JAK1 (IC50 = 10 nM); JAK2 (IC50 = 18 nM); Tyk2 (IC50 = 84 nM); JAK3 (IC50 = 99 nM)

Oclacitinib's efficacy and selectivity against members of the JAK family and cytokines that cause JAK activation in cells are assessed using isolated enzyme systems and in vitro human or canine cell models. In isolated enzyme systems, oclacitinib's inhibitory action against members of the JAK family is ascertained. JAK1, JAK2, JAK3, and TYK2 are all 50% inhibited by oclacitinib at doses (IC50's) of 10, 18, 99, and 84 nM, respectively. With a 1.8-fold selectivity for JAK1 vs. JAK2 and a 9.9-fold selectivity for JAK1 vs. JAK3, oclacitinib exhibits the highest potency against the JAK1 enzyme. Oclacitinib does not inhibit a panel of 38 non-JAK kinases (IC50's >1000 nM), yet it inhibits JAK family members by 50% at doses (IC50's) ranging from 10 to 99 nM. At IC50 values ranging from 36 to 249 nM, oclacitinib also reduces the activity of JAK1-dependent cytokines implicated in allergy, inflammation, and pruritus (IL-2, IL-4, IL-6, and IL-13). Oclacitinib has negligible effects on cytokines (erythropoietin, granulocyte/macrophage colony-stimulating factor, IL-12, IL-23; IC50's >1000 nM) that do not activate the JAK1 enzyme in cells[1]. Topical treatment with Tofacitinib (0.1%) and Oclacitinib (0.1%) significantly reduces cell migration from mouse ear explants when compared to ears treated with vehicle (all P < 0.05). When compared to each epidermis treated with a JAK inhibitor, the numbers of MHC class II positive cells, or Langerhans cells, are significantly lower in the vehicle-treated epidermis (all P<0.01)[2].

Scratching bouts at the high dose in the Oclacitinib group are considerably less than in the vehicle-only group (P<0.01)[2]. Client-owned dogs (n=436) with moderate to severe owner-assessed pruritus and a presumptive diagnosis of allergic dermatitis are included. Dogs are randomized to receive Oclacitinib at 0.4-0.6 mg/kg orally twice day or an excipient-matched placebo. An improved 10 cm visual analog scale (VAS) is used to assess the intensity of pruritus from day 0 to 7 and to assess the severity of dermatitis on days 0 and 7. Dogs can remain on the research for 28 days. Oclacitinib produces a rapid onset of effectiveness within 24 h[3].

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There are considerably fewer scratching episodes at the high dose in the Oclacitinib group (P<0.01) compared to the vehicle-only group. Enrollment of client-owned dogs (n = 436) with a probable diagnosis of allergic dermatitis and moderate to severe owner-assessed pruritus occurs. Dogs are randomized to receive an excipient-matched placebo or oclacitinib at a dose of 0.4–0.6 mg/kg orally twice a day. The intensity of dermatitis is measured on days 0 and 7, and the degree of pruritus is measured from day 0 to day 7 using an improved 10 cm visual analog scale (VAS). Dogs can spend up to 28 days in the trial. Oclacitinib has a 24-hour quick onset of action[3].

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Results: Pretreatment owner and veterinary VAS scores were similar for the two treatment groups. Oclacitinib produced a rapid onset of efficacy within 24 h. Mean oclacitinib Owner Pruritus VAS scores were significantly better than placebo scores (P < 0.0001) on each assessment day. Pruritus scores decreased from 7.58 to 2.59 cm following oclacitinib treatment. The day 7 mean oclacitinib Veterinarian Dermatitis VAS scores were also significantly better (P < 0.0001) than placebo scores. Diarrhoea and vomiting were reported with similar frequency in both groups. Conclusions and clinical importance: In this study, Oclacitinib provided rapid, effective and safe control of pruritus associated with allergic dermatitis, with owners and veterinarians noting substantial improvements in pruritus and dermatitis VAS scores[3].

Janus kinase enzyme activity assays and kinase selectivity panels [1]
Recombinant human active kinase domains for JAK1 (amino acids 852–1142; NP_002218), JAK2 (amino acids 808–1132; NP_004963), JAK3 (amino acids 781–1124; NP_000206), and TYK2 (amino acids 870–1187; NP_003322) were used in isolated enzyme assays using Caliper microfluidics technology to determine potency of Oclacitinib against the JAK family members, as previously described (Meyer et al., 2010). Sequence homology to the analogous sequences in the canine JAK enzymes are 98, 98, 100, and 90%, respectively (Figure S1). Invitrogen kinase panel testing was performed to determine potency of Oclacitinib toward 38 different non-JAK kinases using their SelectScreen™ Kinase Profiling Services. Oclacitinib was evaluated at a concentration of 1 μm. Kinase-specific assay conditions and data analyses are described on their Web site http://www.lifetechnologies.com/us/en/home/products-and-services/services/custom-services/screening-and-profiling-services/selectscreen-profiling-service/selectscreen-kinase-profiling-service.html. This service utilizes the Z'-Lyte technology for all kinase screening. All tests were run in duplicate.

Interleukin-6 cytokine function [1]
The CellSensor® STAT3-bla HEK293T human epithelial cell line from Invitrogen was used. Cells were plated into 384-well assay plates, black-wall, clear bottom at a density of 1.875 × 105 cells per well in DMEM high glucose medium containing 5% FBS and incubated at 37 °C, 5% CO2. Oclacitinib (0.0000954–25 μm) or vehicle control was added to cells for 1 h. Twenty nanograms per milliliter hIL-6 was then added to cell cultures for 5 h. Activation of the STAT3-beta-lactamase reporter gene by IL-6 was determined by detecting beta-lactamase activity with the LiveBLAzer™-FRET B/G substrate (CCF-4 AM). Fluorescence emission values at 460 and 530 nm were obtained using a fluorescent plate reader. The 460/530 nm ratios were expressed as percent control, and dose–response data were analyzed using a 4-parameter logistic equation.

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Interleukin-13 cytokine function [1]
The HT-29 human colonic epithelial cell line from American Type Culture Collection was used. Cells were propagated in McCoy's 5A medium containing 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, and 2 mm l-glutamine. Cells were trypsinized from flasks, washed in fresh medium, and resuspended in 96-well assay plates at a density of 3 × 105 cells per well. Oclacitinib (0.0015–30 μm)) or vehicle control was added to cells while on ice for 30 min. One nanogram per milliliter hIL-13 was then added. Cells were incubated in a 37 °C water bath for 30 min then fixed in 1.75% formaldehyde in PBS, washed in PBS containing 0.5% BSA, and incubated overnight at −20 °C in absolute methanol. Fixed cells were stained with PE-labeled antibody to human pSTAT6. Samples were analyzed with an FACSCalibur equipped with a plate-based autosampler and analyzed using FlowJo software, version 7.6.1. Data were expressed as mean fluorescence and then expressed as percent control. Dose–response data were then analyzed using a 4-parameter logistic equation.

Randomization and masking [3]
Dogs were randomized to one of two treatment groups (i.e. Oclacitinib or placebo) in a 1:1 ratio. Blocking was based on order of enrolment within clinic. The dog was the experimental unit. All clinical trial personnel, the owner and the laboratory were blinded to the treatment group assignments. The placebo and Oclacitinib tablets were identical in size and appearance. An interactive voice response system was used to manage patient treatment assignment and blinded drug dispensing. Upon each dog's enrolment, the sites accessed the IVRS system, and the system then randomized the dogs to the respective treatment group.

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Drug administration [3]
Dogs in the Oclacitinib treatment group were given Oclacitinib maleate caplets orally at a dose of 0.4–0.6 mg/kg twice daily. The scored caplets were provided in three strengths containing 3.6, 5.4 and 16 mg of oclacitinib. Dogs in the placebo treatment group were given the same number of caplets, identical in appearance to Oclacitinib maleate caplets and containing all of the same excipients except oclacitinib maleate. Owners administered the study drug at home, with or without food,21 and were instructed to maintain as close to a 12 h interval between doses as possible.

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Study schedule and variables measured [3]
Following randomization, the dogs were assigned to receive either the excipient placebo or Oclacitinib at a dose of 0.4–0.6 mg/kg, orally twice daily from day 0 to day 7 (+3 days; study phase). If, in the veterinarian's clinical judgment, the pruritic condition resolved or improved to a point that no additional therapy was indicated, day 7 was regarded as the final study day. Dogs in which the underlying diagnosis (presenting complaint) was not resolved at the end of the study phase, but that had responded well to therapy, were permitted to remain on therapy (either placebo or Oclacitinib ) up to day 28 (±2 days; continuation phase). Certain concurrent medications not permitted for use on days 0–7 could be added on or after day 8 (e.g. systemic antimicrobial drugs). Glucocorticoids, antihistamines, ciclosporin or other immunosuppressive drugs were not permitted during either phase of the study. Dogs were withdrawn if the owner or veterinarian felt that their pruritus and/or dermatitis required treatment with a prohibited medication. Owners were free to withdraw their dog at any point.

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Dissolved in a 0.5% methylcellulose/0.25% Tween 20 solution for oral administration and a 7:1 acetone: DMSO solution for topical application; Oral doses are as follows: 30 and 45 mg/kg. Topically administered doses are 0.1, 0.25, and 0.5%.

BALB/cAnN (female, 6 weeks old)

This study aimed to determine the pharmacokinetic parameters of oclavinib maleate as a topical formulation in adult horses. Six adult horses with an average weight of 528 kg received a single dose of 0.5 mg/kg oclavinib maleate. Blood samples were collected before administration and at 15, 30, 45, 1, 2, 4, 6, 8, 12, 24, 48, and 72 hours after administration. Plasma concentrations of oclavinib maleate were determined using liquid chromatography-mass spectrometry (LC-MS). The results showed that a single-compartment model best suited the pharmacokinetic parameters. The mean Cmax was 486 ng/ml (range 423–549 ng/ml), and the estimated Tmax was 1.7 h (range 0.3–3.1 h). The estimated T1/2 was 7.5–8 h. https://pubmed.ncbi.nlm.nih.gov/35098559/

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Background: Oclaptinib, a Janus kinase (JK)1 inhibitor, has been shown to be effective and safe in the treatment of canine atopic dermatitis. Its use in cats is limited due to a lack of pharmacokinetic data.
Objective: To determine the pharmacokinetic parameters of oclaptinib after oral and intravenous administration in cats.
Animals: Six adult domestic shorthaired cats.
Methods and Materials: A two-cycle, two-treatment design was used. Cats received intravenous and oral administration of oclaptinib maleate at doses of 0.5 mg/kg and 1 mg/kg, respectively. A one-week washout period was followed between the two administrations. Each cat received only one dose. Plasma concentrations of oclavinib at 0, 5, 15, 30, 1, 4, 6, 10, and 24 hours after intravenous injection, and at 0, 15, 30, 1, 2, 4, 6, 10, and 24 hours after oral administration, were determined using high-performance liquid chromatography (HPLC). Results: After oral administration, oclavinib was rapidly and almost completely absorbed, with an absolute bioavailability of 87% and a time to peak concentration (Tmax) of 35 minutes. Elimination was also very rapid, with a half-life of 2.3 hours and a clearance rate of 4.45 mL/min/kg (after intravenous injection). Conclusions and Clinical Significance: The pharmacokinetic parameters of oclavinib in cats are similar to those in dogs, but the absorption and elimination rates in cats are slightly faster, and inter-individual variability is also slightly greater. It is recommended that cats use higher doses and/or shorter dosing intervals to achieve plasma concentrations similar to those in dogs. The pharmacokinetics of oclavinib maleate were evaluated in four independent studies. The absolute bioavailability study used a crossover design and included 10 dogs. The effect of food on bioavailability was investigated in a crossover study including 18 dogs. The effect of breed on pharmacokinetics was evaluated in a crossover study including beagles and mongrel dogs. Dose ratio and multiple-dose pharmacokinetics were evaluated in a parallel design study with 8 dogs in each group. Serial plasma samples were collected in all four studies. Following oral administration, oclavinib maleate was rapidly and well absorbed, reaching peak concentration in <1 hour, with an absolute bioavailability of 89%. Fasting status had no significant effect on the rate and extent of absorption of orally administered oclavinib maleate, and there were no significant differences in pharmacokinetic parameters between the fasting and postprandial groups. The pharmacokinetics of oclavinib also showed similarities in the laboratory populations of beagles and mongrel dogs. Following oral administration, exposure to oclavinib maleate increased proportionally with increasing dose from 0.6 mg/kg to 3.0 mg/kg. Furthermore, no significant gender differences in the pharmacokinetics of oclavinib were observed in any of the pharmacokinetic studies. https://pubmed.ncbi.nlm.nih.gov/24330031/

[1]. Oclacitinib is a novel Janus kinase inhibitor with activity against cytokines involved in allergy. J Vet Pharmacol Ther. 2014 Aug;37(4):317-24.

[2]. Topically Administered Janus-Kinase Inhibitors Tofacitinib and Oclacitinib Display Impressive Antipruritic and Anti-Inflammatory Responses in a Model of Allergic Dermatitis. J Pharmacol Exp Ther. 2015 Sep;354(3):394-405.

[3]. Efficacy and safety of oclacitinib for the control of pruritus and associated skin lesions in dogs with canine allergic dermatitis. Vet Dermatol. 2013 Oct;24(5):479-e114.

Janus kinases (JAKs) are involved in multiple cellular signaling pathways that are activated by dysregulated cytokines in allergic reactions. This study aimed to determine whether the novel JAK inhibitor oclacitinib could reduce the activity of cytokines associated with canine allergic dermatitis. Using isolated enzyme systems and in vitro human or canine cell models, we determined the potency and selectivity of oclacitinib against JAK family members and cytokines that can trigger intracellular JAK activation. Oclacitinib inhibited 50% of the activity of JAK family members at concentrations ranging from 10 to 99 nM (IC50) and had no inhibitory effect on 38 non-JAK kinases (IC50 > 1000 nM). Oclacitinib showed the strongest inhibitory effect on JAK1 (IC50 = 10 nM). Oclacitinib also inhibited the function of JAK1-dependent cytokines (IL-2, IL-4, IL-6, and IL-13) involved in allergy and inflammation, as well as pruritus factor (IL-31), with IC50 values ranging from 36 to 249 nM. Oclatinib had little effect on cytokines that do not activate intracellular JAK1 enzymes (erythropoietin, granulocyte/macrophage colony-stimulating factor, IL-12, IL-23; IC50 value >1000 nM). These results suggest that oclatinib is a targeted therapy that selectively inhibits JAK1-dependent cytokines involved in allergy, inflammation, and pruritus, and suggests that these mechanisms may be the mechanisms by which oclatinib effectively controls clinical symptoms associated with canine allergic dermatitis. [1]

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The prevalence of allergic dermatitis is rapidly increasing, and there is an urgent need to develop therapeutic drugs to relieve symptoms. In this study, Janus kinase (JAK) inhibitors tofacitinib and oclatinib were administered orally or topically in a mouse dermatitis model, and their efficacy in relieving pruritus and inflammation was compared. At the same time, the in vitro effects of JAK inhibitors on bone marrow-derived dendritic cells (BMDCs) were analyzed. In the allergic dermatitis model, female BALB/c mice were sensitized and challenged with toluene-2,4-diisocyanate (TDI). JAK inhibitors were administered orally or topically 30 minutes before and 4 hours after TDI stimulation. Scratching frequency and ear thickness were measured in mice, cytokines in the stimulated skin were detected, and cells in draining lymph nodes were analyzed by flow cytometry. In vitro experiments showed that both JAK inhibitors significantly inhibited cytokine production, migration, and maturation in bone marrow-derived dendritic cells (BMDCs). Mice receiving oral JAK inhibitors showed a significant reduction in scratching behavior, but no significant decrease in ear thickness. In contrast, the topically treated group showed significantly lower scratching behavior and ear thickness compared to the control group. However, the regulatory effects of JAK inhibitors on cytokine production differed, with some cytokines significantly decreased and others significantly increased. In summary, oral JAK inhibitors significantly reduced pruritus but had little effect on the inflammatory response; while topically applied JAK inhibitors simultaneously improved both pruritus and the inflammatory response. Although the two JAK inhibitors exhibited different JAK inhibitory profiles in vitro and in vivo, their therapeutic effects were comparable. [2]

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Background: Octlatinib (Apoquel®) inhibits the function of a variety of pro-inflammatory, pro-allergic, and pruritus cytokines that depend on Janus kinase activity. Octlatinib selectively inhibits Janus kinase 1. Hypothesis/Objective: We aimed to evaluate the safety and efficacy of oclatinib for the treatment of pruritus associated with atopic dermatitis in a randomized, double-blind, placebo-controlled trial. Methods: 436 dogs with owners-assessed moderate to severe pruritus and a preliminary diagnosis of atopic dermatitis were enrolled. Dogs were randomly assigned to either the oclatinib group (0.4–0.6 mg/kg, orally twice daily) or the placebo group (using excipient-matched placebo). Owners assessed pruritus severity on days 0–7 using the enhanced 10 cm visual analog scale (VAS), while veterinarians assessed dermatitis severity on days 0 and 7. Dogs were eligible to participate in the study for 28 days. Results: Before treatment, owner and veterinarian VAS scores were similar in both groups. Oclatinib had a rapid onset of action within 24 hours. On each assessment day, the mean VAS score for pruritus in the oclatinib group was significantly better than that in the placebo group (P < 0.0001). After oclatinib treatment, the pruritus score decreased from 7.58 cm to 2.59 cm. On day 7, the mean VAS score for dermatitis in the oclatinib group was also significantly better than that in the placebo group (P < 0.0001). The frequency of diarrhea and vomiting was similar in both groups. Conclusion and clinical significance: This study demonstrates that oclatinib can rapidly, effectively and safely control pruritus associated with atopic dermatitis, and significant improvements in pruritus and dermatitis VAS scores were observed in both pet owners and veterinarians. [3]

C19H27N5O6S

453.512583017349

453.168

C, 50.32; H, 6.00; N, 15.44; O, 21.17; S, 7.07

1640292-55-2

Oclacitinib;1208319-26-9; 1208319-27-0; 1640292-55-2

44631937

White to off-white solid powder

4

10

7

31

606

0

S(C([H])([H])C1([H])C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N(C([H])([H])[H])C1C2C([H])=C([H])N([H])C=2N=C([H])N=1)(N([H])C([H])([H])[H])(=O)=O.O([H])C(/C(/[H])=C(/[H])\C(=O)O[H])=O

VQIGDTLRBSNOBV-BTJKTKAUSA-N

InChI=1S/C15H23N5O2S.C4H4O4/c1-16-23(21,22)9-11-3-5-12(6-4-11)20(2)15-13-7-8-17-14(13)18-10-19-15;5-3(6)1-2-4(7)8/h7-8,10-12,16H,3-6,9H2,1-2H3,(H,17,18,19);1-2H,(H,5,6)(H,7,8)/b;2-1-

N-methyl-1-((1r,4r)-4-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclohexyl)methanesulfonamide maleate

PF 03394197 maleate; Apoquel; OCLACITINIB MALEATE; 1208319-27-0; 1640292-55-2; Oclacitinib (maleate); Apoquel; PF-03394197 maleate; PF03394197 maleate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.51 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.51 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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