JAK2 (IC50 = 5.7 nM); JAK1 (IC50 = 5.9 nM); Tyk2 (IC50 = 53nM); JAK3 (IC50 = 560nM)

Cell-based studies demonstrated the potency of baricitinib (INCB028050) as an inhibitor of JAK signaling and function. Baricitinib has IC50 values of 44 nM and 40 nM, respectively, which prevent IL-6-stimulated phosphorylation of canonical substrate STAT3 (pSTAT3) and the subsequent generation of the chemokine MCP-1 in PBMC. INCB028050 also suppresses pSTAT3 activated by IL-23 (IC50=20 nM) in isolated naïve T cells. This suppression stops Th17 cells from producing the two harmful cytokines, IL-17 and IL-22. Th17 cells have an IC50 value of 50 nM and are a subtype of helper T cells with unique inflammatory and pathogenic characteristics. Even at doses up to 10 μM, the structurally similar but ineffective JAK1/2 inhibitors INCB027753 and INCB029843 exhibited no discernible effect in any of these assay systems [1].

Over the course of a 2-week treatment period, baricitinib (INCB028050) therapy reduced the rise in hindpaw volume by 50% at 1 mg/kg and >95% at 3 or 10 mg/kg. Given that baseline measurements of paw volume were obtained in animals exhibiting clear illness indications on day 0 of treatment, those exhibiting a notable improvement in swelling may exhibit >100% inhibition [1]. Mice given baricitinib (0.7 mg/kg/day) showed markedly decreased MHC class I and class II expression, decreased CD8 infiltration, and dramatically decreased inflammation (measured by H&E staining). When compared to vehicle control animals, the number of CD8+NKG2D+ cells, which are important effector cells in alopecia areata (AA) illness in both people and mice, is markedly lower in mice treated with baricitinib [2].

Biochemical assays[1]
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.

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.

In vivo experiments[1]
Animals 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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Pharmacokinetics.[1]
Female 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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Rat adjuvant-induced arthritis.[1]
Adjuvant-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: In 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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Collagen-induced arthritis.[1]
DBA/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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Anti-collagen Ab-induced arthritis.[1]
BALB/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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Dissolved in 5% dimethyl acetamide, 0.5% methocellulose; 180 mg/kg/day; Oral gavage

JAK2V617F-driven mouse model

Absorption, Distribution and Excretion
The absolute bioavailability of baricitinib is approximately 80%. The Cmax was reached after one hour of oral drug administration. A high-fat meal decreased the mean AUC and Cmax of baricitinib by approximately 11% and 18%, respectively, and delayed Tmax by 0.5 hours.
Baricitinib is predominantly excreted via renal elimination. It is cleared via filtration and active secretion. Approximately 75% of the administered dose was eliminated in the urine, with 20% of that dose being the unchanged drug. About 20% of the dose was eliminated in the feces, with 15% of that dose being an unchanged drug.
Following intravenous administration, the volume of distribution was 76 L, indicating distribution into tissues.
The total body clearance of baricitinib was 8.9 L/h in patients with rheumatoid arthritis. The total body clearance and half-life of baricitinib was 14.2 L/h in intubated patients with COVID-19 who received baricitinib via nasogastric (NG) or orogastric (OG) tube.

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Metabolism / Metabolites
Baricitinib is metabolized by CYP3A4. Approximately 6% of the orally administered dose was identified as metabolites in urine and feces; however, no metabolites of baricitinib were quantifiable in plasma.

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Biological Half-Life
The elimination half-life in patients with rheumatoid arthritis is approximately 12 hours. The elimination half-life was 10.8 hours in intubated patients with COVID-19 who received baricitinib via nasogastric (NG) or orogastric (OG) tube.

Hepatotoxicity
In the large prelicensure clinical trials in rheumatoid arthritis, serum aminotransferase elevations occurred in up to 17% of baricitinib treated subjects compared to 11% in placebo recipients. The elevations were typically mild and transient and values above 3 times the upper limit of normal (ULN) occurred in 1% to 2% of patients. The elevations occasionally led to early discontinuations, but more often resolved even without dose adjustment. In prelicensure studies in rheumatoid arthritis, alopecia areata and other rheumatic and immune-mediated disorders, there were no instances of clinically apparent liver injury attributed to baricitinib. Since approval and more wide scale availability of baricitinib, there have been no published reports of hepatotoxicity associated with its use.
Use of baricitinib in combination with remdesivir for severe COVID-19 pneumonia has been reported but with little information on its potential for causing liver injury. Patients with severe SARS-CoV-2 infection frequently have elevated serum aminotransferase levels and occasionally are jaundiced. Furthermore, remdesivir has been linked to serum aminotransferase elevations during therapy that are generally mild-to-moderate in severity and resolve rapidly once the drug is stopped. Whether baricitinib increases the risk of liver injury during COVID-19 has yet to be shown, but hepatotoxicity was not a prominent feature in these early studies of its use in patients with severe COVID-19.
Finally, baricitinib is an immune modulatory agent and has the potential of causing reactivation of viral infections including hepatitis B. In clinical trials, patients with HBsAg in serum were excluded from enrollment but patients with anti-HBc without HBsAg were allowed. While routine monitoring for reactivation was not performed on all patients, at least 15% of anti-HBc positive persons with rheumatoid arthritis treated with baricitinib developed virologic evidence of reactivation marked by de novo appearance of low levels of HBV DNA in serum. In all cases, the period of viremia was brief and not associated with serum aminotransferase elevations or jaundice. Thus, baricitinib appears to be capable of causing HBV reactivation but it is generally subclinical. Furthermore, the short courses of baricitinib used in the treatment of severe COVID-19 have not been linked to episodes of HBV reactivation.
Likelihood score: E* (unlikely to be a cause of idiosyncratic clinically apparent liver injury but has the potential to cause reactivation of hepatitis B).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of baricitinib during breastfeeding. Most sources recommend that mothers not breastfeed while taking baricitinib. An alternate drug is preferred, especially while nursing a newborn or preterm infant. The manufacturer recommends that women avoid nursing during therapy and for 4 days after the last dose.
◉ 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
Baricitinib is approximately 50% bound to plasma proteins and 45% bound to serum proteins.

[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.

[3]. Intermuscular and perimuscular fat expansion in obesity correlates with skeletal muscle T cell and macrophage infiltration resistance. Int J Obes (Lond). 2015 Nov;39(11):1607-18.

Pharmacodynamics
Baricitinib is a disease-modifying antirheumatic drug (DMARD) used to ameliorate symptoms and slow down the progression of rheumatoid arthritis. In animal models of inflammatory arthritis, baricitinib was shown to have significant anti-inflammatory effects but also led to the preservation of cartilage and bone, with no detectable suppression of humoral immunity or adverse hematologic effects. Baricitinib decreased the levels of immunoglobulins and serum C-reactive protein in patients with rheumatoid arthritis.

C16H17N7O2S

371.42

371.116

C, 51.74; H, 4.61; N, 26.40; O, 8.62; S, 8.63

1187594-09-7

Baricitinib phosphate;1187595-84-1;Baricitinib-d5;1564241-79-7;Baricitinib-d3;1564242-30-3

44205240

Typically exists as white to gray solids at room temperature

1.6±0.1 g/cm3

707.2±70.0 °C at 760 mmHg

381.5±35.7 °C

0.0±2.3 mmHg at 25°C

1.763

-0.06

1

7

5

26

678

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

XUZMWHLSFXCVMG-UHFFFAOYSA-N

InChI=1S/C16H17N7O2S/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/h3,6-8,11H,2,4,9-10H2,1H3,(H,18,19,20)

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: ≥ 2.5 mg/mL (6.73 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 25.0 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: ≥ 2.5 mg/mL (6.73 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 25.0 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: ≥ 2.5 mg/mL (6.73 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 25.0 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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Solubility in Formulation 5: 2.5 mg/mL (6.73 mM) in 0.5% Methylcellulose/saline water (add these co-solvents sequentially from left to right, and one by one), suspension 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.)