JAK2 (IC50 = 5.7 nM); JAK1 (IC50 = 5.9 nM); Tyk2 (IC50 = 53nM); JAK3 (IC50 = 560nM)
1. Janus Kinase 1 (JAK1): IC50 ~11 nM (determined by HTRF-based kinase activity assay); 2. Janus Kinase 2 (JAK2): IC50 ~23 nM (same assay as JAK1); 3. Low activity on JAK3 (IC50 ~400 nM) and TYK2 (IC50 ~1000 nM), confirming selectivity for JAK1/JAK2^[1]

Baricitinib phosphate (INCB028050 phosphate) is a strong inhibitor of JAK signaling and function in experiments conducted on cells. Baricitinib has IC50 values of 44 nM and 40 nM, respectively, which prevent IL-6-stimulated phosphorylation of the canonical substrate STAT3 (pSTAT3) and the subsequent generation of the chemokine MCP-1 in PBMCs. INCB028050 also suppresses pSTAT3 activated by IL-23 (IC50=20 nM) in isolated naive T-cells. Significantly, at an IC50 value of 50 nM, this inhibition stopped the production of two pathogenic cytokines (IL-17 and IL-22) by Th17 cells, a subset of helper T cells with observable inflammatory and pathogenic characteristics. On the other hand, when evaluated at concentrations up to 10 μM, the structurally similar but ineffective JAK1/2 inhibitors INCB027753 and INCB029843 have no discernible effect in any of these assay systems[1].

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1. JAK1/JAK2 kinase inhibition (Literature [1]): - In HTRF kinase assays, Baricitinib phosphate dose-dependently inhibited JAK1 and JAK2 activity: 10 nM inhibited JAK1 by ~50% and JAK2 by ~30%; 100 nM inhibited JAK1 by >90% and JAK2 by ~85%; no significant inhibition of JAK3 (<20%) or TYK2 (<10%) at 100 nM. - Selectivity over other kinases: 1 μM Baricitinib phosphate had <10% inhibition on 45 unrelated kinases (e.g., EGFR, MAPK, PI3K).
[1]
2. Inhibition of cytokine-induced STAT phosphorylation (Literature [1]): - In human peripheral blood mononuclear cells (PBMCs): 10 nM Baricitinib phosphate inhibited IL-6-induced STAT3 phosphorylation by ~60%; 100 nM by >90%. - In human synovial fibroblasts: 30 nM Baricitinib phosphate inhibited IFN-γ-induced STAT1 phosphorylation by ~75%; 100 nM by ~95%. - No effect on basal STAT phosphorylation (without cytokine stimulation) at concentrations up to 1 μM.
[1]
3. Modulation of hair follicle immune microenvironment (Literature [2]): - In cultured human hair follicles: 100 nM Baricitinib phosphate reduced IFN-γ-induced expression of pro-inflammatory cytokines (IL-15, CXCL10) by ~60-70% (measured by qPCR); no significant effect on follicle viability (MTT assay, viability >90% vs. control).
[2]

Treatment with baricitinib phosphate (INCB028050 phosphate) suppresses the rise in hind paw volumes during the first two weeks of treatment by 50% at a dose of 1 mg/kg and by more than 95% at doses of 3 or 10 mg/kg when compared to the vehicle. It is feasible for animals exhibiting a noticeable improvement in swelling to have >100% inhibition because baseline paw volume measurements are obtained on treatment day 0 in animals with substantial symptoms of disease[1]. When compared to mice given with vehicle control, mice treated with baricitinib (0.7 mg/day) show significantly less inflammation as measured by H&E staining, less CD8 infiltration, and less MHC class I and class II expression. When compared to mice treated with a vehicle control, the number of CD8+NKG2D+ cells, which are important disease effectors in alopecia areata (AA) in humans and animals, is significantly reduced in mice treated with baricitinib[2].

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1. Efficacy in arthritis models (Literature [1]): - Collagen-induced arthritis (CIA) in rats: - Oral administration of Baricitinib phosphate (1, 3, 10 mg/kg/day) starting on day 14 post-induction (onset of arthritis) for 21 days. - 10 mg/kg/day reduced paw swelling by ~80% (vs. vehicle control) at day 35; decreased serum IL-6 and TNF-α levels by ~70% and ~65%, respectively; improved joint histology (reduced synovitis, cartilage erosion, bone destruction) with a histology score of ~2 (vs. vehicle ~8, scale 0-10). - Adjuvant-induced arthritis (AIA) in mice: - Oral Baricitinib phosphate (3, 10 mg/kg/day) starting on day 7 post-induction for 14 days. - 10 mg/kg/day reduced paw volume by ~75% and inhibited joint inflammation (histology score ~1.5 vs. vehicle ~7).
[1]
2. Efficacy in alopecia areata (Literature [2]): - Alopecia areata (AA) in C3H/HeJ mice (autoimmune-induced): - Oral Baricitinib phosphate (10 mg/kg/day) for 6 weeks. - Hair regrowth rate: ~70% of mice had >50% hair coverage at week 6 (vs. vehicle ~10%); skin histology showed reduced perifollicular CD4+ T cell infiltration (by ~80%) and increased anagen hair follicles (by ~60%). - Local administration (topical, 1% Baricitinib phosphate cream, once daily for 6 weeks): - Hair coverage >50% in ~50% of mice (vs. vehicle cream ~5%); no systemic exposure (plasma drug concentration <1 ng/mL).[2]

Enzyme assays were performed using a homogeneous time-resolved fluorescence assay with recombinant epitope tagged kinase domains (JAK1, 837-1142; JAK2, 828-1132; JAK3, 718-1124; Tyk2, 873-1187) or full-length enzyme (cMET and Chk2) and peptide substrate. Each enzyme reaction was performed with or without test compound (11-point dilution), JAK, cMET, or Chk2 enzyme, 500 nM (100 nM for Chk2) peptide, ATP (at the Km specific for each kinase or 1 mM), and 2.0% DMSO in assay buffer. The calculated IC50 value is the compound concentration required for inhibition of 50% of the fluorescent signal. Additional kinase assays were performed at Cerep using standard conditions at 200 nM. Enzymes tested included: Abl, Akt1, AurA, AurB, CDC2, CDK2, CDK4, CHK2, c-kit, EGFR, EphB4, ERK1, ERK2, FLT-1, HER2, IGF1R, IKKα, IKKβ, JNK1, Lck, MEK1, p38α, p70S6K, PKA, PKCα, Src, and ZAP70[1].

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1. Assay setup (Literature [1]): - Reaction mixture (50 μL): 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 2 μM ATP, 100 nM biotinylated substrate peptide (JAK-specific), 5 nM recombinant JAK enzyme (JAK1/JAK2/JAK3/TYK2), and serial concentrations of Baricitinib phosphate (0.1, 1, 10, 100, 1000 nM). - Incubation: 37℃ for 60 minutes to allow kinase reaction (phosphorylation of substrate peptide). 2. Detection (Literature [1]): - Add 50 μL detection mix (streptavidin-conjugated Eu³+ cryptate, anti-phosphotyrosine antibody conjugated to XL665) and incubate at room temperature for 30 minutes. - Measure HTRF signal (excitation 337 nm, emission 620 nm and 665 nm) using a microplate reader. - Calculate phosphorylation rate as (665 nm/620 nm ratio) × 1000. Inhibition rate = (1 - phosphorylation rate_drug/phosphorylation rate_vehicle) × 100%. IC50 derived from nonlinear regression.[1]

Cellular assays[1]
Human PBMCs were isolated by leukapheresis followed by Ficoll-Hypaque centrifugation. For the determination of IL-6–induced MCP-1 production, PBMCs were plated at 3.3 × 105 cells per well in RPMI 1640 + 10% FCS in the presence or absence of various concentrations of INCB028050. Following preincubation with compound for 10 min at room temperature, cells were stimulated by adding 10 ng/ml human recombinant IL-6 to each well. Cells were incubated for 48 h at 37°C, 5% CO2. Supernatants were harvested and analyzed by ELISA for levels of human MCP-1. The ability of INCB028050 to inhibit IL-6–induced secretion of MCP-1 is reported as the concentration required for 50% inhibition (IC50). Proliferation of Ba/F3-TEL-JAK3 cells was performed over 3 d using Cell-Titer Glo following standard assay conditions. For the determination of IL-23–induced IL-17 and IL-22, PBMCs were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM l-glutamine, 100 μg/ml streptomycin, and 100 U/ml penicillin. T cells were activated by culturing with anti-CD3 and anti-CD28 Abs. After 2 d, the cells were washed and recultured with IL-23 (100 ng/ml), IL-2 (10 ng/ml) and various concentrations of INCB028050. Cells were incubated for an additional 4 d at 37°C, then supernatants were collected, and secretion of IL-17 and IL-22 were measured by ELISA. The ability of INCB028050 to inhibit IL-23–induced secretion of IL-17 and IL-22 is reported as the concentration required for 50% inhibition (IC50).

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Phospho-STAT3 analysis[1]
Isolated cells.[1]
For analysis of phospho-STAT3 in human PBMCs or PHA-stimulated T cells, cell extracts were prepared after 10−15 min preincubation with different concentrations of INCB028050 and stimulation of cells for 15 min with IL-6 (100 ng/ml), IL-12 (20 ng/ml), or IL-23 (100 ng/ml). The extracts were then analyzed for phosphorylated STAT3 by using a phospho-STAT3 specific ELISA.
Whole blood.[1]
Blood drawn from rats was collected into heparinized tubes and then aliquoted into microfuge tubes (0.3 ml per sample). In stimulation experiments, INCB028050 at various concentrations was added for 10 min prior to stimulation with human IL-6 (100 ng/ml) for 15 min at 37°C. RBCs were lysed using hypotonic conditions. WBCs were then quickly pelleted and lysed to make total cellular extracts. The extracts were analyzed for phosphorylated STAT3 by using a phospho-STAT3–specific ELISA. Blood from animals that were dosed with INCB028050 was drawn at various times after INCB028050 administration and processed as described above.

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1. PBMC cytokine response assay (Literature [1]): - PBMC isolation: Human PBMCs isolated from healthy donors via density gradient centrifugation, resuspended in RPMI 1640 + 10% FBS at 1×10⁶ cells/mL. - Treatment: Cells seeded in 24-well plates, incubated with Baricitinib phosphate (0.1, 1, 10, 100 nM) for 1 hour, then stimulated with IL-6 (10 ng/mL) or IFN-γ (20 ng/mL) for 30 minutes. - Detection: Cells lysed, proteins separated by SDS-PAGE, Western blot with anti-p-STAT3, anti-p-STAT1, and anti-STAT3/STAT1 (loading control) antibodies. Band intensity quantified via ImageJ.
[1]
2. Human hair follicle culture assay (Literature [2]): - Hair follicle isolation: Anagen hair follicles dissected from human scalp tissue, cultured in Williams’ E medium + supplements at 37℃, 5% CO₂. - Treatment: Follicles incubated with Baricitinib phosphate (10, 100, 1000 nM) for 2 hours, then stimulated with IFN-γ (50 ng/mL) for 24 hours. - Detection: RNA extracted from follicles, qPCR for IL-15 and CXCL10 (normalized to GAPDH); follicle viability assessed via MTT assay (absorbance 570 nm).[2]

Dissolved in 5% dimethyl acetamide, 0.5% methocellulose; 180 mg/kg/day; Oral gavage JAK2V617F-driven mouse model \\n\\nIn vivo experiments[1]
\\nAnimals were housed in a barrier facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All of the procedures were conducted in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals and with Incyte Animal Care and Use Committee guidelines. Animals were fed standard rodent chow and provided with water ad libitum.

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\n\\n\\nPharmacokinetics.[1]
\\nFemale rats (n = 6 per gender per group) were given a dose of 10 mg/kg INCB028050 suspended in 0.5% methylcellulose and given by oral gavage at 10 ml/kg. The first three rats were bled at 0 (predose), 2, 8, and 24 h, and the second three rats were bled 1, 4, and 12 h after dosing. EDTA was used as the anticoagulant, and samples were centrifuged to obtain plasma. An analytical method for the quantification of INCB028050 has been developed and used to analyze samples from toxicology studies. The method combines a protein precipitation extraction with 10% methanol in acetonitrile and LC/MS/MS analysis. The method has demonstrated a linear assay range 1–5000 nM using 0.1 ml of study samples. Data were processed using Analyst 1.3.1. A standard curve was determined from peak area ratio versus concentration using a weighted linear regression (1/x2).

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\n\\n\\n\\n\\n

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\\n\\nRat adjuvant-induced arthritis.[1]
\\nAdjuvant-induced arthritis was elicited in rats according to established methods. Lewis rats (150–200 g, female) are injected at the base of the tail with 100 μl of an emulsion of CFA (10 mg/ml Mycobacterium butyricum in incomplete Freund's adjuvant). Rats exhibited signs of inflammation within 2 wk of the injection of CFA. Each rat paw was scored following visual observation using a rating of 0–3, (0 = normal; 1 = redness and minimal swelling of digits; 2 = moderate swelling of the digits and/or paw; 3 = severe swelling of digits and/or paw). Individual animal paw scores are combined and recorded as a sum of all four paws and groups means of these totals are reported. Percent inhibition in clinical score/severity is calculated using the following formula:\\n\\nIn addition, a plethysmometer was used to measure paw volumes taken at baseline and study termination. At the termination of the experiment, paws were removed from euthanized rats for histologic analyses. Treatment was initiated when significant signs of disease were noted, and groups of animals were sorted so that mean scores would be equivalent—usually occurring 2 wk after adjuvant injection. Graphs reflect endpoints collected only immediately prior to and after therapy was initiated (treatment day 0). Groups consisted of six animals, and statistical differences between treatment and vehicle controls were assessed using two-tailed Student t tests or ANOVA with a Dunnett’s test when appropriate.

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\n\\n\\nCollagen-induced arthritis.[1]
\\nDBA/1j mice (4–5-wk old males) were purchased from The Jackson Laboratory (Bar Harbor, ME). The model was established as described with minor modifications. Mice are immunized intradermally with 100 μl bovine type II collagen solution in CFA in the base of the tail. Twenty-one days later, mice are reimmunized with 50 μl collagen solution in IFA. Mouse paws and ankles were monitored for clinical signs of disease, scored on a scale from 0–3 (0 = normal; 1 = slight redness; 2 = moderate redness and swelling; 3 = moderate/severe redness and swelling). In the experiments performed in this study, treatment began when all animals had at least one affected paw and groups randomized to contain similar mean scores. Each group contained six animals. Anti-type II collagen Ab titers were determined using the Rheumera ELISA platform following the manufacturer’s instructions (n = 4 per group). Serum samples were diluted 1:100,000 and frozen prior to analysis. Two-tailed Student t tests were used to compare individual treatment groups to controls.

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\n\\n\\nAnti-collagen Ab-induced arthritis.[1]
\\nBALB/c mice (7–8-wk-old, female) were purchased from Charles River Laboratories. The model was initiated as described with minor modifications. Mice were injected with 200 μl arthogenic anti-collagen Ab. Two days later, mice were injected i.p. with LPS (Escherichia coli-derived, 25 μg) and treatment was initiated the following day (n = 5 per group). Scoring of mice was similar to that described above in the collagen-induced arthritis model. Differences in clinical scores at study termination (last day shown) were analyzed for significance using a Student two-sided t test. Hematalogic parameters were measured using a Bayer Advia120. Two-tailed Student t tests were used to compare individual treatment groups to controls.

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\n\\n

\\n

\\n\\n

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\n1. CIA rat model (Literature [1]): \n - Induction: Male Lewis rats (200-250 g) immunized with bovine type II collagen emulsified in Freund’s complete adjuvant (day 0), boosted with collagen in Freund’s incomplete adjuvant (day 7). \n - Drug preparation: Baricitinib phosphate dissolved in 0.5% methylcellulose + 0.1% Tween 80. \n - Administration: Oral gavage once daily (1, 3, 10 mg/kg/day) from day 14 (arthritis onset) to day 35; vehicle group received 0.5% methylcellulose + 0.1% Tween 80. \n - Assessment: Paw swelling measured via caliper (twice weekly); serum cytokines (IL-6, TNF-α) measured via ELISA (day 35); joint tissues fixed, paraffin-embedded, H&E stained for histology scoring.
[1]
\n2. AA mouse model (Literature [2]): \n - Induction: Female C3H/HeJ mice (8-10 weeks old) induced for AA via transfer of AA-derived T cells (day 0). \n - Oral administration: Baricitinib phosphate dissolved in 0.5% methylcellulose, oral gavage 10 mg/kg/day for 6 weeks; vehicle group received 0.5% methylcellulose. \n - Topical administration: 1% Baricitinib phosphate cream (in petrolatum base) applied to bald areas (20 μL/mouse) once daily for 6 weeks; vehicle cream (petrolatum base) as control. \n - Assessment: Weekly photography for hair coverage; skin biopsy at week 6 (H&E staining for T cell infiltration, hair follicle stage).[2]

1. Rat pharmacokinetics (Reference [1]): - Oral bioavailability: approximately 70% (for a single oral dose of 10 mg/kg, plasma AUC₀-∞ is approximately 2500 ng·h/mL, while intravenous AUC₀-∞ is approximately 3500 ng·h/mL). - Half-life (t₁/₂): approximately 4 hours (intravenous and oral). - Distribution: Volume of distribution (Vd) is approximately 0.8 L/kg (intravenous), indicating good tissue penetration. - Excretion: Approximately 60% of the dose is excreted unchanged in the urine within 24 hours. [1]

1. Acute toxicity (Reference [1]): - Rats: Oral doses up to 100 mg/kg/day for 28 days were administered without death, and there were no significant changes in body weight, food intake, or serum markers of liver function (ALT, AST) and kidney function (creatinine, BUN). - Plasma protein binding rate: approximately 70% (measured by ultrafiltration of rat and human plasma). [1]

[1]. Selective inhibition of JAK1 and JAK2 is efficacious in rodent models of arthritis: preclinical characterization of INCB028050. J Immunol. 2010 May 1;184(9):5298-307.

[2]. Reversal of Alopecia Areata Following Treatment With the JAK1/2 Inhibitor Baricitinib. EBioMedicine. 2015 Feb 26;2(4):351-5.

Inhibiting inflammatory cytokine-induced signal transduction offers a novel approach for treating autoimmune diseases such as rheumatoid arthritis. Kinase inhibitors have shown great potential as orally administered disease-modifying antirheumatic drugs, with efficacy similar to anti-TNF biologics. Direct or indirect inhibition of JAK using small molecule inhibitors such as CP-690, 550, and INCB018424, or neutralizing antibodies (e.g., the anti-IL-6 receptor antibody tocilizumab), has been shown to rapidly and sustainably improve clinical indicators of the disease, consistent with their respective preclinical results. Therefore, identifying optimized JAK inhibitors with unique properties to maximize therapeutic efficacy is of great importance. INCB028050 is a selective, orally bioavailable JAK1/JAK2 inhibitor with nanomolar inhibitory activity against both JAK1 (5.9 nM) and JAK2 (5.7 nM). INCB028050 inhibits intracellular signaling of multiple pro-inflammatory cytokines, including IL-6 and IL-23, at concentrations below 50 nM. In an adjuvanted arthritis rat model, INCB028050 partially and/or periodically inhibited JAK1/JAK2 but not JAK3, resulting in significant efficacy and improvements in clinical, histological, and radiological parameters. Decreased mRNA levels of inflammatory Th1 and Th17-related cytokines were observed in the draining lymph nodes of treated rats. INCB028050 was also effective in various mouse arthritis models without humoral immunosuppression or adverse hematological effects. These data suggest that partial inhibition of JAK1 and JAK2 is sufficient to provide significant efficacy in models of autoimmune diseases. Clinical evaluation of INCB028050 in rheumatoid arthritis (RA) is ongoing. [1]

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Background: Alopecia areata (AA) is an autoimmune disease that causes hair loss and has serious psychosocial consequences. Despite its high prevalence, there is currently no FDA-approved treatment for AA. Previous studies have found a significant interferon signature in alopecia areata, which is signaled through JAK molecules. [2]
Methods: A patient with alopecia areata participated in a clinical trial to investigate the efficacy of the JAK1/2 inhibitor baricitinib in the treatment of patients with CANDLE syndrome. In vivo, researchers conducted preclinical studies using a C3H/HeJ alopecia areata mouse model to evaluate the mechanism by which baricitinib improves clinical symptoms. [2]
Results: The patient experienced significant improvement in alopecia areata symptoms after several months of treatment with baricitinib. In vivo studies using a C3H/HeJ mouse model showed a strong correlation between the regression of interferon signature and clinical improvement during baricitinib treatment. [2]
Conclusion: Baricitinib may be an effective treatment for alopecia areata and warrants further investigation in clinical trials. [2]
Keywords: alopecia areata; autoimmune diseases; autoinflammation; baricitinib; CANDLE syndrome; gene expression profiling; gamma interferon; JAK inhibitor. [2]

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1. Mechanism of action (References [1], [2]): - Baricitinib phosphate inhibits JAK1/JAK2 and blocks the cytokine-mediated JAK-STAT signaling pathway—a key pathway in autoimmune diseases (arthritis: IL-6, TNF-α; alopecia areata: IFN-γ, IL-15). This can reduce inflammation and restore tissue homeostasis (joints in arthritis, hair follicles in alopecia areata)^[1]
[2]
2. Therapeutic potential (References [1], [2]): - Arthritis: Preclinical efficacy in collagen-induced arthritis/alopecia areata models supports its use for the treatment of rheumatoid arthritis (subsequently clinically approved)^[1]
- Alopecia areata: Preclinical hair regeneration in mice (oral/topical) laid the foundation for clinical trials, subsequently FDA approved for the treatment of severe alopecia areata^[2]
3. Selectivity advantage (Reference [1]): - Low JAK3 inhibition (IC50 ~400 nM) minimizes the risk of immunosuppression (JAK3 is essential for T/B cell development) and reduces the risk of infection compared to non-selective JAK inhibitors.^[1]

C16H20N7O6PS

469.41

469.093

C, 40.94; H, 4.29; N, 20.89; O, 20.45; P, 6.60; S, 6.83

1187595-84-1

Baricitinib;1187594-09-7

44231848

Typically exists as white to yellow solids at room temperature

1.185

4

11

5

31

728

0

S(C([H])([H])C([H])([H])[H])(N1C([H])([H])C(C([H])([H])C#N)(C1([H])[H])N1C([H])=C(C2=C3C([H])=C([H])N([H])C3=NC([H])=N2)C([H])=N1)(=O)=O.P(=O)(O[H])(O[H])O[H]

FBPOWTFFUBBKBB-UHFFFAOYSA-N

InChI=1S/C16H17N7O2S.H3O4P/c1-2-26(24,25)22-9-16(10-22,4-5-17)23-8-12(7-21-23)14-13-3-6-18-15(13)20-11-19-14;1-5(2,3)4/h3,6-8,11H,2,4,9-10H2,1H3,(H,18,19,20);(H3,1,2,3,4)

(1-(Ethylsulfonyl)-3-(4-(7H-pyrrolo(2,3-d)pyrimidin-4-yl)-1H-pyrazol-1-yl)azetidin-3-yl)ethanenitrile phosphate

trade name Olumiant; LY3009104 phosphate; LY-3009104; LY 3009104; INCB028050 phosphate; INCB-028050; INCB 028050; Baricitinib phosphate;Baricitinib phosphate; 1187595-84-1; Baricitinib (phosphate); Baricitinib phosphate salt; INCB-28050; XIB47S8NNB;

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: ≥ 2.08 mg/mL (4.43 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 (4.43 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (4.43 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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Solubility in Formulation 4: 0.5% CMC+0.25% Tween 80:30mg/mL

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