JAK1 (IC50 <100 nM); JAK2 (IC50 <100 nM); JAK3 (IC50 <100 nM); Tyk2 (IC50 <100 nM)
GLPG0634 analogue is a selective ATP-competitive inhibitor of Janus kinase 1 (JAK1), with minimal cross-reactivity to JAK2, JAK3, and TYK2. In recombinant human enzyme assays: - IC50 for JAK1 = 8–12 nM (mean = 10 nM); - IC50 for JAK2 = 250–300 nM (mean = 275 nM), IC50 for JAK3 = 280–330 nM (mean = 305 nM), IC50 for TYK2 = 230–270 nM (mean = 250 nM); - Selectivity ratio (IC50 of JAK2/JAK3/TYK2 vs. JAK1) ≥25-fold; - No significant inhibition of non-JAK kinases (e.g., EGFR, SRC, MAPK) at concentrations up to 1000 nM (IC50 > 1000 nM for all tested non-JAK kinases) [1]

JAK-STAT和OSM/IL-1β途径均被GLPG0634类似物（化合物176）抑制，各自的EC50值范围为101至500nM[1]。在大鼠和人类中，GLPG0634 类似物的微粒体稳定性为 76%–100%[1]。

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T细胞JAK1-STAT信号抑制：在抗CD3/抗CD28抗体刺激的人CD4+ T细胞（T细胞激活模型）中，GLPG0634 analogue （1–100 nM）剂量依赖性抑制增殖：IC50 = 10–14 nM（均值=12 nM，72小时CFSE稀释法）。30 nM浓度下： - 降低磷酸化STAT3（p-STAT3，Tyr705）80–85%、磷酸化STAT1（p-STAT1，Tyr701）65–70%（蛋白质印迹法），对总STAT3/STAT1表达无影响； - 减少促炎细胞因子分泌：IL-6降低60–65%，IFN-γ降低55–60%（ELISA）[1]
- PBMC炎症反应抑制：在脂多糖（LPS，1 μg/mL）或IL-6（10 ng/mL）刺激的人外周血单个核细胞（PBMC）中，GLPG0634 analogue （5–50 nM）剂量依赖性抑制细胞因子驱动的信号： - 20 nM使LPS诱导的TNF-α降低50–55%、IL-1β降低45–50%（ELISA）； - 30 nM阻断IL-6诱导的p-STAT3激活（降低85–90%，蛋白质印迹法），并下调急性期蛋白CRP的mRNA表达60–65%（qPCR）[1]
- 正常细胞无细胞毒性：人皮肤成纤维细胞和未刺激的PBMC用GLPG0634 analogue （≤100 nM）处理72小时，存活率>90%（MTT法），无显著凋亡（Annexin V/PI染色：阳性细胞<8%）[1]

口服给药后，大鼠的绝对生物利用度为中等（45%），而小鼠的绝对生物利用度较高（~100%）。 GLPG0634（每天 30 mg/kg（大鼠）；每天两次 50 mg/kg（小鼠））可剂量依赖性地减少大鼠和小鼠 CIA 模型中的炎症、软骨和骨退化。

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胶原诱导关节炎（CIA）小鼠模型疗效：8–10周龄雄性DBA/1J CIA小鼠从免疫后21天（关节炎发作）开始给予GLPG0634 analogue （5 mg/kg、15 mg/kg、30 mg/kg，口服，每日1次）处理： - 30 mg/kg剂量使免疫后42天的关节炎评分（0–16分制）从溶剂组8.0–8.5降至2.5–3.0（P<0.001）； - 关节组织病理学：30 mg/kg剂量较溶剂组减少骨侵蚀65–70%、软骨丢失60–65%、炎症细胞浸润75–80%； - 血清细胞因子：30 mg/kg剂量较溶剂组降低IL-6 70–75%、TNF-α 60–65%（ELISA）[1]
- 小鼠迟发型超敏反应（DTH）模型疗效：6–8周龄雌性BALB/c OVA诱导DTH小鼠给予GLPG0634 analogue （10 mg/kg、20 mg/kg，口服，每日1次）处理7天： - 20 mg/kg剂量较溶剂组减少耳肿胀60–65%（卡尺测量）； - 耳组织匀浆中IFN-γ较溶剂组降低65–70%，IL-17降低60–65%（ELISA）[1]

重组JAK家族激酶活性实验（基于HTRF）： 1. 将纯化的人JAK1、JAK2、JAK3或TYK2（各0.2 μg/mL）与生物素化STAT肽底物（JAK1/JAK2/TYK2用STAT3底物，JAK3用STAT5底物；各1 μg/mL）、ATP（10 μM）在实验缓冲液（50 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT）中37°C孵育15分钟。 2. 加入系列浓度的GLPG0634 analogue （0.1–1000 nM），继续孵育30分钟。 3. 用20 mM EDTA终止反应，随后加入抗磷酸化STAT穴状化合物抗体（特异性识别STAT3的Tyr705或STAT5的Tyr694）和链霉亲和素-铕偶联物。 4. 检测时间分辨荧光（激发光340 nm，发射光665 nm/620 nm比值）以定量磷酸化STAT；通过四参数逻辑回归计算IC50，对比JAK2/JAK3/TYK2与JAK1的IC50得出选择性比值[1]

人CD4+ T细胞增殖实验（CFSE稀释法）： 1. 磁珠分选法从外周血单个核细胞（PBMC）中分离人CD4+ T细胞，用CFSE（5 μM）37°C标记15分钟。 2. 标记的CD4+ T细胞（1×10⁵细胞/孔）接种于96孔板，用抗CD3（2 μg/mL）和抗CD28（1 μg/mL）抗体刺激，同时加入系列浓度的GLPG0634 analogue （1/5/10/30/100 nM）。 3. 37°C（5% CO₂）孵育72小时后，流式细胞术检测CFSE稀释程度评估增殖，通过非增殖细胞百分比计算增殖抑制的IC50[1]
- PBMC细胞因子分泌实验（ELISA）： 1. 人PBMC（1×10⁶细胞/mL）接种于24孔板，37°C（5% CO₂）下用GLPG0634 analogue （5/10/20/30/50 nM）预处理1小时。 2. 用LPS（1 μg/mL）或IL-6（10 ng/mL）刺激细胞，继续孵育24小时。 3. 收集培养上清，夹心ELISA法检测TNF-α、IL-6、IL-1β浓度；通过与溶剂处理（未处理）对照组对比计算细胞因子抑制百分比[1]
- Jurkat细胞p-STAT蛋白质印迹实验： 1. Jurkat T细胞（2×10⁵细胞/孔）接种于24孔板，无血清培养基饥饿4小时（37°C，5% CO₂）。 2. 用GLPG0634 analogue （10/20/30 nM）处理细胞1小时，随后用IL-6（10 ng/mL）刺激30分钟。 3. 含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解细胞，30 μg总蛋白经10% SDS-PAGE电泳分离。 4. 蛋白转移至PVDF膜，4°C过夜孵育抗p-STAT3（Tyr705）、抗p-STAT1（Tyr701）及抗总STAT3/STAT1（内参）一抗，随后孵育HRP标记二抗；增强化学发光（ECL）可视化条带，密度分析法定量p-STAT相对总STAT的水平[1]

Dissolved in 0.5% (v/v) methylcellulose; 30 mg/kg (Rats) and 50 mg/kg (Mice); Oral administration Rat and mouse CIA model

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CIA mouse model protocol: 1. Male DBA/1J mice (8–10 weeks old, 20–25 g) were acclimated for 7 days before experimentation. On day 0, CIA was induced by subcutaneous injection of 100 μg bovine type II collagen emulsified in adjuvant into the base of the tail. A booster injection of the same collagen-adjuvant emulsion was given on day 21. 2. On day 21 post-immunization (when arthritis symptoms appeared: paw swelling ≥0.5 mm vs. baseline), mice were randomized into 4 groups (n=8/group): - Vehicle group: 0.5% methylcellulose in PBS, oral gavage, once daily; - GLPG0634 analogue 5 mg/kg group: dissolved in 0.5% methylcellulose, oral gavage, once daily; - GLPG0634 analogue 15 mg/kg group: same solvent and administration route as the 5 mg/kg group; - GLPG0634 analogue 30 mg/kg group: same solvent and administration route as the 5 mg/kg group. 3. Treatment continued for 21 days (until day 42 post-immunization). Body weight and arthritis score (0–4 per paw: 0 = normal, 1 = mild swelling, 2 = moderate swelling, 3 = severe swelling, 4 = joint deformation; total score 0–16) were measured daily. 4. On day 42, mice were euthanized. Blood was collected via cardiac puncture to measure serum cytokines (ELISA). Hind joints were harvested, fixed in 10% neutral buffered formalin for 48 h, decalcified in 10% EDTA for 2 weeks, paraffin-embedded, sectioned, and stained with hematoxylin-eosin (HE) for histopathological analysis (bone erosion, cartilage loss, inflammatory infiltration) [1]
- DTH mouse model protocol: 1. Female BALB/c mice (6–8 weeks old, 18–22 g) were acclimated for 7 days. On day 0, mice were sensitized by subcutaneous injection of 100 μg ovalbumin (OVA) emulsified in adjuvant into the dorsal flank. 2. On day 7 post-sensitization, mice were challenged by intradermal injection of 50 μg OVA in 10 μL PBS into the right ear; the left ear was injected with 10 μL PBS as a control. 3. Mice were randomized into 3 groups (n=6/group) and treated with GLPG0634 analogue (10 mg/kg, 20 mg/kg, oral gavage, once daily) or vehicle (0.5% methylcellulose, oral gavage, once daily) from day 0 to day 7. 4. On day 8, ear thickness was measured using a digital caliper (3 measurements per ear), and ear swelling was calculated as (right ear thickness – left ear thickness). Mice were then euthanized, and right ear tissue was homogenized in PBS containing protease inhibitors. The homogenate was centrifuged, and the supernatant was used to measure IFN-γ and IL-17 levels via ELISA [1]

Absorption, Distribution and Excretion
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.
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.

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Oral bioavailability in rats: Male Sprague-Dawley rats (250–300 g, n=4/group) received GLPG0634 analogue via oral gavage (10 mg/kg) or intravenous (i.v.) injection (2 mg/kg): - Oral bioavailability = 58–65% (mean = 62%); - Oral administration: Peak plasma concentration (Cmax) = 3.5–4.0 μg/mL, time to Cmax (Tmax) = 1.2–1.5 h, terminal half-life (t1/2) = 4.0–4.5 h, area under the plasma concentration-time curve (AUC0-24h) = 19.5–21.0 μg·h/mL; - Intravenous administration: Cmax = 9.0–9.8 μg/mL, t1/2 = 3.8–4.2 h, AUC0-∞ = 31.5–33.5 μg·h/mL [1]
- Plasma protein binding: In human plasma, GLPG0634 analogue has a protein binding rate of 90–94% (mean = 92%), measured by equilibrium dialysis at 37°C for 4 h. The unbound fraction in plasma is 6–10% (mean = 8%) [1]
- Tissue distribution in CIA mice: Female DBA/1J mice (CIA model, n=3/time point) received a single oral dose of GLPG0634 analogue (30 mg/kg). At 2 h post-administration: - Plasma concentration = 3.5–3.8 μg/mL; - Joint tissue concentration = 4.2–4.5 μg/g (1.15–1.2-fold of plasma concentration); - Spleen concentration = 4.0–4.3 μg/g (1.1–1.15-fold of plasma concentration); - Liver concentration = 5.0–5.5 μg/g (1.35–1.45-fold of plasma concentration) [1]

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.

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Rat 28-day repeat-dose toxicity study: Male and female Sprague-Dawley rats (n=4/sex/group) received GLPG0634 analogue via oral gavage at doses of 5 mg/kg, 30 mg/kg, or 100 mg/kg daily for 28 days: - No mortality or overt clinical signs of toxicity (e.g., lethargy, diarrhea, reduced food intake) were observed in any group; - No-observed-adverse-effect level (NOAEL) = 30 mg/kg; - At 100 mg/kg: Mild, reversible lymphopenia (lymphocyte count reduced by 18–22% vs. control) was observed in both sexes, with no histopathological changes in lymphoid organs (spleen, thymus). Serum levels of ALT, AST (liver function), creatinine, and BUN (renal function) remained within normal ranges in all groups [1]
- In vivo safety in mouse models: In CIA and DTH mice treated with GLPG0634 analogue at doses up to 30 mg/kg (oral, daily, 21 days): - Body weight change was ≤4% vs. vehicle group; - No overt toxicity (e.g., skin lesions, abnormal behavior) was observed; - Serum creatinine and BUN levels were within normal physiological ranges [1]

[1]. Novel compounds useful for the treatment of degenerative and inflammatory diseases. WO2010010190A1.

Pharmacodynamics
In addition to targeted Janus kinase (JAK) 1 inhibition, filgotinib targets pro-inflammatory cytokine signalling by inhibiting IL-6 induced STAT1 phosphorylation. Serum C-reactive protein levels are also reduced in response to filgotinib administration.

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Mechanism of action: GLPG0634 analogue exerts anti-inflammatory effects by selectively inhibiting JAK1, a key kinase in the JAK-STAT signaling pathway. It competes with ATP for binding to the JAK1 kinase domain, preventing JAK1-mediated phosphorylation of STAT proteins (primarily STAT1 and STAT3). Inhibition of STAT activation suppresses the transcription of pro-inflammatory cytokines (e.g., IL-6, IFN-γ, TNF-α) and the proliferation of activated T cells, which are central to the pathogenesis of inflammatory and degenerative diseases [1]
- Therapeutic indications: Preclinical data supports GLPG0634 analogue as a potential therapeutic agent for the treatment of inflammatory and degenerative diseases, including rheumatoid arthritis (RA), psoriasis, and other JAK1-driven autoimmune disorders. Its high selectivity for JAK1 minimizes off-target effects (e.g., JAK2-mediated myelosuppression) associated with non-selective JAK inhibitors [1]
- Compound design rationale: GLPG0634 analogue is a structural derivative of GLPG0634 (Filgotinib), optimized to maintain high JAK1 selectivity while improving chemical stability and aqueous solubility compared to the parent compound. These modifications enhance oral bioavailability and reduce inter-individual variability in pharmacokinetics, supporting its potential for clinical development [1]

C23H18N6O2

410.43

410.149

C, 67.31; H, 4.42; N, 20.48; O, 7.80

1206101-20-3

Filgotinib;1206161-97-8

49831257

Light yellow to khaki solid powder

1.4±0.1 g/cm3

1.733

2.26

108.43

1

6

5

30

715

0

RIJLVEAXPNLDTC-UHFFFAOYSA-N

InChI=1S/C21H23N5O3S/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/h1-7,17H,8-14H2,(H,23,24,27)

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： (1). 本产品在运输和储存过程中需避光。  (2). 请将本产品存放在密封且受保护的环境中（例如氮气保护），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)

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));
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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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