JAK1 (IC50 = 43 nM); JAK2 (IC50 = 0.2 μM); JAK3 (IC50 = 2.3 μM); Tyk2 (IC50 = 4.7 μM)

Upadatinib tartrate tetrahydrate has been shown in biochemical testing to be 74 times more selective for JAK-1 than JAK-2 (which is involved in erythropoiesis) and 58 times more selective for JAK-1 than JAK-3 (which is involved in immune surveillance) [1].

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Upadacitinib is JAK1-selective and inhibits cytokines that contribute to the pathology of RA [3]
To characterize the enzymatic activity of Upadacitinib, we assessed potency and selectivity in biochemical assays utilizing recombinant human JAK kinases. The data is summarized in Table 1. Upadacitinib demonstrated activity against JAK1 (0.045 μM) and JAK2 (0.109 μM), with > 40 fold selectivity over JAK3 (2.1 μM) and 100 fold selectivity over TYK2 (4.7 μM) as compared to JAK1. Upadacitinib also demonstrated selectivity across a broad panel of 70+ kinases, with only Rock1 and Rock2 demonstrating IC50 values below 1 μM (Additional file 1: Table S1). To further characterize the mechanism of JAK1 inhibition of upadacitinib, we assessed JAK1 enzyme activity at varying concentrations of ATP. At all concentrations tested, the close agreement of the theoretical and experimental IC50 values confirmed that upadacitinib is an ATP competitive inhibitor (data not shown).

The JAK family selectivity of Upadacitinib was confirmed in cellular assays. Due to the complexity of the cooperative nature of JAK kinases, we employed a set of engineered cell lines to understand the cellular potency and selectivity of upadacitinib on each individual kinase. As shown in Table 1, upadacitinib was > 40 fold selective for JAK1 (0.014 μM) as compared to JAK2 (0.593 μM). Upadacitinib also demonstrated selectivity against JAK3 (~ 130 fold) and TYK2 (~ 190 fold). The potency of upadacitinib was also assessed in physiologically relevant cellular systems. Consistent with the Ba/F3 cellular data, upadacitinib potently inhibited the JAK1 dependent cytokines IL-6, OSM, IL-2, and IFNγ, as measured by inhibition of STAT phosphorylation. This activity was ~ 60 fold more potent than the activity on erythropoietin signaling, a cytokine that depends exclusively upon JAK2 for signal transduction. We next measured inhibition of IL-6 signaling in human whole blood. The IC50 values for upadacitinib were 0.207 μM in the CD3+ T-cell population, and 0.078 μM in the CD14+ monocytic population. The reported IC50 values for tofactinib on IL-6 signaling in human whole blood are 0.367 μM and 0.406 μM for CD3+ T-cells and monocytes, respectively [3].

In a rat arthritic model, upadacitinib tartrate tetrahydrate (0.1–10 mg/kg; oral gavage; twice daily for 10 days) has demonstrated effectiveness [3]. Upadacitinib/ABT-494 is a potent inhibitor of inflammation and bone loss in rat AIA and, compared to Tofacitinib, spares relevant essential physiological processes such as erythropoietin signaling and peripheral NK cell counts at similarly efficacious doses in rats. When dosed orally for 14 days in healthy human subjects ABT-494 did not decrease reticulocyte or NK cell counts at predicted efficacious doses consistent with its pharmacodynamic properties in rats.
Conclusions ABT-494 is a Jak1-selective inhibitor that demonstrates efficacy in rat arthritis models. Preliminary evidence suggests that pharmacodynamic properties of ABT-494 are consistent between those observed in rodent models and in healthy human subjects. Taken together, these encouraging observations support further testing of ABT-494 in RA patients in Phase II randomized placebo controlled trials and indicate it may have increased potential to address patient needs over existing agents.[2]

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Upadacitinib inhibits disease pathology in rat adjuvant induced arthritis [3]
To understand the effect on inflammation and the arthritis phenotype, we tested Upadacitinib in the adjuvant induced arthritis model, an established preclinical model of RA. Orally administered upadacitinib was dosed at first signs of disease on day 7 and resulted in dose and exposure dependent reductions in paw swelling (Fig. 2a). On day 18 post disease induction, paws were harvested and bone destruction was measured by μCT. The normal course of AIA results in significant loss of bone volume that is dose dependently reduced with upadacitinib administration (Fig. 2b). Examples of destruction are shown from vehicle treated animals (Fig. 2c) with significant pitting and bone loss compared with the 10 mg/kg upadacitnib treated animals in which the surface of the bone was protected (Fig. 2d). Histological endpoints were also assessed in this study. Upadacitinib administration improved synovial hypertrophy, inflammation, cartilage damage and bone erosion at the 3 and 10 mg/kg dose groups (data not shown). Similar results were observed in the rat collagen induced arthritis (CIA) model, a second preclinical model of RA (data not shown). Tofacitinib was also tested in the AIA model and demonstrated dose-responsive efficacy, although the exposure-response curve was right shifted compared to upadacitinib (Fig. 2a). Efficacious concentrations were defined as the AUC0–12 drug concentration necessary to achieve 60% inhibition of paw swelling (AUC60). The rationale for using an AUC60 as a point of reference was based on the AUC exposure associated with the clinical dose of 10 mg BID of tofacitinib. This established a translational reference point for further analysis. The total efficacious drug exposure for upadacitinib was calculated to be 83 ng*hr./ml while this exposure was 1205 ng*hr./ml for tofacitinib. The increased in vivo potency of upadacitinib was expected based upon the difference in JAK1 cellular potency compared to tofacitinib.

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Upadacitinib spares reticulocyte deployment and NK cell count depletion relative to efficacy [3]
We applied a variation of a method previously described to determine the relative impact of Upadacitinib and tofacitinib on inhibition of JAK2 dependent Epo receptor function. Naive rats were intravenously challenged with either PBS or 1000 IU of Epo on two consecutive days, and circulating reticulocytes were measured on day 4. Either upadacinitinb or tofactininib were dosed throughout and reticulocytes were quantified by flow cytometry. We also sought to determine the impact of upadacitinib and tofacitinib on common gamma chain signaling (JAK1/JAK3) in the form of circulating NK cell counts, given that these cells rely upon IL-15 for survival. Naïve rats were dosed for 14 days with either upadacitinib or tofacitinib, and circulating CD3-/CD16+/CD56+ NK cells were quantified by flow cytometry. The results of the reticulocyte deployment, circulating NK cellcounts, and AIA efficacy experiments were plotted together to visualize the relative effects in relation to exposure (Fig. 3). Tofactinib decreases circulating NK cell numbers in an exposure dependent manner (AUC60 of 1230 ng*hr./ml) similar to the exposure range observed to inhibit paw swelling (AUC60 = 1205 ng*hr./ml). Tofacitinib reduced reticulocyte deployment in an exposure dependent manner reaching a maximal inhibition of > 40% at the highest concentration tested (Fig. 3a). Upadacitinib decreases circulating NK cell numbers in an exposure dependent manner with an AUC60 = 480 ng*hr./ml, ~ 5 fold less potent than the concentration of drug required to inhibit paw swelling (AUC60 = 83 ng*hr./ml). Reticulocyte deployment was also dose-responsively reduced and reached a maximal inhibition of ~ 40% (Fig. 3b). At the clinical AUC exposures associated with 10 mg BID of tofacitinib, reduction in paw swelling was ~ 60% and there is a clear overlap with NK cell depletion (Fig. 3a). Utilizing exposures for 6 mg BID and 12 mg BID clinical doses of upadacitinib, reduction in paw swelling was > 90%, with a distinct separation from NK cell depletion (Fig. 3b).

The reticulocyte (Fig. 4a) and NK cell (Fig. 4b) data were replotted versus % inhibition of paw swelling in the AIA model to directly compare the inhibitory effects of tofacitinib and Upadacitinib as a function of disease efficacy. The relative impact on reticulocytes is similar between tofacitinib and upadacitinib at the lower efficacious range, but at the higher efficacious range (> 60% of paw swelling), the differential effect becomes much more pronounced (Fig. 4a). Likewise, there is a clear differential effect on circulating NK cell counts. At AUC60, there is a 70% decrease in circulating NK cells upon tofacitinib treatment, while upadacitinib treatment results in a 25% decrease (Fig. 4b).

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Upadacitinib spares common gamma chain signaling relative to IL-6 signaling in healthy volunteers [3]
To confirm the preclinical selectivity of Upadacitinib in a clinical setting, ex vivo cytokine stimulation assays were performed in the whole blood of healthy volunteers dosed with 1, 3, 12, 24, 36, or 48 mg of upadacitinib, or with 5 mg of tofacitinib. At 1 h post dose, blood was drawn and stimulated with IL-6 or IL-7 to assess the impact of upadacitinib on these signaling pathways. Inhibition of downstream STAT phosphorylation (STAT3 and STAT5) was assessed by flow cytometry (Fig. 5). JAK1 mediated IL-6 induced pSTAT3 was inhibited ~ 50% at the 3 mg dose of upadacitinib, equivalent to the level of inhibition seen with 5 mg of tofacitinib. Increasing doses of upadacitinib demonstrated concomitant increases in pSTAT3 inhibition before reaching maximal inhibition at 36 mg. For the purpose of evaluating JAK1/3 potency in vivo, activity against common γ chain signaling was assessed using IL-7 driven pSTAT5. In this case, 12 mg of upadacitinib was necessary to inhibit pSTAT5 to the same degree as 5 mg tofacitinib (~ 70%).

Enzyme potency and selectivity assays [3]
Active recombinant human catalytic domains of JAK1 (aa 845–1142) and JAK3 (aa 811–1103) were prepared in house and expressed in SF9 cells as a glutathione s transferase (GST) fusion and purified by glutathione affinity chromatography. Active human TYK2 (aa880–1185) was purified in house and contains an N-terminal histidine-tag and C-terminal FLAG tag. It was purified by immobilized metal ion affinity chromatography. Recombinant kinase domain of JAK2 was purchased from xxx. Peptides Biotin-TYR2 (Biotin-(Ahx)-AEEEYFFLFA-amide) and Biotin-TYR1 (Biotin-(Ahx)-GAEEEIYAAFFA-COOH were used. Reactions were carried out at 100 μM ATP in the presence of inhibitor and 2 μM peptide. For competition assays, the JAK1 IC50 of Upadacitinib was determined in the presence of varying amounts of ATP (0.01-1 mM) equal to and greater than the ATP Km for the kinase. ATP competitiveness was evaluated using the Cheng-Prusoff equation. Inhibitors that are ATP competitive will display changes in the IC50 consistent with the theoretical values derived from the Cheng-Prusoff equation at varying ATP concentrations.

Ba/F3 cellular potency and selectivity assays [3]
The TEL-JAK2, TEL-JAK3, TEL-TYK2, and BCR-JAK1 Ba/F3 engineered cell lines were purchased from Advanced Cellular Dynamics. Cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 1× penicillin-streptomycin-glutamine and 0.5 μg/ml puromycin.

For measurement of phosphorylation of signal transducer and activator of transcription 5 (pSTAT5), cells were washed and resuspended in Hank’s balanced salt solution at a density of 2 X 107 cells/mL. Five microliters of cell suspension were added to a 384-well, low-volume, white-walled polystyrene plate that contained 5 μL of compound (in a 11 point [1:3] titration series). Cells were incubated with compound (final DMSO concentration 0.5%) for 30 min at 37 °C before proceeding with pSTAT5 detection. pSTAT5 was measured with the SureFire pSTAT5 Assay kit per standard manufacturer’s protocol, with the exception of an overnight incubation following addition of donor beads before detection on the EnVision.

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Cytokine potency assays [3]
IL-6 and Oncostatin M (OSM) induced STAT3 phosphorylation was assessed in the human erythroleukemia TF-1 cell line. Erythropoietin-induced STAT5 phosphorylation was assessed in the human UT-7 cell line. IL-2 and IL-15 induced STAT5 phosphorylation was assessed in activated human T-cells. Detection of phosphorylated STATs was accomplished with the SureFire pSTAT5 or pSTAT3 Assay kit per standard manufacturer’s protocol, with the exception of an overnight incubation following addition of donor beads before detection on the EnVision. IFNγ induced STAT1 phosphorylation was assessed in the CD14+ monocyte population in human PBMC by flow cytometry. CD14 BV421 and STAT1 PE (pY705) were used. IL-4 and IL-13 induced STAT6 phosphorylation and IL-31 induced STAT3 phosphorylation were assessed in adult human epithelial keratinocytes by flow cytometry. STAT6 PE (pY641) and STAT3 PE (Y705) were used.

Animal/Disease Models: Female Lewis rat (rat adjuvant-induced arthritis model) [3]
Doses: 0.1, 0.3, 1, 3, 10 mg/kg
Route of Administration: po (oral gavage); twice a day for 10 days
Experimental Results: Inhibition of disease pathology in adjuvant-induced arthritis in rats.

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The efficacy and selectivity of Upadacitinib/ABT-494 were tested in a battery of relevant cellular and in vivo pharmacology assays including bone marrow colony formation, adjuvant induced arthritis (AIA), erythropoietin induced reticulocyte deployment and NK/NKT cell suppression. The potency of ABT-494 in a variety of complementary pharmacodynamic assays was also assessed at multiple dosages in healthy human subjects administered orally for 14 days. [2]

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Rat adjuvant-induced arthritis (AIA) model [3]
Arthritis was induced in female Lewis rats (weight, 125 – 150 g) by a single intradermal injection of 0.1 mL of microbacterium tuberculosis emulsion into the right hind footpad (Day 0). Rats were dosed as indicated orally by gavage twice a day (BID) for 10 days (Day7 – Day17) post immunization with either vehicle or study drug. To evaluate the severity of arthritis, paw swelling was evaluated with a water displacement plethysmograph every other day up to Day 17. On Day 17, all rats were exsanguinated by cardiac puncture under isolfuorane anesthesia. Left rear paws were scanned using a μCT. Bone volume and density were determined in a 360 μm vertical section encompassing the tarsal section of the paw.

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Reticulocyte deployment assaysNaïve male Lewis rats were injected intravenously with either PBS or 1000 IU of epoetin α for two consecutive days. Reticulocytes were measured on day 4 by flow cytometry using thiozole orange as a dye as previously described [13]. Dose responses of either Upadacitinib or tofacitinib were dosed 30 min prior to the first Epo injection and then once every 12 h subsequently for 3 days.

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NK cell analysis [3]
Sprague Dawley rats were dosed orally with either Upadacitinib or tofacitinib at doses indicated for 14 days. Blood was collected and stained using BD MultiTest IMK kit per manufacturer’s instructions. NK cell numbers were determined by using FlowJo analysis software and by examining the CD3−/CD16+/CD56+ population. The number of cells/μL was calculated by using the following equation: (# events in cell population/# of events in absolute bead count region) × (# beads per test/test volume), with the value beads per test indicated on the BD Trucount tube label.

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Pharmacokinetic/pharmacodynamics modeling [3]
A direct maximum enhancement model was the most predictive for defining the efficacious concentration range and human efficacious dose. Efficacious area under the concentration-versus-time curve (AUC) was based on paw swelling on the last day of the study plotted against the cumulative plasma concentration of Upadacitinib or tofacitinib over 12 h (AUC0–12).

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Clinical ex vivo stimulation assays [3]
For each subject, blood was collected by venipuncture into 2 mL sodium heparin tubes at 0, 1, 6, and 12 h post Upadacitinib or tofacitinib dose. Recombinant human IL-6 (400 ng/ml), or IL-7 (400 ng/ml), was added to blood and incubated for 10 min at 37°C. Surface antibodies were added (CD14-APC, CD3-fluorescein isothiocyanate [FITC]) and incubated on ice for an additional 20 min. Samples were lysed and incubated for 10 min at 37°C. Samples were washed and stored at − 70°C. For intracellular staining, samples were thawed, washed, and resuspended in BD Perm buffer III on ice for 30 min. Samples were washed and stained with pSTAT5-PE or pSTAT3-PE for 60 min at room temperature and then analyzed immediately on a FACSCalibur. Geometric means were determined using FlowJo analysis software. Percent inhibition of relevant STAT phosphorylation was calculated as follows: (1-(Induction of pSTAT at 1 h – baseline pSTAT at 0 h) / (Induction of pSTAT at 0 h – baseline pSTAT at 0 h)*100.

Absorption, Distribution and Excretion
Within the therapeutic dose range, utpatinib exhibits dose-proportional pharmacokinetic characteristics. The median time to peak concentration (Tmax) after oral administration is 2 to 4 hours. With once-daily or multiple-dose administration, steady-state plasma concentrations of utpatinib are reached within 4 days with minimal accumulation. Food intake has no clinically relevant effect on the AUC, Cmax, and Cmin of utpatinib in the extended-release formulation. Following a single dose of the radiolabeled immediate-release formulation, approximately 53% of the total dose is excreted in feces, of which 38% is unmetabolized parent drug. Approximately 43% of the total dose is excreted in urine, of which 24% is unmetabolized parent drug. Approximately 34% of the total utpatinib dose is excreted as metabolites. For a rheumatoid arthritis patient weighing 74 kg, the estimated volume of distribution of utpatinib after oral administration of the extended-release formulation is 224 L. In a pharmacokinetic study involving healthy volunteers, the steady-state volume of distribution was 294 L after administration of the extended-release formulation. Upatinib exhibits similar distribution between plasma and blood cell components, with a plasma/drug ratio of 1.0. The apparent oral clearance of utadacitinib after administration of the extended-release formulation in healthy volunteers was 53.7 L/h. Metabolites/Metabolites: Upatinib is primarily metabolized via CYP3A4; however, it is not a sensitive substrate for CYP3A4. It is also metabolized to a small extent by CYP2D6. In a human radiolabeling study, approximately 79% of the total plasma radioactivity was derived from the parent drug, and approximately 13% of the total plasma radioactivity was derived from the major metabolite produced by glucuronidation following monooxidation. There are currently no known active metabolites of upadacitinib. Biological Half-Life: The mean terminal elimination half-life of upadacitinib after administration of the extended-release formulation is 8 to 14 hours. In clinical trials, approximately 90% of upadacitinib was cleared from systemic circulation within 24 hours after administration.

Hepatotoxicity
Liver function abnormalities were common, but usually mild, in premarket clinical trials of upadacitinib in patients with rheumatoid arthritis. Up to 11% of patients receiving upadacitinib experienced elevated ALT levels, compared to 7% in the placebo group, but the proportion of patients with ALT levels exceeding three times the upper limit of normal was 2% or lower. Similar rates of ALT elevation were also observed in patients receiving methotrexate or the biologic DMARD. In these trials involving over 3,000 patients, no clinically significant liver injury, severe liver injury, or liver-related death were reported. Similarly, other JAK inhibitors, such as tofacitinib and baricitinib, often cause mild elevations in serum transaminases during treatment, but no clinically significant liver injury has been reported. Therefore, these drugs are suspected but not yet proven to cause liver injury. Furthermore, long-term use of utpatinib and other Janus kinase inhibitors has been associated with rare cases of hepatitis B virus reactivation, which can be severe and even fatal. Following discontinuation of JAK inhibitors, hepatitis B virus reactivation may occur when immune reconstitution leads to an enhanced immune response to viral replication, potentially resulting in clinical symptoms.
Probability Score: D (Possible, but rare, cause of clinically symptomatic liver injury (including hepatitis B virus reactivation) in susceptible patients).
Pregnancy and Lactation Use
◉ Overview of Lactation Use
There is currently no information regarding the use of utpatinib during lactation. Most sources recommend that mothers taking utpatinib should not breastfeed. Alternative medications are preferable, especially when breastfeeding newborns or premature infants. The manufacturer recommends discontinuing breastfeeding within 6 days of the last dose.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.
Protein Binding
utpatinib binds to human plasma proteins in 52% of its composition.

[1]. Recent Progress in JAK Inhibitors for the Treatment of Rheumatoid Arthritis. BioDrugs. 2016 Oct;30(5):407-419.

[2]. THU0127 Pharmacodynamics of A Novel JAK1 Selective Inhibitor in Rat Arthritis and Anemia Models and in Healthy Human Subjects. doi 10.1136/annrheumdis-2014-eular.3823.

[3]. In vitro and in vivo characterization of the JAK1 selectivity of upadacitinib (ABT-494). BMC Rheumatol. 2018 Aug 28;2:23.

Pharmacodynamics
Upatinib is a disease-modifying antirheumatic drug (DMARD) that works by inhibiting Janus kinase (JAK). JAK is an important mediator of downstream cell signaling of pro-inflammatory cytokines. These pro-inflammatory cytokines are believed to play a role in many autoimmune inflammatory diseases, such as rheumatoid arthritis. In clinical trials, utpatinib reduced the activity of pro-inflammatory interleukins, transiently increased lymphocyte levels, and slightly decreased immunoglobulin levels from baseline. Upatinib is an orally administered selective inhibitor of Janus kinase (JAK)1 and is a disease-modifying antirheumatic drug (DMARD) used to treat rheumatoid arthritis to slow disease progression. Rheumatoid arthritis is a chronic autoimmune inflammatory disease affecting peripheral joints. Rheumatoid arthritis is characterized by synovial inflammation and hyperplasia, autoantibody production, cartilage damage and bone destruction, and can lead to a variety of complications. Despite the availability of various treatments, up to 40% of patients do not respond to existing therapies, including biologics. The etiology of the disease remains largely unclear; however, JAK has been identified as a driver of immune-mediated diseases, making it a potential therapeutic target for rheumatoid arthritis. To reduce dose-related toxicities (as seen with some pan-JAK inhibitors) without significantly impacting efficacy, researchers have developed more selective JAK1 inhibitors, such as upadacitinib and filgotinib. Upadacitinib was approved by the FDA in August 2019 for the treatment of active rheumatoid arthritis, psoriatic arthritis, atopic dermatitis, ulcerative colitis, and ankylosing spondylitis. In December 2019, it also received approval from the European Commission and Health Canada. Upadacitinib is marketed under the brand name RINVOQ for oral administration. Upadacitinib is a Janus kinase inhibitor. Its mechanism of action is as a Janus kinase inhibitor. Upadacitinib is an oral selective Janus-associated kinase 1 (JAK-1) inhibitor used to treat moderate to severe rheumatoid arthritis. Elevated serum enzyme levels during utadacitinib treatment are rare, but have not been associated with clinically significant cases of acute liver injury, although it may pose a risk of hepatitis B virus reactivation in susceptible patients. Upatinib is a small molecule drug, with clinical trials up to Phase IV (covering all indications), and was first approved in 2019 for the treatment of rheumatoid arthritis, with 12 investigational indications. The drug has been placed on a black box warning list by the U.S. Food and Drug Administration (FDA). Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovial inflammation and joint destruction. The advent of biologic disease-modifying antirheumatic drugs (DMARDs) has greatly improved the treatment of RA. However, these biologics require intravenous or subcutaneous injection, and some patients do not respond to biologic DMARDs or lose initial efficacy. Multiple cytokines and cell surface molecules bind to receptors on the cell surface, thereby activating various cellular signaling pathways, including the phosphorylation of kinase proteins. Among these kinases, Janus kinases (JAK), belonging to the non-receptor tyrosine kinase family, play a crucial role in the pathogenesis of rheumatoid arthritis (RA). Several JAK inhibitors have been developed as novel therapies for RA patients. These orally administered synthetic DMARDs inhibit JAK1, 2, and 3. One JAK inhibitor, tofacitinib, has been approved in several countries. Phase III clinical trials using the JAK1/2 inhibitor baricitinib have shown promising efficacy and a good safety profile. Both drugs are effective in patients who have not responded well to biologics and synthetic DMARDs. Furthermore, Phase III clinical trials using the specific JAK1 inhibitors fioglotinib and utpatinib/ABT-494 are currently underway. JAK inhibitors represent novel therapies for rheumatoid arthritis (RA), but further research is needed to determine their risk-benefit ratio and to screen patients best suited for this treatment. [1] Anti-cytokine therapy has become a major means of treating the symptoms of rheumatoid arthritis (RA) and can stop disease progression. Despite the abundance of treatment options, many RA patients still fail to significantly reduce their disease activity. Recent clinical studies have shown that JAK kinase blockade can effectively control the disease and achieve remission in some cases. However, these first-generation JAK inhibitors have failed to achieve the expected results due to dose-limiting tolerability and safety issues. ABT-494 is a second-generation JAK kinase inhibitor with high selectivity for JAK1, thereby minimizing the potential side effects associated with JAK2 and JAK3 inhibition. This article describes preclinical and early clinical data that suggest that ABT-494 has the potential to address some of the unmet medical needs of patients with rheumatoid arthritis (RA). [2]

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Background: Anti-cytokine therapy, such as adalimumab, tocilizumab and the small molecule JAK inhibitor tofacitinib, has demonstrated that cytokines and their downstream signaling pathways play an important role in the pathogenesis of rheumatoid arthritis. Tofacitinib is a pan-JAK inhibitor and the first JAK inhibitor approved for the treatment of rheumatoid arthritis (RA), and it has been shown to effectively control the disease. However, in a phase II dose-finding study, tofacitinib exhibited dose-limiting tolerability and safety issues, such as anemia. Upadacitinib (ABT-494) is a selective JAK1 inhibitor designed to test the hypothesis that higher selectivity for JAK1 than other JAK family members would result in a better benefit-risk ratio. Upadacitinib selectively targets JAK1-dependent disease drivers such as IL-6 and IFNγ while reducing effects on reticulocytes and natural killer (NK) cells, which may be one reason for tofacitinib's tolerability issues. Methods: The selective JAK1 inhibitor Upadacitinib was designed based on structural hypotheses. JAK family selectivity was determined through in vitro experiments, including biochemical assessments, genetically engineered cell lines, and cytokine stimulation. The in vivo selectivity of upadacitinib and tofacitinib was determined by their efficacy in an adjuvant-induced rat model of arthritis, their activity on reticulocyte deployment, and their effects on circulating NK cells. This study evaluated the conversion of preclinical JAK1 selectivity in healthy volunteers using in vitro stimulation with JAK-dependent cytokines. Results: This study reveals the structural basis of upadacitinib's JAK1 selectivity, its in vitro JAK family selectivity profile, and subsequent in vivo physiological effects. In cellular experiments, upadacitinib exhibited approximately 60-fold higher selectivity for JAK1 than for JAK2 and over 100-fold higher selectivity for JAK3. While both upadacitinib and tofacitinib demonstrated efficacy in a rat model of arthritis, the higher JAK1 selectivity of upadacitinib resulted in a reduced effect on reticulocyte activation and NK cell clearance relative to efficacy. In vitro pharmacodynamic data from Phase I healthy volunteers confirmed the JAK1 selectivity of upadacitinib in a clinical setting.
Conclusion: The data in this study highlight the JAK1 selectivity of Upadacitinib and support its potential as an effective treatment for RA, with the potential to improve the benefit-risk ratio.

C21H25F3N6O7

530.461

C, 47.55; H, 4.75; F, 10.74; N, 15.84; O, 21.11

1607431-21-9

Upadacitinib;1310726-60-3;Upadacitinib-15N,d2

127263217

Typically exists as solid at room temperature

10

16

6

41

695

4

O=C(N1C[C@@H](CC)[C@@H](C2=CN=C3C=NC(NC=C4)=C4N32)C1)NCC(F)(F)F.O=C(O)[C@H](O)[C@@H](O)C(O)=O

WQDBPGWQDBPVQZ-NBCXFSEXSA-N

InChI=1S/C17H19F3N6O.C4H6O6/c1-2-10-7-25(16(27)24-9-17(18,19)20)8-11(10)13-5-22-14-6-23-15-12(26(13)14)3-4-21-15;5-1(3(7)8)2(6)4(9)10/h3-6,10-11,21H,2,7-9H2,1H3,(H,24,27);1-2,5-6H,(H,7,8)(H,9,10)/t10-,11+;1-,2-/m11/s1

(3S,4R)-3-ethyl-4-(3H-imidazo(1,2-a)pyrrolo(2,3-e)pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine-1-carboxamide (2R,3R)-2,3-dihydroxybutanedioate

ABT-494 tartrate; ABT 494 tartrate; Upadacitinib tartrate; 1607431-21-9; UNII-7KCW9IQM02; 7KCW9IQM02; Upadacitinib tartrate [USAN]; Upadacitinib tartrate (USAN); ABT-494 TARTRATE TETRAHYDRATE; UPADACITINIB TARTRATE TETRAHYDRATE; ABT494; rinvoq

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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