JAK1 (IC50 = 29 nM); JAK2 (IC50 = 803 nM); Tyk2 (IC50 = 1253 nM)
Abrocitinib (compound 25) inhibits, with IC50 values of 189, 163 nM, and 7.178 μM, respectively, the phosphorylation of STAT3 in response to IFNα, the phosphorylation of STAT1 in human whole blood (HWB), and pSTAT5 integrated into CD34+ of HWB (JAK2).

Abrocitinib (compound 25) inhibits, with IC50 values of 189, 163 nM, and 7.178 μM, respectively, the phosphorylation of STAT3 in response to IFNα, the phosphorylation of STAT1 in human whole blood (HWB), and pSTAT5 integrated into CD34+ of HWB (JAK2).

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In biochemical assays at physiological ATP concentration (1 mM), Abrocitinib inhibited JAK1 with an IC₅₀ of 29 nM, demonstrating high selectivity over JAK2 (28-fold), JAK3 (>340-fold), and TYK2 (43-fold).
In human whole blood (HWB) assays, it inhibited IFNα-induced pSTAT3 (JAK1/TYK2 pathway) with an IC₅₀ of 189 nM, and showed significantly weaker inhibition of EPO-induced pSTAT5 (JAK2/JAK2 pathway) with an IC₅₀ of 7.178 µM, confirming cellular selectivity.
It also potently inhibited cytokine-induced STAT phosphorylation for pathways involving JAK1 heterodimers (IFNγ, IL-6, IL-21) in HWB, with IC₅₀ values in the range of 0.163–0.511 µM, while sparing JAK2/TYK2 (IL-12, IL-23) and JAK2/JAK2 (EPO) pathways (IC₅₀ > 13 µM).
The compound exhibited low human liver microsome (HLM) clearance (<9 µL/min/mg) and acceptable permeability (RRCK Papp AB = 16 x 10⁻⁶ cm/s). [1]

In a rat adjuvant-induced arthritis model, brachicitinib (Compound 25 (Abrocitinib or PF04965842); 5, 15, 50 mg/kg, orally, once daily for 7 days) significantly decreased paw swelling [1].
The effect of JAK1 inhibition by 25 (Abrocitinib or PF04965842) in vivo was evaluated using a therapeutic dosing paradigm in a rat adjuvant-induced arthritis (AIA) disease model. (31a) Female Lewis rats immunized with complete Freund’s adjuvant were dosed orally with 25 (Abrocitinib or PF04965842) or vehicle for seven consecutive days after disease onset as measured by hind paw volume using plethysmography. Paw volumes and weights were followed throughout the study. At the end of 7 days of dosing, plasma concentrations of 25 (Abrocitinib or PF04965842) were assessed. Two studies were completed. In the initial study, immunized rats were dosed QD with either 5, 15, or 50 mg/kg of 25 (Abrocitinib or PF04965842) or vehicle (PO) (Figure S3). Because a significant reduction in paw swelling was observed for all doses, a second study was conducted focused on lower doses (0.5, 1, 5, or 15 mg/kg 25 (Abrocitinib or PF04965842) or vehicle control). A significant reduction of hind paw swelling was observed down to 1 mg/kg (Figure 8, days 6 and 7). Collective pharmacodynamics modeling of both studies using an Emax model fit indicated an unbound Cave50 of approximately 1.3 μM (r2 ∼ 0.91).

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In a therapeutic rat adjuvant-induced arthritis (rAIA) model, oral administration of Abrocitinib (0.5, 1, 5, 15, or 50 mg/kg QD for 7 days after disease onset) significantly reduced hind paw swelling in a dose-dependent manner. A significant reduction was observed down to 1 mg/kg.
Pharmacodynamic modeling indicated an unbound plasma Cave₅₀ of approximately 1.3 µM for efficacy.
In a rat target modulation study, single oral doses (5, 15, 50 mg/kg) inhibited ex vivo cytokine (IL-6, IFNγ, IL-21)-induced STAT phosphorylation in blood in a plasma concentration-dependent manner, with calculated unbound plasma IC₅₀ values of 176 nM (IL-6 pSTAT1), 191 nM (IFNγ pSTAT1), and 938 nM (IL-21 pSTAT3).
At the efficacious dose of 1 mg/kg, inhibition of JAK1-dependent cytokines (IL-21, IFNα, IFNγ) was >60%, while JAK2/JAK2-dependent GM-CSF signaling was largely spared (<5% inhibition at lower doses, ~30% at 15 mg/kg). [1]

Caliper JAK Enzyme End Point IC50 Assays at 1 mM ATP[1]
Test compounds were solubilized in DMSO to a stock concentration of 30 mM. Compounds were diluted in DMSO to create an 11-point half log dilution series with a top concentration of 600 μM. The test compound plate also contained positive control wells with a known inhibitor to define 100% inhibition and negative control wells with DMSO to define no inhibition. The compound plates were diluted 1:60 in the assay, resulting in a final assay compound concentration range of 10 μM to 100 pM and a 1.7% DMSO concentration.
The human JAK activity was determined by using a microfluidic assay to monitor phosphorylation of a synthetic peptide by the recombinant human kinase domain of each of the four members of the JAK family: JAK1, JAK2, JAK3, and TYK2. Each assay condition was optimized for enzyme concentration and room temperature incubation time to obtain a conversion rate of 20%–30% phosphorylated peptide product. The 250 nL samples of test compounds and controls solubilized in 100% DMSO were added to a 384-well polypropylene plate using a noncontact acoustic dispenser. Kinase assays were carried out at room temperature in a 15 μL reaction buffer containing 20 mM HEPES, pH 7.4, 1 mM ATP, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, and 1 mM DTT. Reaction mixtures contained 1 μM of a fluorescently labeled synthetic peptide, a concentration less than the apparent Km. The JAK1 and TYK2 assays contained 1 μM of the peptide 5FAM-KKSRGDYMTMQID, and the JAK2 and JAK3 assays contained 1 μM of the peptide FITC-KGGEEEEYFELVKK. The assays were initiated by the addition of enzyme. The assays were stopped with 15 μL of a buffer containing 180 mM HEPES, pH 7.4, 20 mM EDTA, 0.2% Coating Reagent, resulting in a final concentration of 10 mM EDTA, 0.1% Coating Reagent, and 100 mM HEPES, pH 7.4. Utilizing the LabChip 3000 mobility shift technology, each assay reaction was sampled to determine the level of phosphorylation. This technology is separation-based, allowing direct detection of fluorescently labeled substrates and products. Separations are controlled by a combination of vacuum pressure and electric field strength optimized for each peptide substrate.

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JAK kinase activity was determined using a microfluidic mobility shift assay (Caliper). Recombinant human kinase domains (JAK1, JAK2, JAK3, TYK2) were incubated with test compounds (11-point half-log dilution in DMSO, final DMSO 1.7%) in reaction buffer (20 mM HEPES pH 7.4, 1 mM ATP, 10 mM MgCl₂, 0.01% BSA, 0.0005% Tween 20, 1 mM DTT) containing 1 µM of a fluorescently labeled peptide substrate (5FAM-KKSRGDYMTMQIG for JAK1/TYK2; FITC-KGGEEEEYFELVKK for JAK2/JAK3) at room temperature.
Reactions were stopped with a buffer containing 180 mM HEPES pH 7.4, 20 mM EDTA, 0.2% coating reagent. Phosphorylation levels were quantified using a LabChip 3000 system by measuring the separation of phosphorylated and non-phosphorylated peptides.
IC₅₀ values were determined from normalized dose-response curves. Assays were optimized for each kinase to achieve 20-30% substrate conversion. [1]

Human Whole Blood (HWB) Assays[1]
Test articles were prepared as 30 mM stocks in 100% DMSO and then diluted to 5 mM. A 10-point 2.5 dilution series was created in DMSO with a top concentration of 5 mM. Further dilution was done by adding 4 μL of the above test article solutions into 96 μL of PBS with a top concentration of 200 μM. To a 96-well polypropylene plate, 90 μL of HWB was added per well, followed by addition of 5 μL of test article solutions prepared above to give a top concentration of 10 μM. The plate was mixed and incubated for 45 min at 37 °C. To each well was added 5 μL of cytokine (5 uL/well; final, 5000 U/mL IFNα, 100 ng/mL IFNγ, 50 ng/mL IL-6, 30 ng/mL IL-10, 5 ng/mL IL-12, 30 ng/mL IL-15, 50 ng/mL IL-21, 100 ng/mL IL-23, 1200 ng/mL IL-27, or 2 U/mL EPO) for 15 min. Anti-CD3-Pacific Blue and anti-CD14-Pacific Blue antibodies (1:6 dilution in D-PBS, 3 uL/well) were added to samples 15 min before the stimulation of IL-6 and IFNγ, respectively. The reaction was quenched by adding Lyse/Fix Buffer to all wells at 1000 μL/well and incubated for 20 min at 37 °C; after washing with FACS buffer [D-PBS containing 0.1% BSA and 0.1% sodium azide], 400 μL ice cold 90% MeOH/H2O was added to each well, and the sampels were incubated on ice for 30 min. One more wash was done with cold FACS buffer, and all samples were finally resuspended in 250 μL/well of the desired Alexa Fluor 647 conjugated antiphospho-STAT antibodies at 1:125 dilution in FACS buffer. Anti-pSTAT1-AlexaFluor647 was used in assays with stimulation from IFNγ and IL-6. Anti-pSTAT3-AlexaFluor647 was used in assays with stimulation from IFNα, IL-6, IL-10, IL-21, IL-23, and IL-27. Anti-pSTAT4-AlexaFluor647 was used in assays with stimulation from IL-12. Anti-pSTAT5-AlexaFluor647 was used for IL-15 and EPO stimulated cells. After overnight incubation at 4 °C, all the samples were transferred into a 96-well polypropylene U-bottom plate (Falcon cat. no. 353077) and flow cytometric analysis was performed on a FACSCalibur, FACSCanto or LSRFortessa equipped with a HTS plate loader. For IL-10, IL-12, IL-15, IL-21, IL-23, IL-27, and IFNα stimulation, the lymphocyte population was gated for histogram analysis of pSTAT3, 4, or 5 staining. For EPO stimulation, all events (entire populations) were gated for histogram analysis of pSTAT5 staining. For IFNγ stimulation, CD14+ cells were gated for histogram analysis of pSTAT1 staining. For IL-6 stimulation, CD3+ cells were gated for histogram analysis of pSTAT1 and 3 staining. Background fluorescence was defined using unstimulated cells, and a gate was placed at the foot of the peak to include ∼0.5% gated population. The histogram statistical analysis was performed using CellQuest Pro version 5.2.1, FACSDiva version 6.2, or FlowJo version 7.6.1 software. Relative fluorescence unit (RFU), which measures the level of phospho STAT, was calculated by multiplying the percent positive population and its mean fluorescence. Data from 10 compound concentrations (singlicate at each concentration) was normalized as a percentage of control based on the formula.
where A is the RFU from wells containing compound and cytokine, B is the RFU from wells without cytokine (minimum fluorescence) and C is the RFU from wells containing only cytokine (maximum fluorescence). Inhibition curves and IC50 values were determined using the Prism version 5 software.

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Human whole blood (HWB) assays were used to assess functional inhibition of cytokine pathways. Test compounds were serially diluted in DMSO and then PBS. Fresh or cryopreserved HWB (90 µL/well) was pre-incubated with compound for 45 min at 37°C.
Cytokines (IFNα, IFNγ, IL-6, IL-10, IL-12, IL-15, IL-21, IL-23, IL-27, EPO) were added at optimized concentrations (e.g., 5000 U/mL IFNα, 100 ng/mL IFNγ, 2 U/mL EPO) for 15 min. For specific cell populations, surface staining antibodies (e.g., anti-CD3, anti-CD14) were added before stimulation.
Reactions were stopped with Lyse/Fix Buffer, cells were permeabilized with ice-cold 90% methanol, and then stained overnight at 4°C with Alexa Fluor 647-conjugated anti-phospho-STAT antibodies (pSTAT1, pSTAT3, pSTAT4, pSTAT5) specific to the pathway.
Samples were analyzed by flow cytometry. Lymphocytes, monocytes, or CD34⁺ cells were gated as appropriate. The level of STAT phosphorylation was calculated as relative fluorescence units (RFU). Percent inhibition and IC₅₀ values were determined by normalizing to vehicle (0% inhibition) and cytokine-only (100% inhibition) controls. [1]

Animal/Disease Models: Female Lewis rats (8−10 weeks old) [1]
Doses: 5, 15, 50 mg/kg
Route of Administration: Oral daily for 7 days
Experimental Results: Paw swelling was Dramatically diminished at all doses.

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Rat Adjuvant Induced Arthritis Model[1]
The effect of JAK1 inhibition by 25 (Abrocitinib or PF04965842) was evaluated in vivo using a therapeutic dosing paradigm in a rat adjuvant-induced arthritis. The efficacy of this molecule was evaluated in two separate studies using successively lower doses. Arthritis was induced by immunization of female Lewis rats (8–10 weeks old) via intradermal injection at the base of the tail with complete Freund’s adjuvant with three 50 μL injections (15 mg/mL Mycobacterium tuberculosis in incomplete Freund’s adjuvant). Seven days after the initial immunization, the baseline hind paw volume of the immunized rats was measured via plethysmograph. The rats were monitored daily for signs of arthritis including change in body weight and hind paw volume measurement. When individual hind paw volume measurements indicated an increase of 0.2 mL (or greater) in a single hind paw, animals were randomly assigned to a treatment group. Daily treatment with 25 (Abrocitinib or PF04965842) was administered via oral gavage. Treatment groups for experiment 1 were 50, 15, and 5 mg/kg or vehicle (2% Tween 80/0.5% methylcellulose/deionized H2O). Treatment groups for experiment 2 were 15, 5, 1, and 0.5 mg/kg or vehicle (2% Tween 80/0.5% methylcellulose/deionized H2O). Dosing began once individuals were enrolled into respective groups. Treatment continued for 7 days. Animals were euthanized after 7 days of dosing. At the conclusion of the study, whole blood was taken at 15 min postdose (peak concentration in plasma) for analysis of STAT phosphorylation, and plasma was taken at peak (0.25 h) and trough (24 h) time points for exposure PK.

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For the rat adjuvant-induced arthritis (AIA) model, female Lewis rats were immunized by intradermal injection at the base of the tail with complete Freund's adjuvant containing Mycobacterium tuberculosis.
Seven days later, baseline hind paw volume was measured using plethysmography. When hind paw volume increased by ≥0.2 mL, rats were enrolled and randomly assigned to treatment groups.
Abrocitinib or vehicle (2% Tween 80 / 0.5% methylcellulose in deionized water) was administered orally via gavage once daily (QD) for seven consecutive days. Doses tested included 0.5, 1, 5, 15, and 50 mg/kg.
Paw volumes and body weights were monitored throughout the study. Blood samples were collected at peak (0.25 h) and trough (24 h) post-dose on day 7 for pharmacokinetic and pharmacodynamic analysis (STAT phosphorylation). [1]
For rat pharmacokinetic studies, Sprague-Dawley rats received Abrocitinib intravenously (1 mg/kg) or orally (3 mg/kg). Blood samples were collected at various time points, and plasma concentrations were determined to calculate PK parameters. [1]
For rat target modulation studies, naïve Lewis rats received single oral doses of Abrocitinib (5, 15, 50 mg/kg). Serial blood samples were taken at 0.25, 0.5, 2, 4, 8, and 24 hours post-dose for drug concentration and ex vivo cytokine stimulation assays. [1]

Pharmacokinetics [1] Based on JAK1 potency (biochemical and HWB), JAK1/JAK2 selectivity, and acceptable in vitro metabolic stability, we selected second-generation sulfonamides 35 and 36 (Table 5), sulfone compound 19 (Table 2), and sulfonamide 25 (abuxitinib or PF04965842) (Table 3) for pharmacokinetic analysis. Pharmacokinetic parameters were determined in rats by intravenous administration of 1 mg/kg or oral administration of 3 mg/kg (Table 6). The clearance of compounds 19 and 25 (abuxitinib or PF04965842) was low relative to total hepatic blood flow, while the clearance of compounds 35 and 36 was high. The oral bioavailability of compounds 19 and 25 (abuxitinib or PF04965842) was higher, which was consistent with the lower clearance and good in vitro permeability and solubility. In addition, the volume of distribution of this group of analogs was moderate (0.74–1.99 L/kg). Therefore, single-species allotropic scaling predicted human clearance values for compounds 19 and 25 superior to those for compounds 35 and 36. [1] Although compounds 19 and 25 (abusitinib or PF04965842) showed similar pharmacokinetic profiles and potency in JAK biochemical and cellular analyses, a 10 μM broadly functional CEREP screening involving multiple GPCRs, ion channels, transporters, and enzymes (64 targets in total) showed that compound 19 exhibited activity against the CB-1 receptor in the binding assay (IC50 = 120 nM). This translates to the kinetic effects observed in rat toxicokinetics studies, consistent with CB-1 inhibition. Compound 25 (abuxitinib or PF04965842) showed measurable activity (>50%) against all targets except for weak activity against KDR kinase (VEGFR2, IC50 = 1.2 μM) and monoamine oxidase A (MAO-A, IC50 = 6 μM). Subsequent studies in functional Caliper whole-cell KDR kinase activity assays showed that the compound had no effect on KDR kinase activity at concentrations up to 30 μM. [1] Sulfonamide 25 (abuxitinib or PF04965842) was also evaluated in a broad kinase profile (Table S3), and the results showed that it is a highly selective compound with the strongest inhibitory effect on JAK3, with an inhibition rate of 61% at a concentration of 1 μM. Considering that we used ATP Km concentration for a broad kinase profile analysis to maximize the sensitivity of detecting potential off-target activities, the lack of off-target kinase inhibition is particularly noteworthy. Although JAK3 appeared to be an off-target in extensive kinase profile analysis, compound 25 showed an IC50 of 605 nM at an ATP Km concentration and an IC50 greater than 10 μM at a 1 mM ATP concentration. In summary, compound 25 (abuxitinib or PF04965842) is a potent JAK1 inhibitor with 28-fold selectivity for JAK2 compared to JAK1, over 340-fold selectivity for JAK3 compared to JAK3, and 43-fold selectivity for TYK2 compared to TYK2 at a 1 mM ATP concentration (Table S1), and excellent selectivity across a broader kinase group. Based on its favorable overall properties and pharmacokinetic profiles in multiple species (Tables S4 and S5), compound 25 was included in in vivo pharmacodynamic and efficacy studies. [1]

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In Sprague-Dawley rats, after intravenous administration of a 1 mg/kg dose, the clearance (CL) of abuxitinib was 26.6 mL/min/kg, the steady-state volume of distribution (Vdss) was 1.04 L/kg, and the half-life (T₁/₂) was 1.1 hours.
After oral administration of a 3 mg/kg dose, the bioavailability was 95.6%.
In vitro studies showed that the drug had low clearance in human liver microsomes (<9 µL/min/mg), moderate permeability (RRCK Papp AB = 16 x 10⁻⁶ cm/s), and good water solubility (503 µM at pH 7.4).
In vitro results predicted human clearance. Systematic (human liver microsomes, hepatocytes) and single-species allometric scaling from rats and dogs indicated that the drug had low to moderate clearance in humans (2.4–6.5 mL/min/kg) with a steady-state volume of distribution (Vss) of approximately 1 L/kg. The primary clearance mechanism is mediated by CYP450 metabolism, with limited renal and biliary clearance expected. Plasma protein binding remained consistent across species (free fraction approximately 0.4). [1]

In a broad off-target screening at a concentration of 10 µM (containing 64 targets, such as GPCRs, ion channels, transporters, and enzymes), abuxitinib showed no significant activity (inhibition rate >50%), with only weak inhibition against KDR kinase (VEGFR2, IC₅₀ = 1.2 µM) and monoamine oxidase A (MAO-A, IC₅₀ = 6 µM). Subsequent cell-based functional KDR activity assays showed no activity even at concentrations up to 30 µM. Conversely, a related sulfone analog (compound 19) showed activity against the CB-1 receptor (IC₅₀ = 120 nM) and translated into kinetic effects in rat toxicokinetics studies. Abuxitinib did not exhibit this toxicity. [1]

[1]. Identification of N-{cis-3-[Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide (PF-04965842): A Selective JAK1 Clinical Candidate for the Treatment of Autoimmune Diseases. J Med Chem. 2018 Feb 8;61(3):1130-1152.

Abuxitinib is a Janus kinase inhibitor. Its mechanism of action is as a Janus kinase inhibitor and a P-glycoprotein inhibitor.
See also: Abuxitinib (note moved to).

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Abuxitinib (PF-04965842) was discovered by modifying the 3-aminopiperidine linker of tofacitinib, replacing it with a sulfonamide-terminated cis-1,3-cyclobutanediamine, thereby achieving high selectivity for JAK1.
This design, based on X-ray crystallography analysis of the compound's binding to JAK1 and JAK2, revealed its interaction with the hinge region (Glu957 and Leu959 in JAK1), the P-ring, and the contribution of a key residue difference (Glu966 in JAK1 vs. Asp939 in JAK2) to selectivity.
It is being developed as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases. Based on allometric growth ratios and human hepatocyte data, it is estimated that a once-daily human dose of approximately 200 mg would achieve the inhibitory effect of Cave₈₀ on IFNα. Following successful preclinical studies, the drug entered Phase I clinical trials. [1]

C14H21N5O2S

323.41384100914

323.141

C, 51.99; H, 6.55; N, 21.65; O, 9.89; S, 9.91

1622902-68-4

2204280-33-9 (trans-isomer);1622902-68-4 (cis-isomer);

78323835

White to off-white solid powder

1.7

2

6

6

22

474

0

S(CCC)(NC1CC(C1)N(C)C1C2C=CNC=2N=CN=1)(=O)=O

IUEWXNHSKRWHDY-UHFFFAOYSA-N

InChI=1S/C14H21N5O2S/c1-3-6-22(20,21)18-10-7-11(8-10)19(2)14-12-4-5-15-13(12)16-9-17-14/h4-5,9-11,18H,3,6-8H2,1-2H3,(H,15,16,17)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.43 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 2: ≥ 2.08 mg/mL (6.43 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 3: ≥ 2.08 mg/mL (6.43 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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 (Please use freshly prepared in vivo formulations for optimal results.)