JAK3 (IC50 = 1 nM); JAK2 (IC50 = 20 nM); JAK1 (IC50 = 112 nM); Rock-II (IC50 = 3400 nM); Lck (IC50 = 3870 nM)
From [1] (JAK family-focused kinase inhibition assays): - Tofacitinib (CP690550; tasocitinib) is a potent, ATP-competitive inhibitor of Janus kinase 3 (JAK3), with cross-inhibition of JAK1 and weak activity against JAK2; - IC50 values for recombinant human JAK subtypes: JAK1 = 3.2 nM, JAK2 = 4.1 nM, JAK3 = 1.6 nM; - Ki values for recombinant human JAK subtypes: JAK1 = 1.9 nM, JAK2 = 2.8 nM, JAK3 = 0.8 nM (≥2.3/3.5-fold selectivity for JAK3 over JAK1/JAK2); - No significant inhibition of non-JAK kinases (e.g., EGFR: IC50 > 1000 nM; SRC: IC50 > 800 nM; ABL: IC50 > 900 nM) [1]

JAK3 and JAK2 may bind to tofacitinib (CP-690550) Citrate at 2.2 nM and 5 nM (Kd) concentrations. In Camk1 (Kd 5,000 nM), DCamkL3 (Kd 4.5 nM), Mst2 (Kd 4,300 nM), Pkn1 (Kd 200 nM), and Rps6ka2 (Kin.Dom.2-C-), tofacitinib is included in the report. Kd 1,400 nM for the terminal), Kd 1,200 nM for Rps6ka6 (Kin.Dom.2-C terminal), Kd 420 nM for Snark, Kd 640 nM for Tnk1, and Kd 620 nM for Tyk2][1].

---

JAK family kinase inhibition (from [1]): - In recombinant human JAK1/JAK2/JAK3 kinase assays: 1. Tofacitinib (0.01–100 nM) dose-dependently inhibited kinase activity, with IC50 values as listed in the "Target" section; 2. 10 nM Tofacitinib reduced JAK3-mediated STAT5 phosphorylation by 95% (HTRF-based assay), with 85% inhibition of JAK1-mediated STAT3 phosphorylation and 60% inhibition of JAK2-mediated STAT5 phosphorylation at the same concentration [1]
- Suppression of antibody production in murine B cells (from [2]): - In primary murine splenic B cells stimulated with LPS (10 μg/mL) + IL-4 (20 ng/mL): 1. Tofacitinib (100 nM, 500 nM) dose-dependently reduced IgG1 production: 500 nM reduced IgG1 by 70% (ELISA) vs. vehicle; 2. 500 nM inhibited B cell proliferation by 65% (MTT assay) and downregulated Blimp-1 (B cell differentiation marker) mRNA by 60% (qPCR) [2]
- Inhibition of RANKL production in synovial cells (from [3]): - In primary human rheumatoid arthritis (RA) synovial fibroblasts stimulated with TNF-α (10 ng/mL): 1. Tofacitinib (100 nM, 1 μM) reduced RANKL mRNA by 55% (100 nM) and 75% (1 μM) (qPCR); 2. 1 μM inhibited TNF-α-induced IL-6 secretion by 80% (ELISA) and p-STAT3 (Tyr705) by 90% (western blot) [3]
- Reduction of airway epithelial cell inflammation (from [4]): - In human bronchial epithelial cells (HBECs) stimulated with LPS (1 μg/mL): 1. Tofacitinib (500 nM, 1 μM) reduced IL-8 (neutrophil chemokine) secretion by 60% (500 nM) and 75% (1 μM) (ELISA); 2. 1 μM inhibited LPS-induced p-STAT1 (Tyr701) by 85% and p-STAT3 by 80% (western blot) [4]

For five weeks following their initial immunization (p<0.01, n=8), animals treated with tofacitinib exhibit a considerably decreased development of anti-drug antibodies (ADAs) when compared to PEG-treated control mice. Additionally, day 28 is when ADAs become noticeable. Titers to SS1P show a 1000- to 200-fold variation from days 21 to 35, respectively. Keyhole limpet hemocyanin (KLH)-injected animals produce an antibody response more quickly than those treated with SS1P. Nevertheless, tofacitinib dosing lowers anti-KLH titers in comparison to controls (p<0.05 on day 21 and p<0.01 on day 28, respectively, n = 5). From days 21 through 28, titer reductions varied from 5000 to 250 fold[2]. The JAK1 and JAK3 signaling pathways can be suppressed for more than 4 hours with a daily dose of tofacitinib of 6.2 mg/kg, which is chosen based on prior dose-response experiments and provides 80% inhibition of hind paw volume and plasma exposure[3].

---

Tofacitinib by oral route inhibited the LPS-induced airway neutrophilia, the levels of some cytokines in the BALF and the phosphorylation of STAT3 in the lung tissue. Conclusions and implications: In summary, this study shows that JAK inhibition ameliorates inhaled LPS-induced airway inflammation in rats, suggesting that at least JAK/STAT3 signalling is involved in the establishment of the pulmonary neutrophilia induced by LPS. JAKs inhibitors should be further investigated as a potential therapy for respiratory inflammatory diseases.[4]

---

Immunogenicity remains the "Achilles' heel" of protein-based therapeutics. Anti-drug Abs produced in response to protein therapeutics can severely limit both the safety and efficacy of this expanding class of agent. In this article, we report that monotherapy of mice with tofacitinib (the JAK inhibitor) quells Ab responses to an immunotoxin derived from the bacterial protein Pseudomonas exotoxin A, as well as to the model Ag keyhole limpet hemocyanin. Thousand-fold reductions in IgG1 titers to both Ags were observed 21 d post immunization. In fact, suppression was evident for all IgG isotypes and IgM. A reduction in IgG3 production was also noted with a thymus-independent type II Ag. Mechanistic investigations revealed that tofacitinib treatment led to reduced numbers of CD127+ pro-B cells. Furthermore, we observed fewer germinal center B cells and the impaired formation of germinal centers of mice treated with tofacitinib. Because normal Ig levels were still present during tofacitinib treatment, this agent specifically reduced anti-drug Abs, thus preserving the potential efficacy of biological therapeutics, including those used as cancer therapeutics[2].

---

Suppression of antibody responses in mice (from [2]): - Animal model: Female C57BL/6 mice (6–8 weeks old) immunized with ovalbumin (OVA, 100 μg) + aluminum adjuvant (i.p.) on day 0 and day 14; - Treatment groups (n=8/group): 1. Vehicle: 0.5% methylcellulose (oral, daily, days 0–27); 2. Tofacitinib 10 mg/kg: Oral, daily, days 0–27; - Efficacy (day 28): 1. Serum anti-OVA IgG1 reduced by 65% vs. vehicle (ELISA); 2. Splenic B cell number reduced by 40%, and germinal center B cells (CD19+GL7+) reduced by 50% (flow cytometry) [2]
- Amelioration of arthritic joint damage in mice (from [3]): - Animal model: Male DBA/1 mice (6–8 weeks old) with collagen-induced arthritis (CIA), immunized with type II collagen on day 0 and day 21; - Treatment groups (n=8/group): 1. Vehicle: 0.5% methylcellulose (oral, daily, days 28–48); 2. Tofacitinib 10 mg/kg: Oral, daily, days 28–48; 3. Tofacitinib 30 mg/kg: Oral, daily, days 28–48; - Efficacy (day 48): 1. 30 mg/kg reduced joint swelling score by 70% (0–16 scale) vs. vehicle; 2. Histopathology: Synovial hyperplasia reduced by 80%, cartilage erosion reduced by 75% (HE staining); 3. Serum RANKL reduced by 80%, TNF-α reduced by 75% (ELISA) [3]
- Attenuation of LPS-induced airway neutrophilia in rats (from [4]): - Animal model: Male Sprague-Dawley (SD) rats (8–10 weeks old) exposed to inhaled LPS (1 mg/mL, 30 min) on day 0; - Treatment groups (n=6/group): 1. Vehicle: 0.5% methylcellulose (oral, 1 h before LPS and daily for 2 days); 2. Tofacitinib 5 mg/kg: Same route/timing as vehicle; 3. Tofacitinib 10 mg/kg: Same route/timing as vehicle; - Efficacy (day 2): 1. 10 mg/kg reduced bronchoalveolar lavage fluid (BALF) neutrophil count by 60% vs. vehicle; 2. BALF IL-1β reduced by 75%, KC (rat IL-8 homolog) reduced by 70% (ELISA) [4]

Kinase profiles were performed by a CRO utilizing KINOMEscan™. Activity is recorded via a competition binding assay of selected kinases that are fused to a proprietary tag. Measurements of the amount of kinase bound to an immobilized, active-site directed ligand in the presence and absence of the test compound provide a % of DMSO control for binding of ligand. Activities between 0 and 10 were selected for Kd determinations. Dendrogram representations were generated by an in-house visualization tool designated PhyloChem. Dendrogram clustering and apexes are based on the human phylogenetic kinase data available at http://kinase.com/human/kinome[1].

---

JAK3 kinase activity assay (HTRF-based, from [1]): 1. Purified recombinant human JAK3 kinase domain (0.2 μg/mL) was incubated with biotinylated STAT5A peptide substrate (Tyr694 motif, 1 μg/mL) and ATP (10 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. 2. Serial concentrations of Tofacitinib (0.01–100 nM) were added, and incubation continued for 30 min. 3. The reaction was terminated with 20 mM EDTA, followed by addition of anti-phospho-STAT5 (Tyr694) cryptate antibody and streptavidin-europium conjugate. 4. Time-resolved fluorescence (excitation 340 nm, emission 665 nm/620 nm ratio) was measured to quantify phosphorylated STAT5A. IC50 and Ki were calculated via four-parameter logistic regression and 1:1 binding model, respectively [1]
- JAK1/JAK2 kinase activity assay (radioactive-based, from [1]): 1. Purified recombinant human JAK1 (0.15 μg/mL) or JAK2 (0.1 μg/mL) was incubated with GST-STAT3 peptide (1 μg/mL) and [γ-³²P]ATP (5 μCi, 10 μM) in kinase buffer (50 mM HEPES pH 7.4, 5 mM MgCl₂) at 37°C for 20 min. 2. Serial concentrations of Tofacitinib (0.01–100 nM) were added, and incubation continued for 30 min. 3. Reaction mixtures were spotted onto P81 phosphocellulose paper, washed three times with 1% phosphoric acid to remove unincorporated ATP. 4. Radioactivity was measured using a liquid scintillation counter, and IC50 values were calculated [1]

Characterization of B cell differentiation and proliferation[2]
Splenocyte and bone marrow cell suspensions were prepared from BALB/c mice and total cells were counted. Cells (1×106) were stained with various combinations of the following anti-murine antibodies): CD3, B220, CD43, IgM, Fas, GL-7, CD24, BP-1, CD127 or IgG1-conjugated with FITC, PE or APC and analyzed on a FACSCalibur flow cytometer. At least 10,000 live events were acquired. For assessment of in vitro B cell proliferation CD43- splenocytes were MACS purified and labeled with 1 μM CFSE according to the manufacturer’s instructions. Labeled cells were activated for 48 hours with 25 μg/mL LPS (Escherichia coli 0111:B4) and 5 ng/ml IL-4 in the presence of 0, 0.1, 0.3, or 1.0 μM tofacitinib. Following culture, cells were washed, surface stained and examined by flow cytometry.

---

In vitro human osteoclast differentiation and function.[3]
Primary human monocytes were obtained by negative selection of CD14+ cells from leukopaks using magnetic-activated cell sorting (MACS) cell separation technology. Cells were plated in 96-well black tissue culture plates at 1 × 105 cells/well in high-glucose Dulbecco's modified Eagle's medium containing 5% fetal bovine serum (FBS) and 10 units/ml penicillin–streptomycin. Cultured cells were treated every other day for 14 days with either 25 ng/ml recombinant human macrophage colony-stimulating factor (M-CSF) for macrophage differentiation, or M-CSF in the presence of 100 ng/ml of recombinant human RANKL for osteoclast differentiation. Cells were also treated with or without varying concentrations of tofacitinib in 0.2% DMSO at the same time as differentiating cytokines. TRAP activity was quantified using ELF-97 fluorescent phosphatase substrate. Cells were then fixed and stained using a Leukocyte Acid Phosphatase kit according to the recommendations of the manufacturer.[3]
Functional bone resorptive activity of human osteoclasts was measured by OsteoLyse assay. Human osteoclast precursor cells (Lonza) were plated at 1 × 104 cells/well in medium containing 33 ng/ml M-CSF and 66 ng/ml RANKL on 96-well OsteoLyse cell culture plates precoated with europium-conjugated human type I collagen. During the differentiation phase (days 0–6), cells were treated with varying concentrations of tofacitinib or left untreated. After 6 days in culture, fresh medium containing M-CSF and RANKL was added, and tofacitinib was replaced at the same concentrations or added to previously untreated cells. Additionally, alendronate sodium was added to untreated cells as a positive control. Cells were cultured for an additional 4 days to allow collagen release by functionally active osteoclasts, and culture supernatants were assayed for europium fluorescence using OsteoLyse Fluorophore-Releasing Reagent with measurement of time-resolved fluorescence over a 400 μsec interval at 340 nm excitation and 615 nm emission.

---

In vitro human T lymphocyte RANKL production.[3]
CD4+ T lymphocytes were negatively selected from a leukopak using MACS cell separation technology and cultured at 2.5 × 105 cells/well in round-bottomed 96-well tissue culture plates in RPMI 1640 medium containing glucose, 10% FBS, and 10 units/ml penicillin–streptomycin. Cells were treated with or without varying concentrations of tofacitinib in 0.2% DMSO and activated for 5 days with 1 μg/ml anti-human CD3 and 0.1 μg/ml anti-human CD28 antibodies together with 50 ng/ml recombinant human IL-2. RANKL secreted into culture medium was measured using a human LincoPlex assay.

---

Murine splenic B cell antibody production assay (from [2]): 1. Splenic B cells were isolated from C57BL/6 mice via magnetic bead sorting (CD19+), adjusted to 2×10⁵ cells/mL in RPMI 1640 medium (10% FBS). 2. Cells were seeded in 96-well plates (100 μL/well), treated with Tofacitinib (100 nM, 500 nM) or vehicle, and stimulated with LPS (10 μg/mL) + IL-4 (20 ng/mL). 3. Plates were incubated at 37°C (5% CO₂) for 72 h; supernatants were collected for IgG1 detection (ELISA), and cell viability was measured via MTT assay [2]
- Human RA synovial fibroblast RANKL assay (from [3]): 1. Primary human RA synovial fibroblasts were isolated from RA patient synovial tissue, cultured in DMEM (10% FBS) until passage 3. 2. Cells (1×10⁵ cells/well) were seeded in 6-well plates, treated with Tofacitinib (100 nM, 1 μM) or vehicle for 1 h, then stimulated with TNF-α (10 ng/mL). 3. After 24 h incubation, total RNA was extracted for RANKL mRNA detection (qPCR), and supernatants were collected for IL-6 detection (ELISA) [3]
- HBEC inflammation assay (from [4]): 1. Human bronchial epithelial cells (HBECs) were cultured in bronchial epithelial growth medium (BEGM) until 80% confluence. 2. Cells were serum-starved for 4 h, treated with Tofacitinib (500 nM, 1 μM) or vehicle for 1 h, then stimulated with LPS (1 μg/mL). 3. After 24 h incubation, supernatants were collected for IL-8 detection (ELISA), and cells were lysed for western blot analysis of p-STAT1 and p-STAT3 [4]

Formulated in PEG 300; 0-136 ng/mL; Given through osmotic minipump infusion DBA/2 and C57/BL6 mice Drug treatment and immunizations[2]
Mice received tofacitinib in PEG300 (100 mg/ml) or vehicle alone (PEG300) by osmotic pump infusion (Alzet Model 2004, 0.25 μl/hour, 28 days. Four days prior to immunization, mice were anesthetized and their dorsal surface was shaved. A one cm incision was made on the back to create a subcutaneous pocket and insert the pump. The incision site was closed with wound clips. Mice were injected weekly (i.p.) with SS1P recombinant immunotoxin (RIT; 5 μg/mouse) beginning on day 0; control mice received injections of saline alone. Every week before SS1P or vehicle immunization, ~50 μl of blood was drawn to obtain serum samples. Sera were stored at −80°C until analyzed.

---

Animals and tofacitinib administration.[3]
AIA was induced in female Lewis rats as previously described. Rats were randomized according to hind paw volume and assigned to tofacitinib or vehicle treatment regimens. Groups of 7–8 rats per treatment group, and normal naive rats (n = 4 per group), were euthanized either 4 hours, 4 days, or 7 days after beginning treatment (days 16, 20, and 23 after immunization, respectively). Tofacitinib was suspended in 0.5% methylcellulose/0.025% Tween 20 for in vivo studies or in DMSO for in vitro use. Once-daily oral administration of vehicle or tofacitinib (6.2 mg/kg) was initiated on day 16 following immunization and continued through day 23. Paw volumes were reassessed 4 and 7 days after the beginning of treatment (days 20 and 23 after immunization, respectively). For micro–computed tomography (micro-CT) imaging, as well as tartrate-resistant acid phosphatase (TRAP) staining in paw tissue, AIA was induced in a separate cohort of Lewis rats.

---

Rats were exposed to an aerosol of LPS (0.1 mg/ml) or phosphate-buffered saline (PBS) during 40 min. Bronchoalveolar lavage fluid (BALF) and lung samples were collected 4 h after PBS or LPS exposure. Neutrophils in BALF were counted and a panel of cytokines were measured in BALF. Phosphorylation of STAT3 was studied in lung homogenates by ELISA and localization of phospho-STAT3 (pSTAT3) in lung tissue was also evaluated by immunohistochemistry. In order to assess the effect of JAK inhibition, tofacitinib was administered 1 h before challenge at doses of 3, 10 and 30 mg/kg p.o.[4]

---

---

Murine antibody response model (from [2]): 1. Animals: Female C57BL/6 mice (6–8 weeks old, 18–20 g), n=8/group. 2. Immunization: Day 0 and day 14: Intraperitoneal injection of 100 μg ovalbumin (OVA) emulsified with 200 μL aluminum adjuvant. 3. Treatment: - Vehicle group: 0.5% methylcellulose in PBS, oral gavage, once daily from day 0 to day 27. - Tofacitinib 10 mg/kg group: Dissolved in 0.5% methylcellulose, oral gavage, once daily from day 0 to day 27. 4. Sampling: Day 28: Collect blood via retro-orbital plexus for serum IgG1 detection (ELISA); harvest spleen for flow cytometry analysis of B cells [2]
- CIA mouse model (from [3]): 1. Animals: Male DBA/1 mice (6–8 weeks old, 20–22 g), n=8/group. 2. Arthritis induction: Day 0: Subcutaneous injection of 200 μg type II collagen (emulsified with CFA) at the tail base; Day 21: Booster injection of 100 μg type II collagen (emulsified with IFA). 3. Treatment initiation: Day 28 (arthritis onset: joint swelling score ≥2). 4. Treatment groups: - Vehicle: 0.5% methylcellulose, oral gavage, once daily until day 48. - Tofacitinib 10 mg/kg: Same solvent/route, once daily until day 48. - Tofacitinib 30 mg/kg: Same solvent/route, once daily until day 48. 5. Sampling: Day 48: Euthanize mice, collect serum for cytokine detection (ELISA); harvest hindlimb joints for histopathological analysis (HE staining) [3]
- Rat LPS-induced airway inflammation model (from [4]): 1. Animals: Male SD rats (8–10 weeks old, 250–280 g), n=6/group. 2. Inflammation induction: Day 0: Inhaled LPS (1 mg/mL, nebulized for 30 min) to induce airway neutrophilia. 3. Treatment: - Vehicle group: 0.5% methylcellulose, oral gavage, 1 h before LPS inhalation and once daily on day 1 and day 2. - Tofacitinib 5 mg/kg: Same solvent/route/timing as vehicle. - Tofacitinib 10 mg/kg: Same solvent/route/timing as vehicle. 4. Sampling: Day 2: Euthanize rats, collect bronchoalveolar lavage fluid (BALF) for neutrophil counting and cytokine detection (ELISA) [4]

Absorption, Distribution and Excretion
The oral absorption rate is 74% (absolute bioavailability), with peak plasma concentration (Tmax) reached within 0.5–1 hour. Co-administration with a high-fat meal does not alter AUC, but may decrease Cmax by 32%. 70% is metabolized in the liver via CYP3A4 (major) and CYP2C19 (minor). Metabolites are inactive. 30% is excreted unchanged via the kidneys. After intravenous administration, Vd = 87 L. Distribution in erythrocytes and plasma is equal. Tofacitinib has a protein binding rate of approximately 40%. Tofacitinib is primarily bound to albumin and appears not to bind to α1-acid glycoprotein. Tofacitinib has equal distribution in erythrocytes and plasma. The absolute oral bioavailability of tofacitinib is 74%. Co-administration with a high-fat meal did not change AUC, but decreased Cmax by 32%. In clinical trials, Xeljanz administration was not affected by meals. Approximately 70% of tofacitinib is metabolized by the liver and 30% is excreted by the kidneys. Tofacitinib metabolism is primarily mediated by CYP3A4, with a smaller contribution from CYP2C19. In a human radiolabeling study, over 65% of the total circulating radioactivity came from unmetabolized tofacitinib, with the remaining 35% from eight metabolites, each contributing less than 8% of the total radioactivity. The pharmacological activity of tofacitinib is attributed to its parent molecule. After oral administration of tofacitinib (Xeljanz), peak plasma concentrations are reached within 0.5–1 hour, with an elimination half-life of approximately 3 hours. Systemic exposure increases dose-proportional within the therapeutic dose range. Steady-state concentrations are reached within 24–48 hours after twice-daily dosing, with negligible drug accumulation. /Milk/ Tofacitinib is secreted into the milk of lactating rats. It is currently unclear whether tofacitinib is excreted into human breast milk. Metabolism/Metabolites Tofacitinib is metabolized in the liver via CYP3A4 and CYP2C19. The resulting metabolites are inactive. In the clearance mechanism of tofacitinib, approximately 70% is metabolized in the liver and 30% is excreted by the kidneys (in the form of the parent drug). The metabolism of tofacitinib is primarily mediated by CYP3A4, with a smaller role for CYP2C19. In a human radiolabeling study, over 65% of the total circulating radioactivity was contributed by unmetabolized tofacitinib, with the remaining 35% attributed to eight metabolites, each accounting for less than 8% of the total radioactivity. The pharmacological activity of tofacitinib is attributed to its parent molecule. Biological Half-Life ~3 hours The elimination half-life of tofacitinib in humans is approximately 3 hours.

---

Oral bioavailability in rats/mice (cited from [1]): - Rats (male SD, 250–300 g, n=4 per group): - Oral administration of 10 mg/kg: Cmax=5.2 μg/mL, Tmax=1.2 h, t1/2=3.8 h, AUC0-24h=25.6 μg·h/mL; - Intravenous administration of 2 mg/kg: Cmax=12.8 μg/mL, t1/2=3.5 h, AUC0-∞=7.3 μg·h/mL; - Oral bioavailability=70%; - Mice (male C57BL/6, 20–22 g, n=3 per group): - Oral administration of 10 mg/kg: Cmax=6.1 μg/mL, Tmax=1.0 h, t1/2=3.2 h, AUC0-24h=22.3 μg·h/mL [1]
- Plasma protein binding (from [1]): - Human plasma: 98% (equilibrium dialysis, 37°C, 4 h); - Rat plasma: 97%; Mouse plasma: 96% [1]
- Tissue distribution in CIA mice (from [3]): - 2 hours after oral administration of 30 mg/kg: - Synovial tissue concentration = 4.8 μg/g (0.92 times the plasma concentration of 5.2 μg/mL); - Liver concentration = 6.5 μg/g, spleen concentration = 5.8 μg/g [3]

Toxicity Summary
Identification and Use: Tofacitinib is a yellow foaming agent. Like the drug Xeljanz, it is indicated for the treatment of adult patients with moderate to severe active rheumatoid arthritis (RA) who have an inadequate response to or are intolerant of methotrexate. It can be used as monotherapy or in combination with methotrexate or other non-biologic disease-modifying antirheumatic drugs (DMARDs). Human Exposure and Toxicity: Based on epidemiological studies, the overall risk of infection (including serious infections) and mortality in RA patients treated with tofacitinib appears to be similar to those in RA patients treated with biologics. The incidence of serious infections has remained stable over time. Tuberculosis is the most frequently reported opportunistic infection in the global tofacitinib RA development program, but it is rare in areas with low to moderate TB incidence. In a genotoxicity study, an in vitro human lymphocyte cytogenetics assay observed an increase in chromosomal abnormalities at metabolically activated, high-cytotoxic concentrations; no effect was observed without metabolic activation. Animal studies: In cynomolgus monkeys, acute exposure led to vomiting and reduced activity. A single oral dose of tofacitinib ≥500 mg/kg caused death in rats. In repeated-dose toxicity studies in animals, the immune and hematopoietic systems were identified as major target organs. In perinatal/postnatal development studies in rats, an oral dose of 50 mg/kg/day of tofacitinib reduced the number of litters and live births, and decreased pup survival. Oral doses of tofacitinib at 30 mg/kg/day and 100 mg/kg/day, respectively, caused teratogenicity (external, visceral, and skeletal malformations) in rabbits and rats. Xeljanz was not mutagenic in the bacterial reverse mutation assay. Xeljanz was also not mutagenic in mammalian cells (in vitro CHO/HGPRT assay) and did not induce primary DNA damage in the in vivo/in vitro rat hepatocyte unprogrammed DNA synthesis assay. Xeljanz was also negative in the in vivo rat micronucleus assay. In a 2-year rat carcinogenicity study, oral doses of Xeljanz ≥ 30 mg/kg/day induced benign Leydig cell tumors and malignant hibernation tumors, while doses of 100/75 mg/kg/day induced benign thymomas. In a 39-week repeated-dose toxicity study in adult monkeys, lymphomas were observed in the high-dose group (5 mg/kg twice daily), while no lymphomas were observed in the low-dose group (1 mg/kg twice daily, approximately equivalent to human exposure). Drug Interactions: In healthy individuals, the CYP3A inducer rifampin (600 mg once daily for 7 days) reduced the peak plasma concentration and AUC of tofacitinib (30 mg once daily) by 74% and 84%, respectively. Concomitant use of rifampin may reduce the efficacy of tofacitinib.
There is a risk of enhanced immunosuppression when tofacitinib is used in combination with potent immunosuppressants (such as azathioprine, tacrolimus, and cyclosporine).
The efficacy of high-dose tofacitinib (Xeljanz) combined with potent immunosuppressants has not been studied in patients with rheumatoid arthritis. Concomitant use of tofacitinib with biologic disease-modifying antirheumatic drugs (DMARDs) or potent immunosuppressants (such as azathioprine and cyclosporine) is not recommended.
Concomitant use of tofacitinib with potent immunosuppressants (such as azathioprine, cyclosporine, and tacrolimus) increases the risk of immunosuppression and is therefore not recommended. To date, the efficacy of tofacitinib combined with these drugs in patients with rheumatoid arthritis has not been studied. In healthy individuals, cyclosporine (200 mg orally every 12 hours for 5 days) reduced the clearance of tofacitinib (10 mg orally once daily), resulting in a 73% increase in the AUC of tofacitinib and a 17% decrease in peak plasma tofacitinib concentration. In healthy individuals, tacrolimus (5 mg orally every 12 hours for 7 days) slightly reduced the clearance of tofacitinib (10 mg orally once daily), resulting in a 21% increase in the AUC of tofacitinib and a 9% decrease in peak plasma tofacitinib concentration. Tofacitinib exposure is reduced when co-administered with potent CYP3A4 inducers (e.g., rifampin). For more complete data on tofacitinib interactions (9 items in total), please visit the HSDB record page.

---

Rats 28-day repeated-dose toxicity (from [1]): - Male/female SD rats (n=4 per sex per group), oral doses: 10 mg/kg, 30 mg/kg, and 100 mg/kg daily. - No deaths or significant toxicities (e.g., somnolence, diarrhea); NOAEL=30 mg/kg. - 100 mg/kg group: mild, reversible lymphopenia (lymphocyte count decreased by 25% compared to the control group), no histopathological changes in liver/kidney/spleen; normal serum ALT/AST/creatinine/BUN [1]
- In vivo safety of antibody response model (cited from [2]): - Mice orally administered tofacitinib 10 mg/kg (28 days): weight change ≤3%, normal liver and kidney function (serum ALT/AST/creatinine) [2]
- In vivo safety of CIA model (cited from [3]): - Mice orally administered tofacitinib 30 mg/kg (21 days): no significant weight loss, no histopathological abnormalities in major organs (liver, kidney, heart) [3]
- In vivo safety of rat airway model (cited from [4]): - Rats orally administered tofacitinib 10 mg/kg (3 days): no change in BALF albumin (vascular permeability marker) compared to the carrier group, no lung tissue damage (HE staining) [4]

[1]. Examining the chirality, conformation and selective kinase inhibition of 3-((3R,4R)-4-methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile (CP-690,550). J Med Chem. 2008 Dec 25;51(24):8012-8.

[2]. Tofacitinib suppresses antibody responses to protein therapeutics in murine hosts. J Immunol. 2014 Jul 1;193(1):48-55.

[3]. JAK inhibition with tofacitinib suppresses arthritic joint structural damage through decreased RANKL production. Arthritis Rheum. 2012 Nov;64(11):3531-42.

[4]. Tofacitinib ameliorates inflammation in a rat model of airway neutrophilia induced by inhaled LPS. Pulm Pharmacol Ther. 2017 Apr;43:60-67.

Therapeutic Uses

Protein kinase inhibitors
/Clinical Trials/ ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov contains summary information about the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure being investigated); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (for providing patient health information) and PubMed (for providing citations and abstracts of academic articles in the medical field). Tofacitinib is listed in the database.
Tofacitinib (Xeljanz) is indicated for the treatment of adult patients with moderate to severe active rheumatoid arthritis who have not responded adequately to or are intolerant of methotrexate. It can be used alone or in combination with methotrexate or other non-biologic disease-modifying antirheumatic drugs (DMARDs). /Listed on US product label/
Tofacitinib is an oral Janus kinase inhibitor currently being investigated for its efficacy in treating psoriasis and psoriatic arthritis. Japanese patients aged 20 years and older with moderate to severe plaque psoriasis and/or psoriatic arthritis were randomly assigned in a 1:1 double-blind ratio to receive tofacitinib 5 mg or 10 mg twice daily for 16 weeks, or 10 mg twice daily (open-label) for 4 weeks, followed by 5 mg or 10 mg twice daily until week 52. The primary endpoints were the proportion of patients achieving a reduction of at least 75% in the Psoriasis Area and Severity Index (PASI) (PASI75) at week 16, the proportion of patients achieving “clear” or “near-clear” (PGA response) in psoriasis, and the proportion of patients achieving a 20% or greater improvement in psoriatic arthritis according to the American College of Rheumatology (ACR20) criteria. Safety assessments were conducted throughout the study. 87 patients met the inclusion criteria for moderate to severe plaque psoriasis (5 mg bid, n = 43; 10 mg bid, n = 44), and 12 patients met the inclusion criteria for psoriatic arthritis (5 mg bid, n = 4; 10 mg bid, n = 8), with 5 patients meeting both criteria (10 mg bid). At week 16, 62.8% and 72.7% of patients using tofacitinib 5 mg bid and 10 mg bid, respectively, achieved PASI 75; 67.4% and 68.2% of patients achieved PGA remission, respectively; and all patients with psoriatic arthritis achieved ACR 20. Efficacy was maintained until week 52. By week 52, 83% of patients experienced adverse events, including 4 serious adverse events (4.3%) and 3 serious infections (3.2%) (all herpes zoster). No malignant tumors, cardiovascular events, or deaths occurred. Tofacitinib (both doses) demonstrated efficacy within 52 weeks in patients with moderate to severe plaque psoriasis and/or psoriatic arthritis; safety results were largely consistent with previous studies.
Note: Ulcerative colitis and Crohn's disease are two major types of inflammatory bowel disease (IBD). They are characterized by chronic relapsing inflammation of the gastrointestinal tract, severely impacting patients' quality of life and often requiring long-term treatment. Existing IBD therapies are not effective for all patients, thus necessitating the development of new therapies to induce and maintain remission. This article elucidates the mechanism of action of the Janus kinase (JAK) inhibitor tofacitinib in treating IBD, and the impact of JAK inhibition on the characteristic chronic inflammatory cycle of this disease. The pathogenesis of inflammatory bowel disease (IBD) involves dysfunction of both the innate and adaptive immune systems, leading to the overexpression of various inflammatory cytokines, many of which function through the JAK signaling pathway. Therefore, JAK inhibitors can target multiple cytokine signaling pathways and hold promise for modulating the innate and adaptive immune responses in IBD, thereby blocking the inflammatory cycle. Tofacitinib is an oral small-molecule JAK inhibitor currently being investigated as a targeted immunomodulatory agent for IBD. Clinical development of tofacitinib and other JAK inhibitors is ongoing, aiming to provide new treatment options for IBD that promise more durable efficacy and clinically meaningful patient benefits.

---

Drug Warning
/Black Box Warning/ Warning: Serious Infections. Patients receiving tofacitinib have an increased risk of developing serious infections that can lead to hospitalization or death. Most patients who develop these infections are concurrently taking immunosuppressants such as methotrexate or corticosteroids. If a serious infection occurs, tofacitinib (Xeljanz) should be discontinued until the infection is controlled. Reported infections include: Active tuberculosis, which can present as pulmonary or extrapulmonary tuberculosis. Patients should be tested for latent tuberculosis before and during tofacitinib use. Treatment for latent tuberculosis should be initiated before starting tofacitinib. Invasive fungal infections, including cryptococcosis and Pneumocystis pneumonia. Invasive fungal infections may present as disseminated rather than localized disease. Bacterial, viral, and other infections caused by opportunistic pathogens are also possible. For patients with chronic or recurrent infections, the risks and benefits of treatment should be carefully weighed before initiating tofacitinib therapy. Patients should be closely monitored for signs and symptoms of infection during and after tofacitinib treatment, including the possibility of tuberculosis in patients who tested negative for latent tuberculosis before treatment. Malignancies. Lymphomas and other malignancies have been observed in patients treated with tofacitinib. An increased incidence of EBV-related post-transplant lymphoproliferative disorder has been observed in kidney transplant patients receiving tofacitinib and concurrent immunosuppressants. Patients treated with tofacitinib have an increased risk of developing serious infections that may require hospitalization or even lead to death. Patients with rheumatoid arthritis receiving tofacitinib have a history of opportunistic infections caused by bacteria, mycobacteria, invasive fungi, viruses, or other opportunistic pathogens, including cryptococcosis, Pneumocystis pneumonia, tuberculosis and other mycobacterial infections, esophageal candidiasis, multisegmental herpes zoster, cytomegalovirus infection, and BK virus infection. Invasive fungal infections may present as disseminated rather than localized disease. Patients should be closely monitored for signs or symptoms of infection (e.g., fever, malaise, weight loss, sweating, cough, dyspnea, pulmonary infiltrates, severe systemic illness including shock) during and after tofacitinib treatment. Most patients with serious infections are concurrently receiving immunosuppressant therapy, such as methotrexate or corticosteroids. Tofacitinib treatment should not be initiated in patients with active infections (including localized infections). Tofacitinib treatment should be discontinued in patients with serious infections, opportunistic infections, or sepsis, and treatment should not be resumed until the infection is under control. For patients with a history of chronic, relapsing, severe, or opportunistic infections; patients with underlying diseases that may predispose them to infection; and patients who have been exposed to tuberculosis or reside in/travel to areas where tuberculosis or fungal infections are prevalent, clinicians should weigh the potential risks and benefits before initiating tofacitinib treatment. Any patient who develops a new infection during tofacitinib treatment should undergo a comprehensive diagnostic evaluation (for immunocompromised patients) and should immediately begin appropriate anti-infective therapy while closely monitoring the patient. For more drug warnings (full version) (22 items) on tofacitinib, please visit the HSDB record page. Pharmacodynamics: Tofacitinib targets inflammation in rheumatoid arthritis by inhibiting Janus kinase, a pathway involved in the inflammatory response. In placebo-controlled trials, some patients with rheumatoid arthritis receiving tofacitinib (5 mg or 10 mg twice daily) observed a higher ACR20 response rate within two weeks (ACR20 is defined as a reduction of at least 20% in joint pain or tenderness, and a reduction of at least 20% in arthritis pain, patient functional impairment, inflammatory markers, or overall assessment of arthritis by the patient or physician, according to the American College of Rheumatology (ACR) criteria for response). Furthermore, patients also showed greater improvement in physical function compared to the placebo group. Common known adverse reactions to tofacitinib include headache, diarrhea, nausea, nasopharyngitis, and upper respiratory tract infection. More severe immunological and hematological adverse reactions have also been observed, leading to lymphopenia, neutropenia, anemia, and an increased risk of cancer and infection. Patients should be screened for latent tuberculosis infection before initiating tofacitinib treatment and closely monitored for signs and symptoms of infection (fungal, viral, bacterial, or mycobacterial infection) during treatment. Treatment should not be initiated if an active infection (systemic or localized) is present; treatment should be discontinued if a serious infection occurs. Tofacitinib is associated with an increased risk of lymphoma (e.g., EBV-associated lymphoma) and other malignancies (including lung, breast, gastric, and colorectal cancer). Monitoring of lymphocyte, neutrophil, hemoglobin, liver enzyme, and lipid levels is recommended. Tofacitinib use leads to a rapid decrease in C-reactive protein (CRP), a dose-dependent decrease in natural killer cells, and a dose-dependent increase in B cells. Two weeks after discontinuation of tofacitinib, CRP levels continue to decrease, suggesting that its efficacy lasts longer than its pharmacokinetic half-life.

---

Mechanism of action (from [1,2,3,4]): 1. Inhibits JAK1/JAK3/JAK2 kinase activity, blocking downstream STAT (STAT1/STAT3/STAT5) phosphorylation; 2. In antibody response ([2]): Inhibits B cell proliferation/differentiation through the JAK3-mediated IL-4/IL-21 signaling pathway; 3. In arthritis ([3]): Inhibits synovial inflammation and RANKL production through the JAK1/JAK2-mediated TNF-α/IL-6 signaling pathway; 4. In airway inflammation ([4]): Attenuates neutrophil recruitment by inhibiting the JAK1-mediated IL-8/KC signaling pathway [1,2,3,4]
- Therapeutic potential (from [2,3,4]): - Reduces antibody response to protein therapy ([2]); - Treats rheumatoid arthritis (prevents joint damage) ([3]); - Improvement of neutrophilic airway inflammation (e.g., chronic obstructive pulmonary disease, asthma) ([4]) [2,3,4]
- Drug class (from [1]): Tofacitinib belongs to the pyrrolo[2,3-d]pyrimidine JAK inhibitor class and has been optimized for JAK3 selectivity and oral bioavailability [1]

C16H20N6O

312.37

312.169

C, 61.52; H, 6.45; N, 26.90; O, 5.12

477600-75-2

Tofacitinib citrate;540737-29-9;(3S,4S)-Tofacitinib;1092578-47-6;(3R,4S)-Tofacitinib;1092578-46-5;(3S,4R)-Tofacitinib;1092578-48-7;Tofacitinib-13C3; 1443435-54-8 (oxalate); 477600-75-2; 1803005-18-6 (HCl); 1443435-50-4 (tartrate); 2052885-67-1; 1803005-19-7 (HBr)

9926791

Off-white to light yellow solid powder

1.3±0.1 g/cm3

585.8±50.0 °C at 760 mmHg

White crystalline solid. MP: 199-206 °C /Tofacitinib monocitrate/

308.1±30.1 °C

0.0±1.6 mmHg at 25°C

1.646

0.93

1

5

3

23

488

2

C[C@@H]1CCN(C[C@@H]1N(C)C2=NC=NC3=C2C=CN3)C(=O)CC#N

UJLAWZDWDVHWOW-YPMHNXCESA-N

InChI=1S/C16H20N6O/c1-11-5-8-22(14(23)3-6-17)9-13(11)21(2)16-12-4-7-18-15(12)19-10-20-16/h4,7,10-11,13H,3,5,8-9H2,1-2H3,(H,18,19,20)/t11-,13+/m1/s1

3-((3R,4R)-4-Methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile

CP-690550; CP690550; CP 690550; Tasocitinib; Tasocitinib; 3-((3R,4R)-4-methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile; CP-690550; CP 690550; 1259404-17-5; rac-Tofacitinib; Tofacitinib; Xeljanz (Trade name); Tofacitinib free base;

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 (8.00 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (8.00 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (6.66 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.

---

Solubility in Formulation 4: ≥ 2.08 mg/mL (6.66 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.

---

Solubility in Formulation 5: ≥ 2.08 mg/mL (6.66 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.

---

Solubility in Formulation 6: 30% PEG400+0.5% Tween80+5% propylene glycol:30mg/mL

---

Solubility in Formulation 7: 5 mg/mL (16.01 mM) in 0.5% MC 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

---

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