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)

The objective of this study was to determine the pharmacokinetic parameters of oclacitinib maleate as a top dress given to adult horses. Six adult horses with a mean weight of 528 kg were administered a single dose of 0.5 mg/kg oclacitinib maleate. Blood was collected prior to drug administration and at 15 min, 30 min, 45 min, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h after treatment. Oclacitinib maleate plasma concentrations were measured by liquid chromatography/mass spectrometry. Pharmacokinetic parameters were found best to fit a one-compartment model. Mean Cmax was 486 ng/ml (range 423-549 ng/ml), and Tmax was estimated to be 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: Oclacitinib is a Janus kinase (JK)1 inhibitor that has been shown to be effective and safe for the treatment of allergic dermatitis in dogs. Its use in cats has been limited by the absence of pharmacokinetic data.
Objective: To determine the pharmacokinetic parameters of oclacitinib in cats after oral and intravenous administration.
Animals: Six adult domestic short hair cats.
Methods and materials: A two period, two treatment design was used in which cats received oclacitinib maleate i.v. and p.o., at a dose of 0.5 mg/kg and 1 mg/kg, respectively. There was a one-week interval of washout between the two treatments. Cats received each treatment only once. The plasma concentration of oclacitinib was determined by high-performance liquid chromatography at 0 min, 5 min, 15 min, 30 min, 1 h, 4 h, 6 h, 10 h and 24 h after intravenous.v administration, at 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 10 h and 24 h after p.o. administration.
Results: After p.o. administration, oclacitinib was absorbed rapidly and almost completely, as shown by an absolute bioavailability of 87% and a Tmax of 35 min. The elimination of the drug also was very rapid as shown by a half-life of 2.3 h and a clearance calculated as 4.45 mL/min/kg (after i.v. administration).
Conclusions and clinical importance: The pharmacokinetic parameters of oclacitinib in the cat are similar to those described for the dog, although absorption and elimination are somewhat faster and variability between individuals is somewhat greater. Larger doses and/or shorter dosing intervals would be recommended in cats to achieve similar blood concentrations to those in dogs. https://pubmed.ncbi.nlm.nih.gov/31769185/

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The pharmacokinetics of oclacitinib maleate was evaluated in four separate studies. The absolute bioavailability study used a crossover design with 10 dogs. The effect of food on bioavailability was investigated in a crossover study with 18 dogs. The breed effect on pharmacokinetics was assessed in a crossover study in beagles and mongrels dogs. Dose proportionality and multiple dose pharmacokinetics were evaluated in a parallel design study with eight dogs per group. In all four studies, serial blood samples for plasma were collected. Oclacitinib maleate was rapidly and well absorbed following oral administration, with a time to peak plasma concentration of <1 h and an absolute bioavailability of 89%. The prandial state of dogs did not significantly affect the rate or extent of absorption of oclacitinib maleate when dosed orally, as demonstrated by the lack of significant differences in pharmacokinetic parameters between the oral fasted and oral fed treatment groups. The pharmacokinetics of oclacitinib in laboratory populations of beagles and mixed breed dogs also appeared similar. Following oral administration, the exposure of oclacitinib maleate increased dose proportionally from 0.6 to 3.0 mg/kg. Additionally, across the pharmacokinetic studies, there were no apparent differences in oclacitinib pharmacokinetics attributable to sex. 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 kinase (JAK) enzymes are involved in cell signaling pathways activated by various cytokines dysregulated in allergy. The objective of this study was to determine whether the novel JAK inhibitor oclacitinib could reduce the activity of cytokines implicated in canine allergic skin disease. Using isolated enzyme systems and in vitro human or canine cell models, potency and selectivity of oclacitinib was determined against JAK family members and cytokines that trigger JAK activation in cells. Oclacitinib inhibited JAK family members by 50% at concentrations (IC50 's) ranging from 10 to 99 nm and did not inhibit a panel of 38 non-JAK kinases (IC50 's > 1000 nM). Oclacitinib was most potent at inhibiting JAK1 (IC50 = 10 nM). Oclacitinib also inhibited the function of JAK1-dependent cytokines involved in allergy and inflammation (IL-2, IL-4, IL-6, and IL-13) as well as pruritus (IL-31) at IC50 's ranging from 36 to 249 nM. Oclacitinib had minimal effects on cytokines that did not activate the JAK1 enzyme in cells (erythropoietin, granulocyte/macrophage colony-stimulating factor, IL-12, IL-23; IC50 's > 1000 nM). These results demonstrate that oclacitinib is a targeted therapy that selectively inhibits JAK1-dependent cytokines involved in allergy, inflammation, and pruritus and suggests these are the mechanisms by which oclacitinib effectively controls clinical signs associated with allergic skin disease in dogs. [1]

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The prevalence of allergic skin disorders has increased rapidly, and development of therapeutic agents to alleviate the symptoms are still needed. In this study, we orally or topically administered the Janus kinase (JAK) inhibitors, tofacitinib and oclacitinib, in a mouse model of dermatitis, and compared the efficacy to reduce the itch and inflammatory response. In vitro effects of JAK inhibitors on bone marrow-derived dendritic cells (BMDCs) were analyzed. For the allergic dermatitis model, female BALB/c mice were sensitized and challenged with toluene-2,4-diisocyanate (TDI). Each JAK inhibitor was orally or topically applied 30 minutes before and 4 hours after TDI challenge. After scratching bouts and ear thickness were measured, cytokines were determined in challenged skin and the cells of the draining lymph node were analyzed by means of flow cytometry. In vitro, both JAK inhibitors significantly inhibited cytokine production, migration, and maturation of BMDCs. Mice treated orally with JAK inhibitors showed a significant decrease in scratching behavior; however, ear thickness was not significantly reduced. In contrast, both scratching behavior and ear thickness in the topical treatment group were significantly reduced compared with the vehicle treatment group. However, cytokine production was differentially regulated by the JAK inhibitors, with some cytokines being significantly decreased and some being significantly increased. In conclusion, oral treatment with JAK inhibitors reduced itch behavior dramatically but had only little effect on the inflammatory response, whereas topical treatment improved both itch and inflammatory response. Although the JAK-inhibitory profile differs between both JAK inhibitors in vitro as well as in vivo, the effects have been comparable.[2]

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Background: Oclacitinib (Apoquel(®) ) inhibits the function of a variety of pro-inflammatory, pro-allergic and pruritogenic cytokines that are dependent on Janus kinase enzyme activity. Oclacitinib selectively inhibits Janus kinase 1. Hypothesis/objectives: We aimed to evaluate the safety and efficacy of oclacitinib for the control of pruritus associated with allergic dermatitis in a randomized, double-blinded, placebo-controlled trial. Methods: Client-owned dogs (n = 436) with moderate to severe owner-assessed pruritus and a presumptive diagnosis of allergic dermatitis were enrolled. Dogs were randomized to either oclacitinib at 0.4-0.6 mg/kg orally twice daily or an excipient-matched placebo. An enhanced 10 cm visual analog scale (VAS) was used by the owners to assess the severity of pruritus from day 0 to 7 and by veterinarians to assess the severity of dermatitis on days 0 and 7. Dogs could remain on the study for 28 days. 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]

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.)