JAK1 (IC50 = 10 nM); JAK2 (IC50= 28 nM); Tyk2 (IC50= 116 nM); JAK3 (IC50= 810 nM)

Filgotinib (GLPG0634) 剂量依赖性地抑制由 IL-4（一种通过 JAK1 和 JAK3 发出信号的细胞因子）介导的 Th2 细胞分化。此外，filgotinib 还在 1 μM 或更低的浓度下有效抑制 Th1 分化 [1]。 PRL 或 EPO 产生的 JAK2 同二聚体介导的信号传导 (IC50 > 10 μM) 不受 filgotinib (GLPG0634) 抑制 [2]。

Filgotinib（马来酸盐）（1–10 mg/mL；口服和静脉注射；患有胶原诱导性关节炎的大鼠）具有良好的药代动力学特征，可减少炎症细胞、骨和软骨退化以及爪子肿胀。因素大小[1]。
在经过修改的大鼠 CIA 模型中，filgotinib（GLPG0634；3、10、30 mg/kg，口服）剂量依赖性地抑制病程。 Filgotinib（50 mg/kg，op）抑制骨骼和软骨的恶化，有效减少足部T细胞（CD3+细胞）和巨噬细胞（F4/80+细胞）的浸润，并降低血液中细胞因子和趋化因子的水平，例如 IL-6、IP-10、XCL1 和 MCP-1[1]。在 CIA 大鼠模型中，filgotinib（GLPG0634；0.1 和 0.3 mg/kg）显示出有效性 [2]。

生化实验[1]
IC50测定。[1]
重组JAK1、TYK2、JAK2和JAK3 分别在50 mM HEPES （pH 7.5）、1 mM EGTA、10 mM MgCl2、2 mM DTT和0.01% Tween 20中进行活性测定。测定每组分中JAK蛋白的量，保持初始速度和随时间的线性。ATP浓度相当于实验Km值的4倍，底物浓度（光共轭的JAK-1（Tyr1023）肽）与实验测定的Km值对应。室温孵育90 min后，在Lance检测缓冲液中加入2 nM的euroium -anti-phosphotyrosine Ab 和10 mM的EDTA，测定磷酸化底物的量。在加入ATP之前，将酶与化合物在RT下预孵育60分钟，测定化合物的IC50值。

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Kd的测定。[1]
解离常数在一家CRO公司测定。将具有快速解离率的荧光标记ATP模拟物（分别为JAK1、JAK2和JAK3的PRO13、PRO14和PRO13）与纯化的jak的JH1结构域一起在20 mM MOPS （pH 7.5）、1 mM DTT、0.01% Tween 20和500 mM hydroxyectoine（仅限JAK3）中孵育30分钟。将化合物（浓度范围为520 pM至1.1 μM）添加到100% DMSO中，并测量报告位移的时间依赖性。得到探针位移50%时对应的IC50值，并根据Cheng-Prusoff方程计算Kd值。

细胞分析[1]
IL-4诱导STAT6磷酸化[1]
将THP-1细胞（ATCC TIB-202）与化合物在室温下预孵育1 h，与IL-4 （10 ng/ml）在室温下孵育60 min，并进行流式细胞术处理。细胞在Cytofix/Cytoperm缓冲液中固定，在Phosflow perm缓冲液III中冰透30分钟。阻断（Fc阻断试剂）后，用小鼠抗人pe标记的抗pSTAT6 Ab检测pSTAT6。

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IL-2、IL-3和促红细胞生成素诱导STAT5磷酸化[1]
NK-92细胞（ATCC CRL-2407） IL-2饥饿过夜，与化合物在37℃预孵育1小时，RT下IL-2 （1 ng/ml）刺激20分钟，并进行alphasgreen分析。将TF1细胞在含0.1% FBS的RPMI 1640中饥饿过夜，在室温下用化合物预孵育1小时，在室温下用IL-3 （30 ng/ml）刺激20分钟，并进行AlphaScreen分析。UT-7-红细胞生成素（EPO）细胞(UT-7的EPO依赖性衍生物；Centocor)与化合物在RT下预孵育1小时，用EPO （1 U/ml）刺激20分钟，然后进行alphasgreen分析。pSTAT5的测量基本上是根据制造商的协议使用AlphaScreen技术。

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IFN-α和IFN-γ诱导STAT1磷酸化[1]
STAT1 U2OS细胞(Invitrogen，目录号：K1469)与化合物在37℃下预孵育1 h，用30,000 U/ml IFN-αB2 (PBL IFN来源，目录号：11115-1)或20 ng/ml IFN-γ在37℃下裂解1小时（裂解缓冲液含有2 nM Tb-Ab），根据制造商的方案，在RT下孵育60分钟。pSTAT1通过时间分辨荧光共振能量转移检测。

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催乳素诱导STAT5磷酸化[1]
22Rv1细胞（ATCC CW22Rv）饥饿过夜，用化合物预孵育，用催乳素（PRL）触发；500 ng/ml人PRL 20 min)，用10 mM Tris-HCl （pH 7.5）、5 mM EDTA、150 mM NaCl、0.5% Triton X-100、50 mM NaF、30 mM焦磷酸钠、10%甘油缓冲液（含磷酸酶/蛋白酶抑制剂鸡尾酒）裂解，离心。细胞裂解液（180 μg）用于STAT5免疫沉淀(anti-STAT5 polyclonal Abs, C-17；蛋白A-Sepharose珠)。Western blotting后用密度分析法测定总STAT5和磷酸化STAT5。

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IL-3/ jak2诱导Ba/F3细胞增殖[1]
Ba/F3细胞（由V. Lacronique, Paris， France提供）依赖于IL-3和JAK2信号，与化合物在37℃孵育40 h，之后通过测量ATP含量来分析细胞增殖。

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肿瘤抑制素m诱导的HeLa细胞STAT1报告基因检测[1]
用pSTAT1报告基因构建体转染HeLa细胞（ATCC CCL-2）。LR0127)。转染24 h后，用化合物孵育1 h，用抑癌素M （OSM）触发；33 ng / ml)。孵育20 h后，裂解细胞，根据供应商推荐使用荧光素酶SteadyLite试剂盒测定荧光素酶活性。同时，测定4 mg/ml 2-硝基苯β-d-半乳糖苷存在时β-半乳糖苷酶活性。

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可拆卸的实验。[1]
从American Type Culture Collection中获得的HeLa和HCT116细胞用50 nM的ON-TARGETplus SMARTpool小干扰RNA （siRNA）转染人JAK1、JAK2、JAK3或TYK2，或用非靶向或gapdh阴性对照siRNA转染Invitrogen公司的Lipofectamine RNAiMAX转染试剂。转染4天后，将细胞饥饿过夜，用IL-6/sIL-6R（均为250 ng/ml）刺激20分钟，并根据制造商的方案使用alphasgreen技术检测pSTAT1水平。

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T细胞分化的研究进展[1]
利用淋巴细胞密度梯度离心从健康供体的肉色被毛中分离PBMCs。使用初始CD4+ T细胞分离试剂盒II，通过消耗非T辅助细胞和记忆CD4+ T细胞进一步分离初始CD4+ T细胞。分离的初始CD4+ T细胞在细胞因子存在的情况下，用板结合的抗cd3 （3 μg/ml）和抗cd28 （5 μg/ml）抗体刺激其分化为Th1、Th2或Th17 Th亚群。在10 μg/ml抗il -4 Ab、10 ng/ml IL-2和10 ng/ml IL-12的作用下培养Th1细胞。在10 μg/ml抗ifn -γ Ab （Becton Dickinson）、25 ng/ml IL-4和10 ng/ml IL-2的作用下培养Th2细胞极化。对于Th17细胞极化，使用以下细胞因子的混合物：10 ng/ml IL-6, 10 ng/ml IL-1β， 1 ng/ml TGF-β和100 ng/ml IL-23。为了监测化合物对T细胞分化的影响，在T细胞分化开始时按指定浓度添加化合物。5 d后，使用RNeasy Mini试剂盒提取RNA，进行逆转录，并通过实时检测IFN-γ （Th1标记物）、IL-13 （Th2标记物）或IL-17F （Th17标记物）的表达来监测Th亚群分化程度。

Pharmacokinetics[1]
Formulations.[1]
GLPG0634 was formulated in polyethyleneglycol 200/0.9% NaCl (60/40; v/v) for i.v. administration and in 0.5% (v/v) methylcellulose for oral administration for all in vivo studies described. Compound purity was >95% as measured by HPLC. Animals.[1]
Male Sprague Dawley rats (180–200 g) and CD1 mice (23–25 g) were obtained from Janvier and Harlan, respectively. Two days before administration of compound, rats underwent surgery to place a catheter in the jugular vein under isoflurane anesthesia. Animals were deprived of food for at least 16 h before oral dosing until 4–6 h after. Before oral dosing, animals were deprived of food for at least 12 h before compound administration until 4 h after administration. All in vivo experiments were carried out in a dedicated pathogen-free facility (22°C).

Pharmacokinetic studies.[1]
GLPG0634 was orally dosed as a single esophageal gavage at 5 mg/kg (dosing volume of 5 ml/kg) and i.v. dosed as a bolus via the caudal vein at 1 mg/kg (dosing volume of 5 ml/kg). In the rat study, each group consisted of three rats and blood samples were collected via the jugular vein. In the mouse study, each group consisted of 21 mice (n = 3/time point) and blood samples were collected by intracardiac puncture under isoflurane anesthesia. Lithium heparin was used as anticoagulant and blood was taken at 0.05, 0.25, 0.5, 1, 3, 5, and 8 h (i.v. route) and 0.25, 0.5, 1, 3, 5, 8, and 24 h (by mouth).

GLPG0634 plasma concentrations were determined by liquid chromatography–tandem mass spectrometry with a lower limit of quantification of 2 ng/ml. Pharmacokinetic parameters were calculated by noncompartmental analysis using WinNonlin software.

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In vivo pharmacology[1]
Rodent CIA models.[1]
Animals.[1]
Dark Agouti rats (females, 7–8 wk old) and DBA/1J mice (male, 6 wk old) were obtained from Janvier.

Materials.[1]
CFA and IFA were purchased from Difco (Detroit, MI). Bovine collagen type II (CII) was used. All other reagents used were of reagent grade and all solvents were of analytical grade.

CIA.[1]
One day before the start of the experiment, CII solution (2 mg/ml) was prepared with 0.05 M acetic acid and stored at 4°C. Just before the immunization, equal volumes of IFA and CII were mixed by a homogenizer in a precooled glass bottle in an ice water bath. For rat CIA experiments, the emulsion (0.2 ml) was injected intradermally at the base of the tail at day 1 and again at day 8. This immunization method was modified from published methods. The in vivo efficacy of GLPG0634 was determined after daily oral administration for a period of 14 d after onset of disease (average clinical score at onset, 2.5 ± 0.3; 10 rats/treatment group) over the dose range 0.1–30 mg/kg. The TNF-α blocker etanercept was administered three times per week at 10 mg/kg by i.p. injection. A fully active dose was reported to require repeated dosing in the 3–9 mg/kg range. In our model of Dark Agouti female rats, disease normalization was reached for 10 mg/kg etanercept dosed three times a week i.p. as measured by clinical score, inflammation, bone resorption, pannus, and cartilage damage. At day 7 or 11, 200 μl blood was collected by retro-orbital puncture with lithium heparin as anticoagulant at predose and 1, 3, and 6 h (n = 2 or 3/time point) for steady-state pharmacokinetics analysis. At sacrifice, hind paws were removed for x-ray analysis and histological examination. A Tukey multiple comparison test was used to perform a meta-analysis of three studies carried out for GLPG0634. The score of each rat was divided by the average score obtained for vehicle in the same readout and study and multiplied by 100. Relative scores were averaged per readout for all animals present in all studies that received the same dose. For mouse CIA experiments, the IFA/CII emulsion (0.2 ml) was injected intradermally at the base of the tail at day 1 and again at day 21. This immunization method was modified from published methods. The in vivo efficacy of GLPG0634 was determined after daily oral administration for a period of 14 d after onset of disease (average clinical score at onset, 2.4 ± 0.6; 10 mice/treatment group) over the dose range 50 mg/kg twice daily. Administration of etanercept and pharmacodynamic and pharmacokinetic analyses were essentially carried out as described for the rat CIA model.

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30 mg/kg daily in Rats); 50 mg/kg twice daily in Mice

In the rat model of collagen-induced arthritis (CIA), oral administration of GLPG0634 shows a marked protection from bone damage at dose of 3 mg/kg. It reduces the infiltration of inflammatory cells significantly from 1 mg/kg onward

Absorption
Filgotinib is rapidly absorbed after oral administration. Median peak plasma concentrations occurred 2-3 hours post-dose for filgotinib and 5 hours post-dose for GS-829845. Steady-state concentrations can be observed in 2-3 days for filgotinib and in 4 days for GS-829845. Food does not appear to have a significant effect on the absorption of filgotinib; therefore, the medication can be administered without regard to food. After repeated oral dosing of filgotinib 200 mg, the reported Cmax and AUCτ values of filgotinib were 2.15 ug/mL and 6.77 ugxh/mL, respectively. For GS-829845 (the major metabolite) the reported Cmax was 4.43 ug/mL and the reported AUCτ was 83.2 ugxh/mL.

Route of Elimination
Of the total administered dose of filgotinib, approximately 87% undergoes renal elimination while 15% undergoes faecal elimination.

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Metabolism / Metabolites
Carboxylesterase enzymes are involved in the metabolism of filgotinib. The carboxylesterase 2 (CES2) isoform is chiefly responsible for metabolizing filgotinib to its major metabolite, GS-829845. Although carboxylesterase 1 (CES1) plays a less prominent role in the biotransformation of filgotinib, in vitro studies have demonstrated that CES1 will partially compensate in the event of CES2 saturation. GS-829845 is thus far the only major circulating metabolite to have been identified.

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Biological Half-Life
The half-life of filgotinib is estimated to be 7 hours, while the half-life of its active metabolite GS-829845 is estimated to be 19 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Filgotinib is not approved in the United States by the Food and Drug Administration. No information is available on the clinical use of filgotinib during breastfeeding. The European manufacturer recommends that breastfeeding be discontinued during filgotinib therapy.

◉ 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
Approximately 55-59% of filgotinib is protein-bound, while 39-44% of the active metabolite GS-829845 is protein-bound.

[1]. Preclinical characterization of GLPG0634, a selective inhibitor of JAK1, for the treatment of inflammatory diseases. J Immunol. 2013 Oct 1;191(7):3568-77.

[2]. Filgotinib for the treatment of Crohn's disease. Expert Opin Investig Drugs. 2018 Mar;27(3):295-300.

Drug Indication
Rheumatoid arthritis Jyseleca 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). Jyseleca may be used as monotherapy or in combination with methotrexate (MTX). Ulcerative colitisJyseleca is indicated for the treatment of adult patients with moderately to severely active ulcerative colitis who have had an inadequate response with, lost response to, or were intolerant to either conventional therapy or a biologic agent.

C25H27N5O7S

541.5762

541.163

C, 55.44; H, 5.03; N, 12.93; O, 20.68; S, 5.92

1802998-75-9

Filgotinib;1206161-97-8

131801100

White to off-white solid powder

180

3

10

7

38

834

0

C1CC1C(=O)NC2=NN3C(=N2)C=CC=C3C4=CC=C(C=C4)CN5CCS(=O)(=O)CC5.C(=C\C(=O)O)\C(=O)O

BFENHEAPFWQJFL-BTJKTKAUSA-N

InChI=1S/C21H23N5O3S.C4H4O4/c27-20(17-8-9-17)23-21-22-19-3-1-2-18(26(19)24-21)16-6-4-15(5-7-16)14-25-10-12-30(28,29)13-11-25;5-3(6)1-2-4(7)8/h1-7,17H,8-14H2,(H,23,24,27);1-2H,(H,5,6)(H,7,8)/b;2-1-

(Z)-but-2-enedioic acid;N-[5-[4-[(1,1-dioxo-1,4-thiazinan-4-yl)methyl]phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxamide

Filgotinib maleate; 1802998-75-9; Filgotinib (maleate); JG8OB4UL9Y; Filgotinib maleate [USAN]; GS-6034; (Z)-but-2-enedioic acid;N-[5-[4-[(1,1-dioxo-1,4-thiazinan-4-yl)methyl]phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxamide; Cyclopropanecarboxamide, N-(5-(4-((1,1-dioxido-4-thiomorpholinyl)methyl)phenyl)(1,2,4)triazolo(1,5-a)pyridin-2-yl)-, (2Z)-2-butenedioate (1:1);

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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：
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;
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