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

使用分离的酶系统和体外人类或犬细胞模型评估奥拉替尼针对 JAK 家族成员和导致细胞中 JAK 激活的细胞因子的效力和选择性。使用分离的酶系统评估 Oclacitinib 对 JAK 家族成员的抑制活性；浓度（IC50）分别为 10、18、99 和 84 nM 时，oclacitinib 可抑制 JAK1、JAK2、JAK3 和 TYK2 50%。 oclacitinib 对 JAK1 的选择性是 JAK2 的 1.8 倍，对 JAK1 的选择性是 JAK3 的 9.9 倍，因此对 JAK1 酶表现出最高的效力。 Oclacitinib 不会抑制 38 种非 JAK 激酶（IC50 > 1000 nM），但在 10 至 99 nM 的剂量（IC50）下可抑制 JAK 家族成员 50%。 oclacitinib 的 IC50 值范围为 36 至 249 nM，还降低与过敏、炎症和瘙痒相关的 JAK1 依赖性细胞因子（IL-2、IL-4、IL-6 和 IL-13）的活性。 Oclacitinib 对不激活细胞中 JAK1 酶的细胞因子（促红细胞生成素、粒细胞/巨噬细胞集落刺激因子、IL-12、IL-23；IC50 > 1000 nM）的影响可以忽略不计[1]。与用媒介物处理的耳朵相比，用托法替尼 (0.1%) 和奥拉替尼 (0.1%) 局部治疗显着减少了小鼠耳外植体的细胞迁移（所有 P < 0.05）。当将用 JAK 抑制剂处理的每个表皮与用媒介物处理的表皮进行比较时，MHC II 类阳性细胞或朗格汉斯细胞的数量显着减少（所有 P < 0.01）[2]。

与仅使用载体组相比，高剂量 Oclacitinib 组的抓挠次数明显减少 (P<0.01)。登记的客户拥有的狗（n = 436）可能被诊断为过敏性皮炎，并且主人评估患有中度至重度瘙痒。狗被随机接受赋形剂匹配的安慰剂或奥拉替尼，剂量为 0.4-0.6 mg/kg，每天口服两次。在第0天和第7天测量皮炎的强度，并使用改进的10cm视觉模拟量表(VAS)从第0天到第7天测量瘙痒程度。狗可以接受长达 28 天的试验。 Oclacitinib 24 小时快速起效[3]。

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结果：两个治疗组的预处理所有者和兽医VAS评分相似Oclacitini在24小时内迅速起效。在每个评估日，平均Oclacitinib所有者Pruritus VAS评分明显优于安慰剂评分（P<0.0001）。经oclacitinib治疗后，瘙痒评分从7.58 cm降至2.59 cm。第7天的平均oclacitinib兽医皮炎VAS评分也明显优于安慰剂评分（P<0.0001）。两组报告的腹泻和呕吐频率相似。 结论和临床重要性：在这项研究中，奥拉西替尼对与过敏性皮炎相关的瘙痒提供了快速、有效和安全的控制，业主和兽医注意到瘙痒和皮炎VAS评分有了显著改善[3]。

Janus激酶活性测定和激酶选择性面板[1]
如前所述（Meyer等人，2010），使用Caliper微流体技术在分离的酶测定中使用JAK1（氨基酸852-1142；NP_002218）、JAK2（氨基酸808-1132；NP_004963）、JAK3（氨基酸781-1124；NP_000206）和TYK2（氨基酸870-1187；NP_003322）的重组人活性激酶结构域，以确定Oclacitinib对JAK家族成员的效力。与犬JAK酶中类似序列的序列同源性分别为98、98、100和90%（图S1）。使用SelectScreen™激酶分析服务进行Invitrogen激酶面板测试，以确定奥拉西替尼对38种不同非JAK激酶的效力奥拉西替尼的浓度为1μm。激酶特异性测定条件和数据分析在他们的网站上进行了描述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.该服务利用Z'-Lyte技术进行所有激酶筛查。所有测试都进行了两次。

白细胞介素-6细胞因子功能[1]
使用Invitrogen的CellSensor®STAT3-bla-HEK293T人上皮细胞系。将细胞在含有5%FBS的DMEM高糖培养基中以每孔1.875×105个细胞的密度铺入384孔、黑壁、透明底部的测定板中，并在37°C、5%CO2下孵育Oclacitinib（0.0000954-25μm）或载体对照加入细胞1小时。然后将每毫升20纳克hIL-6加入细胞培养物5小时。通过用LiveBLAzer™-FRET B/G底物（CCF-4 AM）检测β-内酰胺酶活性来确定IL-6对STAT3β-内酰酶报告基因的激活。使用荧光板读数器获得460和530nm处的荧光发射值。460/530 nm比值表示为对照百分比，剂量反应数据使用4参数逻辑斯谛方程进行分析。

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白细胞介素-13细胞因子功能[1]
使用美国典型培养物保藏中心的HT-29人结肠上皮细胞系。细胞在含有10%FBS、50 U/mL青霉素、50μg/mL链霉素和2 mm l-谷氨酰胺的McCoy 5A培养基中增殖。从培养瓶中胰蛋白酶消化细胞，在新鲜培养基中洗涤，并以每孔3×105个细胞的密度重新悬浮在96孔的测定板上Oclacitini（0.0015-30μm））或载体对照加入细胞中，同时在冰上放置30分钟。然后加入每毫升1纳克hIL-13。细胞在37°C水浴中孵育30分钟，然后在1.75%甲醛的PBS中固定，在含有0.5%BSA的PBS中洗涤，并在-20°C的无水甲醇中孵育过夜。固定细胞用PE标记的人pSTAT6抗体染色。使用配备板式自动取样器的FACSCalibur对样品进行分析，并使用FlowJo软件7.6.1版进行分析。数据表示为平均荧光，然后表示为对照百分比。然后使用4参数逻辑斯谛方程分析剂量反应数据。

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 (APOQUEL) 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]

C15H23N5O2S

337.44

337.157

C, 53.39; H, 6.87; N, 20.75; O, 9.48; S, 9.50

1208319-26-9

Oclacitinib maleate;1640292-55-2;Oclacitinib-13C-d3

44631938

White to off-white solid powder

1.3±0.1 g/cm3

1.618

1.46

99.36

2

6

5

23

487

0

HJWLJNBZVZDLAQ-HAQNSBGRSA-N

HJWLJNBZVZDLAQ-UHFFFAOYSA-N

InChI=1S/C15H23N5O2S/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/h7-8,10-12,16H,3-6,9H2,1-2H3,(H,17,18,19)

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

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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