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).

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).

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.

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.

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.

Pharmacokinetics[1]
On the basis of JAK1 potency (biochemical and HWB), JAK1/JAK2 selectivity, and acceptable in vitro metabolic stability, second generation sulfonamides 35 and 36 (Table 5) were selected for pharmacokinetic profiling along with sulfone 19 (Table 2) and sulfonamide 25 (Abrocitinib or PF04965842) (Table 3). Pharmacokinetic parameters were determined in rats at 1 mg/kg iv or 3 mg/kg po (Table 6). Clearances of 19 and 25 (Abrocitinib or PF04965842) were low relative to total liver blood flow, whereas 35 and 36 showed higher clearance values. Oral bioavailability was better for compounds 19 and 25 (Abrocitinib or PF04965842), consistent with lower clearance as well as good in vitro permeability and solubility. In addition, volumes of distribution were moderate (0.74–1.99 L/kg) for this cohort of analogs. Accordingly, single species allometric scaling predicted more favorable human clearance values for compounds 19 and 25 compared with 35 and 36.[1]
Although compounds 19 and 25 (Abrocitinib or PF04965842) showed similar pharmacokinetic profiles and similar potencies in JAK biochemical and cellular assays, a broad functional CEREP screen at 10 μM consisting of a diverse set of GPCR, ion channel, transporter, and enzymes (64 targets in total) for off-target effects indicated that 19 showed activity against the CB-1 receptor in a binding assay (IC50 = 120 nM). This translated into observed motor effects consistent with CB-1 inhibition in a rat toxicokinetic study. Compound 25 (Abrocitinib or PF04965842) showed no measurable activity (>50%) at any targets profiled with the exception of KDR kinase (VEGFR2, IC50 = 1.2 μM) and weak activity on monoamine oxidase A (MAO-A, IC50 = 6 μM). A follow-up study in a functional Caliper whole cell KDR kinase assay showed no effects on KDR kinase activity at concentrations up to 30 μM.[1]
Sulfonamide 25 (Abrocitinib or PF04965842) was also evaluated in a broad kinase panel (Table S3), which indicated that it is a highly selective compound, crossing over most potently onto JAK3 with 61% inhibition at 1 uM. The lack of off-target kinase inhibition is particularly notable, given that the broad kinase panel was run at the Km of ATP to maximize assay sensitivity for the detection of potential off-target activity. Although JAK3 showed up as an off target in the broad kinase panel, 25 had an IC50 of 605 nM when measured at the Km of ATP and >10 μM when measured at 1 mM ATP. In summary, compound 25 (Abrocitinib or PF04965842) is a potent JAK1 inhibitor with 28-fold selectivity over JAK2, >340-fold over JAK3, and 43-fold over TYK2 (Table S1) at 1 mM ATP and has an excellent selectivity profile over the broader kinome. Based on its favorable overall properties and pharmacokinetic profile across multiple species (Tables S4 and S5), 25 progressed into pharmacodynamic and efficacy studies in vivo.[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.

Abrocitinib is a Janus Kinase Inhibitor. The mechanism of action of abrocitinib is as a Janus Kinase Inhibitor, and P-Glycoprotein Inhibitor.
See also: Abrocitinib (annotation moved to).

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.)