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

Upadacitinib exhibited 74-fold better selectivity for JAK-1 compared to JAK-2, which is involved in erythropoiesis, and 58-fold higher selectivity for JAK-1 compared to JAK-3, which is involved in immune surveillance, in biochemical experiments. [1]. Because of upadacitinib's superior selectivity for JAK-1 over JAK-2 and JAK-3, patients' benefit-risk profiles may be improved across the spectrum of RA [2].

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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 (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 assays Naïve 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
Upadacitinib displays a dose-proportional pharmacokinetic profile over the therapeutic dose range. Following oral administration, the median time to reach Cmax (Tmax) ranges from 2 to 4 hours. The steady-state plasma concentrations of upadacitinib are reached within 4 days following multiple once-daily administrations, with minimal accumulation. Food intake has no clinically relevant effect on the AUC, Cmax, and Cmin of upadacitinib from the extended-release formulation.
Following administration of a single radio-labelled dose from the immediate-release formulation, approximately 53% of the total dose was excreted in the feces where 38% of the excreted dose was an unchanged parent drug. About 43% of the total dose was excreted in the urine, where 24% of that dose was in the unchanged parent drug form. Approximately 34% of the total dose of upadacitinib dose was excreted as metabolites.
The volume of distribution of upadacitinib in a patient with rheumatoid arthritis and a body weight of 74 kg is estimated to be 224 L following oral administration of an extended-release formula. In a pharmacokinetic study consisting of healthy volunteers receiving the extended-release formulation, the steady-state volume of distribution was 294 L. Upadacitinib partitions similarly between plasma and blood cellular components with a blood to plasma ratio of 1.0.
The apparent oral clearance of upadacitinib in healthy volunteers receiving the extended-release formulation was 53.7 L/h.

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Metabolism / Metabolites
Upadacitinib predominantly undergoes CYP3A4-mediated metabolism; however, upadacitinib is a nonsensitive substrate of CYP3A4. It is also metabolized by CYP2D6 to a lesser extent. In a human radio-labelled study, about 79% of the total plasma radioactivity accounted for the parent drug, and about 13% of the total plasma radioactivity accounted for the main metabolite produced from mono-oxidation, followed by glucuronidation. There are no known active metabolites of upadacitinib.

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Biological Half-Life
The mean terminal elimination half-life of upadacitinib ranged from 8 to 14 hours following administration of the extended-release formulation. In clinical trials, approximately 90% of upadacitinib in the systemic circulation was eliminated within 24 hours of dosing.

Hepatotoxicity
In the prelicensure clinical trials of upadacitinib in patients with rheumatoid arthritis, liver test abnormalities were frequent although usually mild. Some degree of ALT elevation arose in up to 11% of upadacitinib treated patients compared to 7% treated with placebo, but were above 3 times the upper limit of normal (ULN) in 2% or less. Furthermore, similar rates of ALT elevations arose in patients treated with methotrexate or biologic DMARDs. In these trials that enrolled over 3000 patients, there were no reports of clinically apparent liver injury, serious cases of liver injury or liver-related deaths. Similarly, other JAK inhibitors such as tofacitinib and baricitinib have been associated with frequent minor serum aminotransferase elevations during treatment, but episodes of clinically apparent liver injury have not been reported. Thus, this class of agents is suspected but not proven capable of causing liver injury.
In addition, long term treatment with upadacitinib and other Janus kinase inhibitors has been linked to rare instances of reactivation of hepatitis B that can be severe and has been linked to fatal outcomes. Reactivation can become clinically apparent after the JAK inhibitor is discontinued, when immune restoration results in an immunologic response to the heightened viral replication.
Likelihood score: D (possible, rare cause of clinically apparent liver injury including reactivation of hepatitis B in susceptible patients).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of upadacitinib during breastfeeding. Most sources recommend that mothers not breastfeed while taking upadacitinib. An alternate drug is preferred, especially while nursing a newborn or preterm infant. The manufacturer recommends that breastfeeding be withheld for 6 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
Upadacitinib is 52% bound to human plasma proteins.

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Toxicity Summary
Upadacitinib was found to be teratogenic in animal studies, although no human studies during pregnancy have been reported. Use during pregnancy is not recommended. Contraception is advised during treatment and for 4 weeks after completing treatment with upadacitinib.

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Hepatotoxicity
The pattern of liver injury associated with upadacitinib indicates a potential for low-level, direct hepatotoxicity. During the initial weeks of treatment, ALT levels may slightly increase, typically returning to baseline upon discontinuation of the drug. Serum aminotransferase elevations above 5 times the upper limit of normal should prompt consideration of dose reduction or temporary cessation of upadacitinib treatment. Managing potential hepatotoxicity carefully is essential for minimizing further liver injury. Close monitoring of liver function tests is crucial during this period to ensure timely intervention and appropriate management.
There is no information regarding overdose of upadacitinib in the FDA-approved product labeling.

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Adverse Effects
Upadacitinib has various adverse effects, which are listed below.
* Upper respiratory tract infections (URTI) (14%)
* Nausea (4%)
* Elevated liver enzymes (2%)
* Fever (1%)
* Cough (2%)

Upper respiratory tract infections (URTI) encompass:
* Acute sinusitis
* Laryngitis
* Nasopharyngitis
* Oropharyngeal pain
* Pharyngitis
* Pharyngotonsillitis
* Rhinitis
* Sinusitis
* Tonsillitis
* Viral upper respiratory tract infection
These adverse effects were observed during placebo-controlled studies where subjects were administered 15 mg of oral upadacitinib. More severe adverse effects, such as herpes zoster virus (HZV) and serious infections, were seen in subjects administered 30 mg in a double-blind, randomized, controlled phase 3 clinical trial (Moraxella infection.

Drug-Drug Interactions: Upadacitinib is metabolized in the liver by the cytochrome P450 (CYP) system, primarily through the CYP3A4 enzyme, and is eliminated in the feces and urine as metabolites, with a drug half-life of 8 to 14 hours. The concomitant use of CYP3A4 inhibitors and CYP3A4 inducers should be approached with caution and is generally not recommended, as it may alter the drug's pharmacokinetics, potentially increasing or decreasing drug plasma concentrations. Clinical studies have also reported malignancy, thrombosis, and gastrointestinal (GI) perforations with concomitant use of non-steroidal anti-inflammatory drugs (NSAIDs).

* Upadacitinib exposure may increase when used concurrently with strong CYP3A4 inhibitors such as grapefruit, ketoconazole, and clarithromycin, potentially exacerbating the risk of adverse reactions. Patients should avoid consuming grapefruit or grapefruit-containing products during upadacitinib treatment.

* For patients with atopic dermatitis or Crohn disease who are prescribed strong CYP3A4 inhibitors, the induction dosage of upadacitinib should be reduced to 30 mg once daily while maintaining the maintenance dosage at 15 mg once daily. Conversely, strong CYP3A4 inducers such as rifampin may decrease upadacitinib exposure, potentially diminishing its therapeutic effects. Therefore, co-administration of upadacitinib with strong CYP3A4 inducers is not advised.

[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
Upadacitinib is a DMARD that works by inhibiting the Janus Kinases (JAKs), which are essential downstream cell signalling mediators of pro-inflammatory cytokines. It is believed that these pro-inflammatory cytokines play a role in many autoimmune inflammatory conditions, such as rheumatoid arthritis. In clinical trials, upadacitinib decreased the activity of pro-inflammatory interleukins, transiently increased the levels of lymphocytes, and insignificantly decreased the levels of immunoglobulins from the baseline.

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Upadacitinib is an oral Janus kinase (JAK)1-selective inhibitor and a disease-modifying antirheumatic drug (DMARD) used in the treatment of rheumatoid arthritis to slow down disease progression. Rheumatoid arthritis is a chronic autoimmune inflammatory disease affecting the peripheral joints. It is characterized by synovial inflammation and hyperplasia, autoantibody production, cartilage damage and bone destruction, leading to co-morbidities. Despite a variety of therapeutic agents available for treatment, up to 40% of the patients do not respond to current therapies, including biological therapies. The etiology of the disease is mostly unknown; however, the role of JAK as a driver of immune-mediated conditions was discovered, leading to the use of JAK as therapeutic targets for rheumatoid arthritis. To reduce dose-related toxicity (as seen with some pan-JAK inhibitors) without significantly affecting efficacy, more selective JAK1 inhibitors, upadacitinib and [filgotinib], were developed. The FDA approved upadacitinib in August 2019 and it is used for the treatment of active rheumatoid arthritis, psoriatic arthritis, atopic dermatitis, ulcerative colitis, and ankylosing spondylitis. In December 2019, it was additionally approved by the European Commission and Health Canada. Upadacitinib is marketed under the brand name RINVOQ for oral administration.

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Upadacitinib is a Janus Kinase Inhibitor. The mechanism of action of upadacitinib is as a Janus Kinase Inhibitor.
Upadacitinib is an oral selective inhibitor of Janus associated kinase 1 (JAK-1) that is used in the therapy of moderate-to-severe rheumatoid arthritis. Upadacitinib has been associated with a low rate of serum enzyme elevations during therapy, but has not been linked to cases of clinically apparent acute liver injury although it may pose a risk for reactivation of hepatitis B in susceptible patients.
Upadacitinib is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2019 and is indicated for rheumatoid arthritis and has 12 investigational indications. This drug has a black box warning from the FDA.

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Drug Indication
Upadacitinib is indicated for the treatment of moderately to severely active **rheumatoid arthritis** or active **psoriatic arthritis** in adult patients who have had an inadequate response or intolerance to one or more disease-modifying anti-rheumatic drugs (DMARDs), such as TNF blockers. In Europe, upadacitinib may be used as monotherapy or in combination with [methotrexate] for rheumatoid or psoriatic arthritis. Upadacitinib is indicated for use in patients 12 years of age and older with refractory, moderate-to-severe **atopic dermatitis** whose disease is inadequately controlled with other systemic therapies or when other therapies are inadvisable. Upadacitinib is indicated for the treatment of active **ankylosing spondylitis** or radiographic axial spondyloarthritis in adult patients who have an inadequate response to conventional therapy. It is also indicated to treat non-radiographic axial spondyloarthritis with objective signs of inflammation in adults who have had an inadequate response or intolerance to TNF blocker therapy. Upadacitinib is also indicated to treat moderately to severely active **ulcerative colitis** in adults who have had an inadequate response or intolerance to either conventional therapy or a biologic agent, such as to one or more TNF blockers. Upadacitinib is indicated to treat moderately to severely active Crohn’s disease in adults who have had an inadequate response or intolerance to one or more TNF blockers. Combining upadacitinib with other JAK inhibitors, biologic DMARDs, or other potent immunosuppressive agents is not recommended.

Rheumatoid arthritis RINVOQ is indicated for the treatment of moderate to severe active rheumatoid arthritis in adult patients who have responded inadequately to, or who are intolerant to one or more disease-modifying anti-rheumatic drugs (DMARDs). RINVOQ may be used as monotherapy or in combination with methotrexate. Psoriatic arthritis RINVOQ is indicated for the treatment of active psoriatic arthritis in adult patients who have responded inadequately to, or who are intolerant to one or more DMARDs. RINVOQ may be used as monotherapy or in combination with methotrexate. Axial spondyloarthritis Non-radiographic axial spondyloarthritis (nr-axSpA)RINVOQ is indicated for the treatment of active non-radiographic axial spondyloarthritis in adult patients with objective signs of inflammation as indicated by elevated C-reactive protein (CRP) and/or magnetic resonance imaging (MRI), who have responded inadequately to nonsteroidal anti-inflammatory drugs (NSAIDs). Ankylosing spondylitis (AS, radiographic axial spondyloarthritis )RINVOQ is indicated for the treatment of active ankylosing spondylitis in adult patients who have responded inadequately to conventional therapy. Atopic dermatitisRINVOQ is indicated for the treatment of moderate to severe atopic dermatitis in adults and adolescents 12 years and older who are candidates for systemic therapy. Ulcerative colitisRINVOQ is indicated for the treatment of adult patients with moderately to severely active ulcerative colitis who have had an inadequate response, lost response or were intolerant to either conventional therapy or a biologic agent. Â Crohn's diseaseRINVOQ is indicated for the treatment of adult patients with moderately to severely active Crohn's disease who have had an inadequate response, lost response or were intolerant to either conventional therapy or a biologic agent.

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Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovial inflammation and joint destruction. Considerable advance in the treatment of RA has been made following the advent of biological disease-modifying anti-rheumatic drugs (DMARDs). However, these biologics require intravenous or subcutaneous injection and some patients fail to respond to biological DMARDs or lose their primary response. Various cytokines and cell surface molecules bind to receptors on the cell surface, resulting in the activation of various cell signaling pathways, including phosphorylation of kinase proteins. Among these kinases, the non-receptor tyrosine kinase family Janus kinase (JAK) plays a pivotal role in the pathological processes of RA. Several JAK inhibitors have been developed as new therapies for patients with RA. These are oral synthetic DMARDs that inhibit JAK1, 2, and 3. One JAK inhibitor, tofacitinib, has already been approved in many countries. Results of phase III clinical trials using a JAK1/2 inhibitor, baricitinib, have shown feasible efficacy and tolerable safety. Both drugs are effective in patients who showed inadequate response to biological DMARDs as well as synthetic DMARDs. In addition, clinical phase III trials using filgotinib and Upadacitinib/ABT-494, specific JAK1 inhibitors, are currently underway. JAK inhibitors are novel therapies for RA, but further studies are needed to determine their risk-benefit ratio and selection of the most appropriate patients for such therapy. [1]

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Anti-cytokine therapies have become the mainstay of treatment for rheumatoid arthritis (RA) disease symptoms and can arrest disease progression. Despite numerous treatment options there are still many RA patients who fail to experience substantial decreases in disease activity. Recently, Jak kinase blockade was shown clinically to be effective in managing disease and in some cases achieving remission. However, these first generation Jak inhibitors have failed to meet expectations 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 for side effects related to Jak2 and Jak3 inhibition. Here we describe preclinical and early clinical data that suggest ABT-494 has potential to address some of the current unmet medical needs of RA patients.[2]

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Background: Anti-cytokine therapies such as adalimumab, tocilizumab, and the small molecule JAK inhibitor tofacitinib have proven that cytokines and their subsequent downstream signaling processes are important in the pathogenesis of rheumatoid arthritis. Tofacitinib, a pan-JAK inhibitor, is the first approved JAK inhibitor for the treatment of RA and has been shown to be effective in managing disease. However, in phase 2 dose-ranging studies tofacitinib was associated with dose-limiting tolerability and safety issues such as anemia. Upadacitinib (ABT-494) is a selective JAK1 inhibitor that was engineered to address the hypothesis that greater JAK1 selectivity over other JAK family members will translate into a more favorable benefit:risk profile. Upadacitinib selectively targets JAK1 dependent disease drivers such as IL-6 and IFNγ, while reducing effects on reticulocytes and natural killer (NK) cells, which potentially contributed to the tolerability issues of tofacitinib.

Methods: Structure-based hypotheses were used to design the JAK1 selective inhibitor Upadacitinib. JAK family selectivity was defined with in vitro assays including biochemical assessments, engineered cell lines, and cytokine stimulation. In vivo selectivity was defined by the efficacy of upadacitinib and tofacitinib in a rat adjuvant induced arthritis model, activity on reticulocyte deployment, and effect on circulating NK cells. The translation of the preclinical JAK1 selectivity was assessed in healthy volunteers using ex vivo stimulation with JAK-dependent cytokines.

Results: Here, we show the structural basis for the JAK1 selectivity of Upadacitinib, along with the in vitro JAK family selectivity profile and subsequent in vivo physiological consequences. Upadacitinib is ~ 60 fold selective for JAK1 over JAK2, and > 100 fold selective over JAK3 in cellular assays. While both upadacitinib and tofacitinib demonstrated efficacy in a rat model of arthritis, the increased selectivity of upadacitinib for JAK1 resulted in a reduced effect on reticulocyte deployment and NK cell depletion relative to efficacy. Ex vivo pharmacodynamic data obtained from Phase I healthy volunteers confirmed the JAK1 selectivity of upadactinib in a clinical setting.

Conclusions: The data presented here highlight the JAK1 selectivity of Upadacitinib and supports its use as an effective therapy for the treatment of RA with the potential for an improved benefit:risk profile.

C17H19F3N6O

380.367573022842

380.157

C, 53.68; H, 5.04; F, 14.98; N, 22.09; O, 4.21

1310726-60-3

Upadacitinib-15N,d2;Upadacitinib tartrate tetrahydrate;1607431-21-9

58557659

White to yellow solid powder

1.6±0.1 g/cm3

16-19

1.678

3.06

2

6

3

27

561

2

CC[C@@H]1CN(C[C@@H]1C2=CN=C3N2C4=C(NC=C4)N=C3)C(=O)NCC(F)(F)F

WYQFJHHDOKWSHR-MNOVXSKESA-N

InChI=1S/C17H19F3N6O/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/h3-6,10-11,21H,2,7-9H2,1H3,(H,24,27)/t10-,11+/m1/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

ABT-494; Upadacitinib; ABT-494; Rinvoq; Upadacitinib anhydrous; UNII-4RA0KN46E0; 4RA0KN46E0; ABT 494; ABBV-599 COMPONENT UPADACITINIB; ABT494

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)

DMSO : ~100 mg/mL (~262.90 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.75 mg/mL (7.23 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.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (7.23 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.

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Solubility in Formulation 3: ≥ 1.67 mg/mL (4.39 mM) 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 16.7 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 4: ≥ 1.67 mg/mL (4.39 mM) 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 16.7 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 5: ≥ 1.67 mg/mL (4.39 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: (saturation unknown) in (add these co-solvents sequentially from left to right, and one by one),
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.)