administration (FDA) for use in the treatment of these conditions. HUMAN EXPOSURE AND TOXICITY: Adverse effects reported in more than 10% of patients receiving ruxolitinib include thrombocytopenia, anemia, neutropenia, bruising, dizziness, and headache. Ruxolitinib-treated patients achieved clinically meaningful improvements in myelofibrosis-related symptoms and quality of life, but patients receiving placebo reported worsening of symptoms and other patient-reported outcomes. Ruxolitinib, as compared with placebo, provided significant clinical benefits in patients with myelofibrosis by reducing spleen size, ameliorating debilitating myelofibrosis-related symptoms, and improving overall survival. These benefits came at the cost of more frequent anemia and thrombocytopenia in the early part of the treatment period. In vitro data indicate that neither ruxolitinib nor its M18 metabolite is an inhibitor of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion-transporting polypeptides (OATP) 1B1 and 1B3, organic cation transporters (OCT) 1 and 2, and organic anion transporters (OAT) 1 and 3 at clinically relevant concentrations. Ruxolitinib also is not a substrate for P-gp. It was not clastogenic in in vitro chromosomal aberration assay (cultured human peripheral blood lymphocytes). ANIMAL STUDIES: Ruxolitinib was not mutagenic in a bacterial mutagenicity assay (Ames test) or clastogenic in vivo in a rat bone marrow micronucleus assay. Ruxolitinib was not carcinogenic in the 6-month Tg.rasH2 transgenic mouse model study or in a 2-year carcinogenicity study in the rat. In a pre- and post-natal development study in rats, pregnant animals were dosed with ruxolitinib from implantation through lactation at doses up to 30 mg/kg/day. There were no drug-related adverse findings in pups for fertility indices or for maternal or embryofetal survival, growth and development parameters at the highest dose evaluated (34% the clinical exposure at the maximum recommended dose of 25 mg twice daily). Ruxolitinib was administered orally to pregnant rats or rabbits during the period of organogenesis, at doses of 15, 30 or 60 mg/kg/day in rats and 10, 30 or 60 mg/kg/day in rabbits. There was no evidence of teratogenicity. However, decreases of approximately 9% in fetal weights were noted in rats at the highest and maternally toxic dose of 60 mg/kg/day. This dose results in an exposure (AUC) that is approximately 2 times the clinical exposure at the maximum recommended dose of 25 mg twice daily. In rabbits, lower fetal weights of approximately 8% and increased late resorptions were also noted at the highest and maternally toxic dose of 60 mg/kg/day.

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Hepatotoxicity
In the large clinical trials, serum ALT elevations occurred in 25% to 48% of ruxolitinib treated subjects versus 7% to 9% of placebo recipients. The ALT elevations were generally self-limited, asymptomatic and mild and were above 5 times ULN in only 1.3% of patients. In the prelicensure clinical trials, no cases of clinically apparent liver injury were reported. Among causes of death in one trial of ruxolitinib for myelofibrosis, one was attributed to hepatic failure; this instance, however, was considered unrelated to ruxolitinib. Since its approval and more wide scale use, rare cases of clinically apparent ruxolitinib induced acute liver injury have been reported, but without documentation of clinical features or careful exclusion of other causes. Importantly, there also have been several published reports of reactivation of hepatitis B, in patients with and without HBsAg (but with anti-HBc) in serum. A rise in HBV DNA levels was identified within 1 to 6 months of starting ruxolitinib and was associated with elevations in ALT levels and jaundice in some patients. HBV DNA levels decreased rapidly upon starting anti-HBV therapy with entecavir and all patients recovered. In one instance, HBV DNA levels declined with lowering of the dose of ruxolitinib, but then rose again when the dose was increased.
So far, reports of the use of ruxolitinib for COVID-19 have included only small numbers of patients and have provided little information on hepatic adverse events or risk of reactivation of hepatitis B.
Likelihood score: C (probable cause of reactivation of hepatitis B in susceptible patients).

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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.

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Protein Binding
Ruxolitinib is approximately 97% bound to plasma proteins, mostly to albumin.

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Interactions
Fluconazole: The AUC of ruxolitinib is predicted to increase by approximately 100% to 300% following concomitant administration with the combined CYP3A4 and CYP2C9 inhibitor fluconazole at doses of 100 mg to 400 mg once daily, respectively. Avoid the concomitant use of Jakafi with fluconazole doses of greater than 200 mg daily.
Concomitant administration of ruxolitinib (single 50-mg dose) with rifampin (600 mg once daily for 10 days) decreased ruxolitinib peak plasma concentrations and AUC by 32 and 61%, respectively. No dosage adjustment is recommended when ruxolitinib is administered with a CYP3A4 inducer.
Concomitant administration of ruxolitinib (single 10-mg dose) with erythromycin (500 mg twice daily for 4 days) increased ruxolitinib peak plasma concentrations and AUC by 8 and 27%, respectively. No dosage adjustment is recommended when ruxolitinib is administered with weak or moderate CYP3A4 inhibitors (e.g., erythromycin). In patients receiving a stable dosage of ruxolitinib, clinicians should use caution when initiating treatment with a moderate CYP3A4 inhibitor, especially in patients with low platelet counts.
Concomitant administration of ruxolitinib (single 10-mg dose) with ketoconazole (200 mg twice daily for 4 days) increased peak plasma concentrations and AUC of ruxolitinib by 33 and 91%, respectively. Half-life of ruxolitinib also was prolonged from 3.7 to 6 hours with concomitant use of ketoconazole. Dosage reduction is recommended when ruxolitinib is administered with potent CYP3A4 inhibitors (e.g., ketoconazole).
Concomitant use of ruxolitinib with potent inhibitors of CYP3A4 (e.g., boceprevir, clarithromycin, conivaptan, grapefruit juice, indinavir, itraconazole, ketoconazole, lopinavir/ritonavir, mibefradil [no longer commercially available in the US], nefazodone, nelfinavir, posaconazole, ritonavir, saquinavir, telaprevir, telithromycin, voriconazole) has resulted in increased peak plasma concentrations and area under the serum concentration-time curve (AUC) of ruxolitinib. Dosage reduction of ruxolitinib is recommended when the drug is used concomitantly with potent CYP3A4 inhibitors.

[1]. Quintas-Cardama A, et al. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood, 2010, 115(15), 3109-3117.
[2]. Verstovsek S, et al. A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med, 2012, 366(9), 799-807.
[3]. Tavallai M, et al. Rationally Repurposing Ruxolitinib (Jakafi (®)) as a Solid Tumor Therapeutic.Front Oncol. 2016 Jun 13;6:14

Therapeutic Uses
/CLINICAL TRIALS/ ClinicalTrials.gov is a registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. The Web site is maintained by the National Library of Medicine (NLM) and the National Institutes of Health(NIH). Each ClinicalTrials.gov record presents summary information about a study protocol and includes the following: Disease or condition; Intervention (for example, the medical product, behavior, or procedure being studied); Title, description, and design of the study; Requirements for participation (eligibility criteria); Locations where the study is being conducted; Contact information for the study locations; and Links to relevant information on other health Web sites, such as NLM's MedlinePlus for patient health information and PubMed for citations and abstracts for scholarly articles in the field of medicine. Ruxolitinib is included in the database.
Jakafi is indicated for treatment of patients with polycythemia vera who have had an inadequate response to or are intolerant of hydroxyurea. /Included in US product label/
Jakafi is indicated for treatment of patients with intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis. /Included in US product label/
Ruxolitinib phosphate is used for the treatment of intermediate- or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis. Ruxolitinib is designated an orphan drug by the US Food and Drug Administration (FDA) for use in the treatment of these conditions.
Ruxolitinib, a Janus kinase (JAK) 1 and 2 inhibitor, was shown to have a clinical benefit in patients with polycythemia vera in a phase 2 study. We conducted a phase 3 open-label study to evaluate the efficacy and safety of ruxolitinib versus standard therapy in patients with polycythemia vera who had an inadequate response to or had unacceptable side effects from hydroxyurea. We randomly assigned phlebotomy-dependent patients with splenomegaly, in a 1:1 ratio, to receive ruxolitinib (110 patients) or standard therapy (112 patients). The primary end point was both hematocrit control through week 32 and at least a 35% reduction in spleen volume at week 32, as assessed by means of imaging. The primary end point was achieved in 21% of the patients in the ruxolitinib group versus 1% of those in the standard-therapy group (P<0.001). Hematocrit control was achieved in 60% of patients receiving ruxolitinib and 20% of those receiving standard therapy; 38% and 1% of patients in the two groups, respectively, had at least a 35% reduction in spleen volume. A complete hematologic remission was achieved in 24% of patients in the ruxolitinib group and 9% of those in the standard-therapy group (P=0.003); 49% versus 5% had at least a 50% reduction in the total symptom score at week 32. In the ruxolitinib group, grade 3 or 4 anemia occurred in 2% of patients, and grade 3 or 4 thrombocytopenia occurred in 5%; the corresponding percentages in the standard-therapy group were 0% and 4%. Herpes zoster infection was reported in 6% of patients in the ruxolitinib group and 0% of those in the standard-therapy group (grade 1 or 2 in all cases). Thromboembolic events occurred in one patient receiving ruxolitinib and in six patients receiving standard therapy. In patients who had an inadequate response to or had unacceptable side effects from hydroxyurea, ruxolitinib was superior to standard therapy in controlling the hematocrit, reducing the spleen volume, and improving symptoms associated with polycythemia vera.

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Drug Warnings
Ruxolitinib can cause adverse hematologic reactions, including thrombocytopenia, anemia, and neutropenia. A complete blood cell count (CBC) must be performed before initiating therapy with ruxolitinib.
Patients should be assessed for the risk of developing serious bacterial, mycobacterial, fungal, and viral infections. Active serious infections should have resolved prior to initiating therapy with ruxolitinib. Clinicians should carefully observe patients receiving ruxolitinib for signs and symptoms of infection and should promptly initiate appropriate treatment.
Herpes zoster infection occurred in 1.9% of patients receiving ruxolitinib in a clinical study. Clinicians should inform patients about the early signs and symptoms of herpes zoster and advise patients to seek treatment as soon as possible for this condition.
Following interruption or discontinuance of ruxolitinib therapy, symptoms of myelofibrosis generally return to pretreatment levels within approximately 1 week. Withdrawal manifestations, characterized by acute relapse of disease symptoms, accelerated splenomegaly, worsening of cytopenias, and occasional hemodynamic decompensation (including septic shock-like syndrome with severe hypoxia, hypotension, fever, and confusion), have been reported in some patients following discontinuance of ruxolitinib. Some experts recommend that ruxolitinib dosage should be tapered gradually over a 2-week period under close medical supervision.
For more Drug Warnings (Complete) data for RUXOLITINIB (9 total), please visit the HSDB record page.

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Pharmacodynamics
Ruxolitinib is an antineoplastic agent that inhibits cell proliferation, induces apoptosis of malignant cells, and reduces pro-inflammatory cytokine plasma levels by inhibiting JAK-induced phosphorylation of signal transducer and activator of transcription (STAT). Inhibition of STAT3 phosphorylation, which is used as a marker of JAK activity, by ruxolitinib is achieved at two hours after dosing which returned to near baseline by 10 hours in patients with myelofibrosis and polycythemia vera. In clinical trials, ruxolitinib reduced splenomegaly and improved symptoms of myelofibrosis. In a mouse model of myeloproliferative neoplasms, administration of ruxolitinib was associated with prolonged survival. Ruxolitinib inhibits both mutant and wild-type JAK2; however, JAK2V617F mutation, which is often seen in approximately 50% of patients with myelofibrosis, was shown to reduce ruxolitinib sensitivity, which may also be associated with possible resistance to JAK inhibitor treatment.

C17H18N6

306.3650

306.159

C, 66.65; H, 5.92; N, 27.43

941678-49-5

Ruxolitinib (S enantiomer);941685-37-6;Ruxolitinib phosphate;1092939-17-7;(Rac)-Ruxolitinib-d9;2469553-67-9;Deuruxolitinib-d8;1513883-39-0;Ruxolitinib sulfate;1092939-16-6

25126798

Colorless oil

1.4±0.1 g/cm3

592.6±50.0 °C at 760 mmHg

312.2±30.1 °C

0.0±1.7 mmHg at 25°C

1.747

1.69

1

4

4

23

453

1

[C@@H](C1CCCC1)(N1N=CC(C2N=CN=C3NC=CC=23)=C1)CC#N

HFNKQEVNSGCOJV-OAHLLOKOSA-N

InChI=1S/C17H18N6/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/h6,8-12,15H,1-5H2,(H,19,20,21)/t15-/m1/s1

(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile

INCB-018424, INCB 018424, INCB 18424, INCB-18424; INCB018424; INC424, INC424, INC-424; INCB18424, 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

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 61 mg/mL (199.1 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility in Formulation 1: 5 mg/mL (16.32 mM) in 5% DMAC in 0.5% methylcellulose aqueous solution (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.79 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 3: ≥ 2.08 mg/mL (6.79 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 4: ≥ 2.08 mg/mL (6.79 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 corn oil and mix evenly.

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

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Solubility in Formulation 6: 5 mg/mL (16.32 mM) in 0.5% Methylcellulose/saline water (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
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.)