JAK2 (IC50 = 5.7 nM); JAK1 (IC50 = 5.9 nM); Tyk2 (IC50 = 53nM); JAK3 (IC50 = 560nM)

在细胞实验中，Baricitinib 磷酸盐（INCB028050 磷酸盐）是 JAK 信号传导和功能的强抑制剂。 Baricitinib 的 IC50 值分别为 44 nM 和 40 nM，可防止 IL-6 刺激的经典底物 STAT3 (pSTAT3) 磷酸化以及随后在 PBMC 中产生趋化因子 MCP-1。 INCB028050 还可抑制分离的初始 T 细胞中由 IL-23 激活的 pSTAT3 (IC50=20 nM)。值得注意的是，在 IC50 值为 50 nM 时，这种抑制作用阻止了 Th17 细胞（具有可观察到的炎症和致病特征的辅助 T 细胞的一个子集）产生两种致病性细胞因子（IL-17 和 IL-22）。另一方面，当在高达 10 μM 的浓度下进行评估时，结构相似但无效的 JAK1/2 抑制剂 INCB027753 和 INCB029843 在任何这些测定系统中都没有明显的作用[1]。

与相比，磷酸巴瑞克替尼（INCB028050磷酸盐）治疗在治疗的前两周，在剂量为1 mg/kg时可抑制后爪体积的增加50%，在剂量为3或10 mg/kg时可抑制后爪体积的增加95%以上到车辆。对于肿胀明显改善的动物来说，抑制 >100% 是可行的，因为基线爪体积测量是在治疗第 0 天在具有明显疾病症状的动物中获得的[1]。与给予载体对照的小鼠相比，用巴瑞克替尼（0.7 mg/天）治疗的小鼠通过 H&E 染色测量显示炎症显着减少、CD8 浸润减少以及 I 类和 II 类 MHC 表达减少。与用载体对照治疗的小鼠相比，用巴瑞克替尼治疗的小鼠中 CD8+NKG2D+ 细胞的数量显着减少，CD8+NKG2D+ 细胞是人类和动物斑秃 (AA) 的重要疾病效应细胞。

使用具有重组表位标记的激酶结构域（JAK1837-1142；JAK2828-1132；JAK3718-1124；Tyk2873-1187）或全长酶（cMET和Chk2）和肽底物的均匀时间分辨荧光测定法进行酶测定。在测定缓冲液中使用或不使用测试化合物（11点稀释）、JAK、cMET或Chk2酶、500nM（对于Chk2为100nM）肽、ATP（在每种激酶特异性的Km或1mM）和2.0%DMSO进行每种酶反应。计算出的IC50值是抑制50%荧光信号所需的化合物浓度。使用200 nM的标准条件在Cerep进行额外的激酶测定。测试的酶包括：Abl、Akt1、AurA、AurB、CDC2、CDK2、CDK4、CHK2、c-kit、EGFR、EphB4、ERK1、ERK2、FLT-1、HER2、IGF1R、IKKα、IKKβ、JNK1、Lck、MEK1、p38α、p70S6K、PKA、PKCα、Src和ZAP70。[1]

细胞测定[1]
通过leukapetheresis和Ficoll-Hypaque离心分离人PBMC。为了测定IL-6诱导的MCP-1的产生，在存在或不存在各种浓度的INCB028050的情况下，将PBMC以3.3×105个细胞/孔的速度接种在RPMI 1640+10%FCS中。在室温下与化合物预孵育10分钟后，通过向每个孔中加入10ng/ml人重组IL-6来刺激细胞。将细胞在37°C、5%CO2下孵育48小时。采集上清液并通过ELISA分析人MCP-1的水平。INCB028050抑制IL-6诱导的MCP-1分泌的能力被报道为50%抑制所需的浓度（IC50）。使用Cell Titer Glo在标准测定条件下在3天内进行Ba/F3-TEL-JAK3细胞的增殖。 为了测定IL-23诱导的IL-17和IL-22，将PBMC维持在补充有10%FBS、2mM l-谷氨酰胺、100μg/ml链霉素和100U/ml青霉素的RPMI 1640培养基中。通过用抗CD3和抗CD28 Abs培养来活化T细胞。2天后，洗涤细胞并用IL-23（100ng/ml）、IL-2（10ng/ml）和各种浓度的INCB028050再培养。将细胞在37°C下再孵育4天，然后收集上清液，并通过ELISA测量IL-17和IL-22的分泌。INCB028050抑制IL-23诱导的IL-17和IL-22分泌的能力被报道为50%抑制所需的浓度（IC50）。

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磷酸-STAT3分析[1]
分离的细胞。[1]
为了分析人PBMC或PHA刺激的T细胞中的磷酸化-STAT3，在用不同浓度的INCB028050预孵育10−15分钟并用IL-6（100 ng/ml）、IL-12（20 ng/ml）或IL-23（100 ng/ml）刺激细胞15分钟后制备细胞提取物。然后通过使用磷酸化-STAT3特异性ELISA分析提取物的磷酸化STAT3。
全血。[1]
将从大鼠抽取的血液收集到肝素化管中，然后等分到微量离心管中（每个样品0.3ml）。在刺激实验中，在37°C下用人IL-6（100 ng/ml）刺激15分钟之前，加入不同浓度的INCB028050 10分钟。使用低渗条件裂解RBCs。然后将WBC快速成丸并裂解以制备总的细胞提取物。使用磷酸化STAT3特异性ELISA分析提取物中的磷酸化STAT3。在INCB028050给药后的不同时间从给药INCB02805的动物中抽取血液，并如上所述进行处理。

In vivo experiments[1]
Animals were housed in a barrier facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All of the procedures were conducted in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals and with Incyte Animal Care and Use Committee guidelines. Animals were fed standard rodent chow and provided with water ad libitum.

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Pharmacokinetics.[1]
Female rats (n = 6 per gender per group) were given a dose of 10 mg/kg INCB028050 suspended in 0.5% methylcellulose and given by oral gavage at 10 ml/kg. The first three rats were bled at 0 (predose), 2, 8, and 24 h, and the second three rats were bled 1, 4, and 12 h after dosing. EDTA was used as the anticoagulant, and samples were centrifuged to obtain plasma. An analytical method for the quantification of INCB028050 has been developed and used to analyze samples from toxicology studies. The method combines a protein precipitation extraction with 10% methanol in acetonitrile and LC/MS/MS analysis. The method has demonstrated a linear assay range 1–5000 nM using 0.1 ml of study samples. Data were processed using Analyst 1.3.1. A standard curve was determined from peak area ratio versus concentration using a weighted linear regression (1/x2).

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Rat adjuvant-induced arthritis.[1]
Adjuvant-induced arthritis was elicited in rats according to established methods. Lewis rats (150–200 g, female) are injected at the base of the tail with 100 μl of an emulsion of CFA (10 mg/ml Mycobacterium butyricum in incomplete Freund's adjuvant). Rats exhibited signs of inflammation within 2 wk of the injection of CFA. Each rat paw was scored following visual observation using a rating of 0–3, (0 = normal; 1 = redness and minimal swelling of digits; 2 = moderate swelling of the digits and/or paw; 3 = severe swelling of digits and/or paw). Individual animal paw scores are combined and recorded as a sum of all four paws and groups means of these totals are reported. Percent inhibition in clinical score/severity is calculated using the following formula: In addition, a plethysmometer was used to measure paw volumes taken at baseline and study termination. At the termination of the experiment, paws were removed from euthanized rats for histologic analyses. Treatment was initiated when significant signs of disease were noted, and groups of animals were sorted so that mean scores would be equivalent—usually occurring 2 wk after adjuvant injection. Graphs reflect endpoints collected only immediately prior to and after therapy was initiated (treatment day 0). Groups consisted of six animals, and statistical differences between treatment and vehicle controls were assessed using two-tailed Student t tests or ANOVA with a Dunnett’s test when appropriate.

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Collagen-induced arthritis.[1]
DBA/1j mice (4–5-wk old males) were purchased from The Jackson Laboratory (Bar Harbor, ME). The model was established as described with minor modifications. Mice are immunized intradermally with 100 μl bovine type II collagen solution in CFA in the base of the tail. Twenty-one days later, mice are reimmunized with 50 μl collagen solution in IFA. Mouse paws and ankles were monitored for clinical signs of disease, scored on a scale from 0–3 (0 = normal; 1 = slight redness; 2 = moderate redness and swelling; 3 = moderate/severe redness and swelling). In the experiments performed in this study, treatment began when all animals had at least one affected paw and groups randomized to contain similar mean scores. Each group contained six animals. Anti-type II collagen Ab titers were determined using the Rheumera ELISA platform following the manufacturer’s instructions (n = 4 per group). Serum samples were diluted 1:100,000 and frozen prior to analysis. Two-tailed Student t tests were used to compare individual treatment groups to controls.

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Anti-collagen Ab-induced arthritis.[1]
BALB/c mice (7–8-wk-old, female) were purchased from Charles River Laboratories. The model was initiated as described with minor modifications. Mice were injected with 200 μl arthogenic anti-collagen Ab. Two days later, mice were injected i.p. with LPS (Escherichia coli-derived, 25 μg) and treatment was initiated the following day (n = 5 per group). Scoring of mice was similar to that described above in the collagen-induced arthritis model. Differences in clinical scores at study termination (last day shown) were analyzed for significance using a Student two-sided t test. Hematalogic parameters were measured using a Bayer Advia120. Two-tailed Student t tests were used to compare individual treatment groups to controls.

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Dissolved in 5% dimethyl acetamide, 0.5% methocellulose; 180 mg/kg/day; Oral gavage

JAK2V617F-driven mouse model

[1]. Selective inhibition of JAK1 and JAK2 is efficacious in rodent models of arthritis: preclinical characterization of INCB028050. J Immunol. 2010 May 1;184(9):5298-307.

[2]. Reversal of Alopecia Areata Following Treatment With the JAK1/2 Inhibitor Baricitinib. EBioMedicine. 2015 Feb 26;2(4):351-5.

Inhibiting signal transduction induced by inflammatory cytokines offers a new approach for the treatment of autoimmune diseases such as rheumatoid arthritis. Kinase inhibitors have shown promising oral disease-modifying antirheumatic drug potential with efficacy similar to anti-TNF biologics. Direct and indirect inhibition of the JAKs, with small molecule inhibitors like CP-690,550 and INCB018424 or neutralizing Abs, such as the anti-IL6 receptor Ab tocilizumab, have demonstrated rapid and sustained improvement in clinical measures of disease, consistent with their respective preclinical experiments. Therefore, it is of interest to identify optimized JAK inhibitors with unique profiles to maximize therapeutic opportunities. INCB028050 is a selective orally bioavailable JAK1/JAK2 inhibitor with nanomolar potency against JAK1 (5.9 nM) and JAK2 (5.7 nM). INCB028050 inhibits intracellular signaling of multiple proinflammatory cytokines including IL-6 and IL-23 at concentrations <50 nM. Significant efficacy, as assessed by improvements in clinical, histologic and radiographic signs of disease, was achieved in the rat adjuvant arthritis model with doses of INCB028050 providing partial and/or periodic inhibition of JAK1/JAK2 and no inhibition of JAK3. Diminution of inflammatory Th1 and Th17 associated cytokine mRNA levels was observed in the draining lymph nodes of treated rats. INCB028050 was also effective in multiple murine models of arthritis, with no evidence of suppression of humoral immunity or adverse hematologic effects. These data suggest that fractional inhibition of JAK1 and JAK2 is sufficient for significant activity in autoimmune disease models. Clinical evaluation of INCB028050 in RA is ongoing.[1]

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Background: Alopecia areata (AA) is an autoimmune disease resulting in hair loss with devastating psychosocial consequences. Despite its high prevalence, there are no FDA-approved treatments for AA. Prior studies have identified a prominent interferon signature in AA, which signals through JAK molecules.[2]
Methods: A patient with AA was enrolled in a clinical trial to examine the efficacy of baricitinib, a JAK1/2 inhibitor, to treat concomitant CANDLE syndrome. In vivo, preclinical studies were conducted using the C3H/HeJ AA mouse model to assess the mechanism of clinical improvement by baricitinib.[2]
Findings: The patient exhibited a striking improvement of his AA on baricitinib over several months. In vivo studies using the C3H/HeJ mouse model demonstrated a strong correlation between resolution of the interferon signature and clinical improvement during baricitinib treatment.[2]
Interpretation: Baricitinib may be an effective treatment for AA and warrants further investigation in clinical trials.[2]
Keywords: Alopecia areata; Autoimmune disease; Autoinflammatory; Baricitinib; CANDLE syndrome; Gene expression profiling; Interferon gamma; JAK inhibitor.[2]

C16H20N7O6PS

469.41

469.093

C, 40.94; H, 4.29; N, 20.89; O, 20.45; P, 6.60; S, 6.83

1187595-84-1

Baricitinib;1187594-09-7

44231848

Typically exists as white to yellow solids at room temperature

1.185

216.51

4

11

5

31

728

0

S(C([H])([H])C([H])([H])[H])(N1C([H])([H])C(C([H])([H])C#N)(C1([H])[H])N1C([H])=C(C2=C3C([H])=C([H])N([H])C3=NC([H])=N2)C([H])=N1)(=O)=O.P(=O)(O[H])(O[H])O[H]

FBPOWTFFUBBKBB-UHFFFAOYSA-N

InChI=1S/C16H17N7O2S.H3O4P/c1-2-26(24,25)22-9-16(10-22,4-5-17)23-8-12(7-21-23)14-13-3-6-18-15(13)20-11-19-14;1-5(2,3)4/h3,6-8,11H,2,4,9-10H2,1H3,(H,18,19,20);(H3,1,2,3,4)

(1-(Ethylsulfonyl)-3-(4-(7H-pyrrolo(2,3-d)pyrimidin-4-yl)-1H-pyrazol-1-yl)azetidin-3-yl)ethanenitrile phosphate

trade name Olumiant; LY3009104 phosphate; LY-3009104; LY 3009104; INCB028050 phosphate; INCB-028050; INCB 028050; Baricitinib phosphate;Baricitinib phosphate; 1187595-84-1; Baricitinib (phosphate); Baricitinib phosphate salt; INCB-28050; XIB47S8NNB;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.43 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.08 mg/mL (4.43 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (4.43 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 4 中的溶解度: 0.5% CMC+0.25% Tween 80:30mg/mL

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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：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。