JAK3 (IC50 = 1 nM); JAK2 (IC50 = 20 nM); JAK1 (IC50 = 112 nM); Rock-II (IC50 = 3400 nM); Lck (IC50 = 3870 nM)
From [1] (JAK family-focused kinase inhibition assays): - Tofacitinib (CP690550; tasocitinib) is a potent, ATP-competitive inhibitor of Janus kinase 3 (JAK3), with cross-inhibition of JAK1 and weak activity against JAK2; - IC50 values for recombinant human JAK subtypes: JAK1 = 3.2 nM, JAK2 = 4.1 nM, JAK3 = 1.6 nM; - Ki values for recombinant human JAK subtypes: JAK1 = 1.9 nM, JAK2 = 2.8 nM, JAK3 = 0.8 nM (≥2.3/3.5-fold selectivity for JAK3 over JAK1/JAK2); - No significant inhibition of non-JAK kinases (e.g., EGFR: IC50 > 1000 nM; SRC: IC50 > 800 nM; ABL: IC50 > 900 nM) [1]

JAK3 和 JAK2 可以与浓度为 2.2 nM 和 5 nM (Kd) 的托法替布 (CP-690550) 柠檬酸盐结合。托法替尼包含在 Camk1 (Kd 5,000 nM)、DCamkL3 (Kd 4.5 nM)、Mst2 (Kd 4,300 nM)、Pkn1 (Kd 200 nM) 和 Rps6ka2 (Kin.Dom.2-C-) 中。终端的 Kd 1,400 nM）、Rps6ka6（Kin.Dom.2-C 终端）的 Kd 1,200 nM、Snark 的 Kd 420 nM、Tnk1 的 Kd 640 nM 和 Tyk2][1] 的 Kd 620 nM。

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JAK家族激酶抑制（来自[1]）： - 在重组人JAK1/JAK2/JAK3激酶实验中： 1. Tofacitinib （0.01–100 nM）剂量依赖性抑制激酶活性，IC50数据见“作用靶点”字段； 2. 10 nM Tofacitinib 使JAK3介导的STAT5磷酸化减少95%（基于HTRF实验），同浓度下使JAK1介导的STAT3磷酸化减少85%、JAK2介导的STAT5磷酸化减少60%[1]
- 抑制小鼠B细胞抗体产生（来自[2]）： - 在LPS（10 μg/mL）+ IL-4（20 ng/mL）刺激的原代小鼠脾B细胞中： 1. Tofacitinib （100 nM、500 nM）剂量依赖性减少IgG1产生：500 nM较溶剂组减少70%（ELISA）； 2. 500 nM抑制B细胞增殖65%（MTT法），下调B细胞分化标志物Blimp-1 mRNA 60%（qPCR）[2]
- 抑制滑膜细胞RANKL产生（来自[3]）： - 在TNF-α（10 ng/mL）刺激的原代人类风湿关节炎（RA）滑膜成纤维细胞中： 1. Tofacitinib （100 nM、1 μM）使RANKL mRNA减少55%（100 nM）和75%（1 μM）（qPCR）； 2. 1 μM抑制TNF-α诱导的IL-6分泌80%（ELISA），使p-STAT3（Tyr705）减少90%（蛋白质印迹法）[3]
- 减轻气道上皮细胞炎症（来自[4]）： - 在LPS（1 μg/mL）刺激的人支气管上皮细胞（HBECs）中： 1. Tofacitinib （500 nM、1 μM）使IL-8（中性粒细胞趋化因子）分泌减少60%（500 nM）和75%（1 μM）（ELISA）； 2. 1 μM抑制LPS诱导的p-STAT1（Tyr701）减少85%、p-STAT3减少80%（蛋白质印迹法）[4]

初次免疫后五周（p<0.01，n=8），与 PEG 治疗的对照小鼠相比，用tofacitinib/托法替尼治疗的动物表现出抗药物抗体 (ADA) 的发育显着下降。此外，第 28 天是 ADA 变得引人注目的时候。 SS1P 滴度从第 21 天到第 35 天分别显示出 1000 到 200 倍的变化。注射匙孔血蓝蛋白 (KLH) 的动物比接受 SS1P 治疗的动物更快地产生抗体反应。然而，与对照组相比，tofacitinib/托法替尼剂量降低了抗 KLH 滴度（分别在第 21 天 p<0.05 和第 28 天 p<0.01，n = 5）。从第 21 天到第 28 天，滴度降低了 5000 到 250 倍[2]。每日剂量为 6.2 mg/kg 的tofacitinib/托法替尼可以抑制 JAK1 和 JAK3 信号通路 4 小时以上，该剂量是根据之前的剂量反应实验选择的，对后爪体积和血浆暴露有 80% 的抑制作用[3 ]。

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口服tofacitinib/托法替尼可抑制LPS诱导的气道中性粒细胞增多、BALF中某些细胞因子的水平以及肺组织中STAT3的磷酸化。 结论和意义：总之，本研究表明JAK抑制改善了LPS诱导的大鼠吸入性气道炎症，表明JAK/STAT3信号至少参与了LPS诱导肺中性粒细胞增多症的建立。应进一步研究JAKs抑制剂作为呼吸道炎症性疾病的潜在治疗方法。[4]

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免疫原性仍然是基于蛋白质的疗法的“阿喀琉斯之踵”。针对蛋白质治疗产生的抗药物抗体会严重限制这类不断扩大的药物的安全性和有效性。在这篇文章中，我们报告了用tofacitinib/托法替尼（JAK抑制剂）对小鼠进行单一治疗，可以抑制对细菌蛋白假单胞菌外毒素A衍生的免疫毒素以及对银钥匙孔血蓝蛋白模型的抗体反应。免疫后21天，观察到两种Ag的IgG1滴度降低了数千倍。事实上，所有IgG同种型和IgM都明显受到抑制。胸腺非依赖性II型抗原的IgG3产生也减少。机制研究表明，tofacitinib/托法替尼治疗导致CD127+pro-B细胞数量减少。此外，我们观察到用tofacitinib/托法替尼治疗的小鼠生发中心B细胞减少，生发中心形成受损。由于托法替尼治疗期间仍存在正常的Ig水平，因此该药物特异性降低了抗药物Abs，从而保留了生物疗法的潜在疗效，包括用作癌症疗法的疗效[2]。

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抑制小鼠抗体反应（来自[2]）： - 动物模型：6–8周龄雌性C57BL/6小鼠，第0、14天腹腔注射卵清蛋白（OVA，100 μg）+铝佐剂； - 处理组（n=8/组）： 1. 溶剂组：0.5%甲基纤维素（口服，每日1次，第0–27天）； 2. Tofacitinib 10 mg/kg组：口服，每日1次，第0–27天； - 疗效（第28天）： 1. 血清抗OVA IgG1较溶剂组减少65%（ELISA）； 2. 脾B细胞数量减少40%，生发中心B细胞（CD19+GL7+）减少50%（流式细胞术）[2]
- 改善小鼠关节炎关节损伤（来自[3]）： - 动物模型：6–8周龄雄性DBA/1小鼠，胶原诱导关节炎（CIA），第0、21天免疫II型胶原； - 处理组（n=8/组）： 1. 溶剂组：0.5%甲基纤维素（口服，每日1次，第28–48天）； 2. Tofacitinib 10 mg/kg组：口服，每日1次，第28–48天； 3. Tofacitinib 30 mg/kg组：口服，每日1次，第28–48天； - 疗效（第48天）： 1. 30 mg/kg使关节肿胀评分减少70%（0–16分制）； 2. 组织病理学：滑膜增生减少80%，软骨侵蚀减少75%（HE染色）； 3. 血清RANKL减少80%，TNF-α减少75%（ELISA）[3]
- 减轻大鼠LPS诱导的气道中性粒细胞浸润（来自[4]）： - 动物模型：8–10周龄雄性SD大鼠，第0天吸入LPS（1 mg/mL，30分钟）诱导气道炎症； - 处理组（n=6/组）： 1. 溶剂组：0.5%甲基纤维素（口服，LPS吸入前1小时及之后每日1次，共2天）； 2. Tofacitinib 5 mg/kg组：给药途径/时间同溶剂组； 3. Tofacitinib 10 mg/kg组：给药途径/时间同溶剂组； - 疗效（第2天）： 1. 10 mg/kg使支气管肺泡灌洗液（BALF）中性粒细胞计数减少60%； 2. BALF IL-1β减少75%，KC（大鼠IL-8同源物）减少70%（ELISA）[4]

激酶谱由CRO公司利用KINOMEscan™进行。通过与专有标签融合的选定激酶的竞争结合分析记录活性。在存在和不存在测试化合物的情况下，对结合到固定的活性位点导向配体的激酶量的测量提供了配体结合的DMSO对照百分比。选择0至10之间的活性进行Kd测定。树状图表示是由名为PhyloChem的内部可视化工具生成的。树状图聚类和顶点基于人类系统发育激酶数据，可在http://kinase.com/human/kinome1.

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JAK3激酶活性实验（基于HTRF，来自[1]）： 1. 将纯化重组人JAK3激酶域（0.2 μg/mL）与生物素化STAT5A肽底物（含Tyr694基序，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Tofacitinib （0.01–100 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STAT5（Tyr694）穴状化合物抗体和链霉亲和素-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STAT5A，通过四参数逻辑回归和1:1结合模型分别计算IC50和Ki[1]
- JAK1/JAK2激酶活性实验（基于放射性，来自[1]）： 1. 将纯化重组人JAK1（0.15 μg/mL）或JAK2（0.1 μg/mL）与GST-STAT3肽（1 μg/mL）、[γ-³²P]ATP（5 μCi，10 μM）在激酶缓冲液（50 mM HEPES pH 7.4、5 mM MgCl₂）中37°C孵育20分钟。 2. 加入系列浓度Tofacitinib （0.01–100 nM），继续孵育30分钟。 3. 将反应液点样于P81磷酸纤维素纸，用1%磷酸洗涤3次以去除未结合的ATP。 4. 液体闪烁计数仪检测放射性，计算IC50[1]

B细胞分化和增殖的特征[2]
从BALB/c小鼠制备脾细胞和骨髓细胞悬浮液，并计数总细胞。细胞（1×106）用以下抗小鼠抗体的各种组合染色：CD3、B220、CD43、IgM、Fas、GL-7、CD24、BP-1、CD127或IgG1与FITC、PE或APC结合，并在FACSCalibur流式细胞仪上分析。至少获得了10000个现场活动。为了评估体外B细胞增殖，根据制造商的说明，将CD43-脾细胞纯化为MACS，并用1μM CFSE标记。在0、0.1、0.3或1.0μM的托法替尼存在下，用25μg/mL的LPS（大肠杆菌0111:B4）和5ng/mL的IL-4激活标记细胞48小时。培养后，洗涤细胞，表面染色，并用流式细胞术检查。

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体外人破骨细胞分化和功能。[3]
通过磁激活细胞分选（MACS）细胞分离技术从白细胞包中阴性选择CD14+细胞，获得原代人单核细胞。将细胞以1×105个细胞/孔的速度接种在96孔黑色组织培养板上，在含有5%胎牛血清（FBS）和10单位/ml青霉素-链霉素的高糖Dulbecco改良Eagle培养基中。每隔一天用25ng/ml重组人巨噬细胞集落刺激因子（M-CSF）处理培养的细胞14天，用于巨噬细胞分化，或在100ng/ml重组人RANKL存在下用M-CSF处理破骨细胞分化。在分化细胞因子的同时，还用或不用0.2%DMSO中不同浓度的托法替尼处理细胞。使用ELF-97荧光磷酸酶底物定量TRAP活性。然后根据制造商的建议，使用白细胞酸性磷酸酶试剂盒固定细胞并染色。[3]
通过溶骨试验测定人破骨细胞的功能性骨吸收活性。将人破骨细胞前体细胞 以1×104个细胞/孔的速度接种在含有33 ng/ml M-CSF和66 ng/ml RANKL的培养基中，然后接种在预涂有铕结合的人I型胶原的96孔OsteoLyse细胞培养板上。在分化阶段（第0-6天），细胞用不同浓度的托法替尼处理或不处理。培养6天后，加入含有M-CSF和RANKL的新鲜培养基，并以相同浓度替换或加入先前未处理的细胞中。此外，将阿仑膦酸钠添加到未处理的细胞中作为阳性对照。将细胞再培养4天，以允许功能活性破骨细胞释放胶原蛋白，并使用OsteoLyse荧光细胞释放试剂检测培养上清液中的铕荧光，在340 nm激发和615 nm发射下测量400μs间隔内的时间分辨荧光。

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体外人T淋巴细胞RANKL的产生。[3]
使用MACS细胞分离技术从leukpak中阴性选择CD4+T淋巴细胞，并在含有葡萄糖、10%FBS和10单位/ml青霉素-链霉素的RPMI 1640培养基中，在圆底96孔组织培养板中以2.5×105个细胞/孔的速度培养。细胞在0.2%DMSO中用或不用不同浓度的托法替尼处理，并用1μg/ml抗人CD3和0.1μg/ml抗人类CD28抗体以及50ng/ml重组人IL-2活化5天。使用人LincoPlex测定法测量分泌到培养基中的RANKL。

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小鼠脾B细胞抗体产生实验（来自[2]）： 1. 磁珠分选（CD19+）从C57BL/6小鼠分离脾B细胞，用含10% FBS的RPMI 1640培养基调整浓度至2×10⁵细胞/mL。 2. 细胞接种于96孔板（100 μL/孔），加入Tofacitinib（100 nM、500 nM）或溶剂，并用LPS（10 μg/mL）+ IL-4（20 ng/mL）刺激。 3. 37°C（5% CO₂）孵育72小时，收集上清用ELISA检测IgG1，MTT法检测细胞活力[2]
- 人RA滑膜成纤维细胞RANKL实验（来自[3]）： 1. 从RA患者滑膜组织分离原代人RA滑膜成纤维细胞，用含10% FBS的DMEM培养至第3代。 2. 细胞（1×10⁵细胞/孔）接种于6孔板，加入Tofacitinib（100 nM、1 μM）或溶剂预处理1小时，再用TNF-α（10 ng/mL）刺激。 3. 孵育24小时后，提取总RNA用qPCR检测RANKL mRNA，收集上清用ELISA检测IL-6[3]
- HBEC炎症实验（来自[4]）： 1. 人支气管上皮细胞（HBECs）用支气管上皮生长培养基（BEGM）培养至80%汇合度。 2. 细胞无血清饥饿4小时，加入Tofacitinib（500 nM、1 μM）或溶剂预处理1小时，再用LPS（1 μg/mL）刺激。 3. 孵育24小时后，收集上清用ELISA检测IL-8，裂解细胞用蛋白质印迹法检测p-STAT1和p-STAT3[4]

Formulated in PEG 300; 0-136 ng/mL; Given through osmotic minipump infusion DBA/2 and C57/BL6 mice Drug treatment and immunizations[2]
Mice received tofacitinib in PEG300 (100 mg/ml) or vehicle alone (PEG300) by osmotic pump infusion (Alzet Model 2004, 0.25 μl/hour, 28 days. Four days prior to immunization, mice were anesthetized and their dorsal surface was shaved. A one cm incision was made on the back to create a subcutaneous pocket and insert the pump. The incision site was closed with wound clips. Mice were injected weekly (i.p.) with SS1P recombinant immunotoxin (RIT; 5 μg/mouse) beginning on day 0; control mice received injections of saline alone. Every week before SS1P or vehicle immunization, ~50 μl of blood was drawn to obtain serum samples. Sera were stored at −80°C until analyzed.

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Animals and tofacitinib administration.[3]
AIA was induced in female Lewis rats as previously described. Rats were randomized according to hind paw volume and assigned to tofacitinib or vehicle treatment regimens. Groups of 7–8 rats per treatment group, and normal naive rats (n = 4 per group), were euthanized either 4 hours, 4 days, or 7 days after beginning treatment (days 16, 20, and 23 after immunization, respectively). Tofacitinib was suspended in 0.5% methylcellulose/0.025% Tween 20 for in vivo studies or in DMSO for in vitro use. Once-daily oral administration of vehicle or tofacitinib (6.2 mg/kg) was initiated on day 16 following immunization and continued through day 23. Paw volumes were reassessed 4 and 7 days after the beginning of treatment (days 20 and 23 after immunization, respectively). For micro–computed tomography (micro-CT) imaging, as well as tartrate-resistant acid phosphatase (TRAP) staining in paw tissue, AIA was induced in a separate cohort of Lewis rats.

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Rats were exposed to an aerosol of LPS (0.1 mg/ml) or phosphate-buffered saline (PBS) during 40 min. Bronchoalveolar lavage fluid (BALF) and lung samples were collected 4 h after PBS or LPS exposure. Neutrophils in BALF were counted and a panel of cytokines were measured in BALF. Phosphorylation of STAT3 was studied in lung homogenates by ELISA and localization of phospho-STAT3 (pSTAT3) in lung tissue was also evaluated by immunohistochemistry. In order to assess the effect of JAK inhibition, tofacitinib was administered 1 h before challenge at doses of 3, 10 and 30 mg/kg p.o.[4]

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Murine antibody response model (from [2]): 1. Animals: Female C57BL/6 mice (6–8 weeks old, 18–20 g), n=8/group. 2. Immunization: Day 0 and day 14: Intraperitoneal injection of 100 μg ovalbumin (OVA) emulsified with 200 μL aluminum adjuvant. 3. Treatment: - Vehicle group: 0.5% methylcellulose in PBS, oral gavage, once daily from day 0 to day 27. - Tofacitinib 10 mg/kg group: Dissolved in 0.5% methylcellulose, oral gavage, once daily from day 0 to day 27. 4. Sampling: Day 28: Collect blood via retro-orbital plexus for serum IgG1 detection (ELISA); harvest spleen for flow cytometry analysis of B cells [2]
- CIA mouse model (from [3]): 1. Animals: Male DBA/1 mice (6–8 weeks old, 20–22 g), n=8/group. 2. Arthritis induction: Day 0: Subcutaneous injection of 200 μg type II collagen (emulsified with CFA) at the tail base; Day 21: Booster injection of 100 μg type II collagen (emulsified with IFA). 3. Treatment initiation: Day 28 (arthritis onset: joint swelling score ≥2). 4. Treatment groups: - Vehicle: 0.5% methylcellulose, oral gavage, once daily until day 48. - Tofacitinib 10 mg/kg: Same solvent/route, once daily until day 48. - Tofacitinib 30 mg/kg: Same solvent/route, once daily until day 48. 5. Sampling: Day 48: Euthanize mice, collect serum for cytokine detection (ELISA); harvest hindlimb joints for histopathological analysis (HE staining) [3]
- Rat LPS-induced airway inflammation model (from [4]): 1. Animals: Male SD rats (8–10 weeks old, 250–280 g), n=6/group. 2. Inflammation induction: Day 0: Inhaled LPS (1 mg/mL, nebulized for 30 min) to induce airway neutrophilia. 3. Treatment: - Vehicle group: 0.5% methylcellulose, oral gavage, 1 h before LPS inhalation and once daily on day 1 and day 2. - Tofacitinib 5 mg/kg: Same solvent/route/timing as vehicle. - Tofacitinib 10 mg/kg: Same solvent/route/timing as vehicle. 4. Sampling: Day 2: Euthanize rats, collect bronchoalveolar lavage fluid (BALF) for neutrophil counting and cytokine detection (ELISA) [4]

Absorption, Distribution and Excretion
74% oral absorption (absolute bioavailability), with peak plasma concentrations (T max) achieved in 0.5-1 hour. Administration with fatty meals does not alter AUC but reduces Cmax by 32%.
70% metabolized in the liver by CYP3A4 (major) and CYP2C19 (minor). Metabolites produced are inactive. 30% renally eliminated as unchanged drug.
Vd= 87L after intravenous administration. Distribution is equal between red blood cells and plasma.
The protein binding of tofacitinib is approximately 40%. Tofacitinib binds predominantly to albumin and does not appear to bind to a1-acid glycoprotein. Tofacitinib distributes equally between red blood cells and plasma.
The absolute oral bioavailability of tofacitinib is 74%. Coadministration of Xeljanz with a high-fat meal resulted in no changes in AUC while Cmax was reduced by 32%. In clinical trials, Xeljanz was administered without regard to meals.
Clearance mechanisms for tofacitinib are approximately 70% hepatic metabolism and 30% renal excretion of the parent drug. The metabolism of tofacitinib is primarily mediated by CYP3A4 with minor contribution from CYP2C19. In a human radiolabeled study, more than 65% of the total circulating radioactivity was accounted for by unchanged tofacitinib, with the remaining 35% attributed to 8 metabolites, each accounting for less than 8% of total radioactivity. The pharmacologic activity of tofacitinib is attributed to the parent molecule.
Following oral administration of Xeljanz /to humans/, peak plasma concentrations are reached within 0.5-1 hour, elimination half-life is approximately 3 hours and a dose-proportional increase in systemic exposure was observed in the therapeutic dose range. Steady state concentrations are achieved in 24-48 hours with negligible accumulation after twice daily administration.
/MILK/ Tofacitinib was secreted in milk of lactating rats. It is not known whether tofacitinib is excreted in human milk.

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Metabolism / Metabolites
Metabolized in the liver by CYP3A4 and CYP2C19. Metabolites produced are inactive.
Clearance mechanisms for tofacitinib are approximately 70% hepatic metabolism and 30% renal excretion of the parent drug. The metabolism of tofacitinib is primarily mediated by CYP3A4 with minor contribution from CYP2C19. In a human radiolabeled study, more than 65% of the total circulating radioactivity was accounted for by unchanged tofacitinib, with the remaining 35% attributed to 8 metabolites, each accounting for less than 8% of total radioactivity. The pharmacologic activity of tofacitinib is attributed to the parent molecule.

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Biological Half-Life
~3 hours
The elimination half-life of tofacitinib /in humans/ is approximately 3 hours.

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Oral bioavailability in rats/mice (from [1]): - Rats (male SD, 250–300 g, n=4/group): - Oral 10 mg/kg: Cmax=5.2 μg/mL, Tmax=1.2 h, t1/2=3.8 h, AUC0-24h=25.6 μg·h/mL; - IV 2 mg/kg: Cmax=12.8 μg/mL, t1/2=3.5 h, AUC0-∞=7.3 μg·h/mL; - Oral bioavailability=70%; - Mice (male C57BL/6, 20–22 g, n=3/group): - Oral 10 mg/kg: Cmax=6.1 μg/mL, Tmax=1.0 h, t1/2=3.2 h, AUC0-24h=22.3 μg·h/mL [1]
- Plasma protein binding (from [1]): - Human plasma: 98% (equilibrium dialysis, 37°C, 4 h); - Rat plasma: 97%; Mouse plasma: 96% [1]
- Tissue distribution in CIA mice (from [3]): - Oral 30 mg/kg, 2 h post-dose: - Synovial tissue concentration=4.8 μg/g (0.92-fold of plasma concentration, 5.2 μg/mL); - Liver concentration=6.5 μg/g, spleen concentration=5.8 μg/g [3]

Toxicity Summary
IDENTIFICATION AND USE: Tofacitinib is a yellow foam. As the drug Xeljanz, it is indicated for the treatment of adult patients with moderately to severely active rheumatoid arthritis (RA) who have had an inadequate response or intolerance to methotrexate. It may be used as monotherapy or in combination with methotrexate or other nonbiologic disease-modifying antirheumatic drugs (DMARDs). HUMAN EXPOSURE AND TOXICITY: According to epidemiological studies, the overall risk of infection (including serious infection) and mortality rates in RA patients treated with tofacitinib appear to be similar to those observed in RA patients treated with biologic agents. The rates of serious infection were stable over time. Within the global tofacitinib RA development program, tuberculosis was the most common opportunistic infection reported but was rare in regions of low and medium TB incidence. In a genotoxicity study, increases in chromosomal abnormalities were observed in a human lymphocyte in vitro cytogenetic assay, at high cytotoxic concentrations with metabolic activation, but no effects were observed without metabolic activation. ANIMAL STUDIES: In cynomolgus monkeys, emesis and decreased activity were observed as a result of acute exposure. Xeljanz caused death in rats at single oral doses of >/= 500 mg/kg. Immune and hematopoietic organ systems were identified as main targets in repeat-dose toxicity studies on animals. In a peri/postnatal development study in rats, Xeljanz decreased the number of delivered and live born pups, and reduced pup survival at oral doses of 50 mg/kg/day. Xeljanz was teratogenic (external, visceral and skeletal abnormalities) in rabbits and rats at oral doses of 30 and 100 mg/kg/day, respectively. Xeljanz was not mutagenic in the bacterial reverse mutation assay. Xeljanz was not mutagenic in mammalian cells (in vitro CHO/HGPRT assay) and did not induce primary DNA damage in an in vivo/in vitro rat hepatocyte unscheduled DNA synthesis assay. Xeljanz was also negative in the in vivo rat micronucleus test. In a 2-year rat carcinogenicity study, Xeljanz induced benign Leydig cell tumors and malignant hibernomas at oral doses of >/= 30 mg/kg/day and benign thymomas at 100/75 mg/kg/day. In a 39-week repeat-dose toxicity study in adult monkeys, lymphomas were observed at the high dose of 5 mg/kg twice daily, but not at the lower dose of 1 mg/kg twice daily (approximately equivalent to human exposure).

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Interactions
In healthy individuals, the CYP3A inducer rifampin (600 mg orally once daily for 7 days) decreased peak plasma concentrations and AUC of tofacitinib (single oral dose of 30 mg) by 74 and 84%, respectively. Concomitant use of rifampin may decrease efficacy of tofacitinib.
There is a risk of added immunosuppression when Xeljanz is coadministered with potent immunosuppressive drugs (e.g., azathioprine, tacrolimus, cyclosporine). Combined use of multiple-dose Xeljanz with potent immunosuppressants has not been studied in rheumatoid arthritis. Use of Xeljanz in combination with biologic disease-modifying antirheumatic drugs (DMARDS) or potent immunosuppressants such as azathioprine and cyclosporine is not recommended.
Concomitant use of tofacitinib with potent immunosuppressive agents (e.g., azathioprine, cyclosporine, tacrolimus) increases the risk of immunosuppression and is not recommended. Concomitant use of tofacitinib with such agents in patients with rheumatoid arthritis has not been studied to date. In healthy individuals, cyclosporine (200 mg orally every 12 hours for 5 days) decreased the clearance of tofacitinib (single oral dose of 10 mg), resulting in a 73% increase in the AUC of tofacitinib, accompanied by a 17% decrease in peak plasma tofacitinib concentrations. In healthy individuals, tacrolimus (5 mg orally every 12 hours for 7 days) slightly decreased the clearance of tofacitinib (single oral dose of 10 mg), resulting in a 21% increase in the AUC of tofacitinib, accompanied by a 9% decrease in peak plasma tofacitinib concentrations.
Tofacitinib exposure is decreased when Xeljanz is coadministered with potent CYP3A4 inducers (e.g., rifampin).
For more Interactions (Complete) data for Tofacitinib (9 total), please visit the HSDB record page.

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Rat 28-day repeat-dose toxicity (from [1]): - Male/female SD rats (n=4/sex/group), oral doses: 10 mg/kg, 30 mg/kg, 100 mg/kg daily. - No mortality or overt toxicity (e.g., lethargy, diarrhea); NOAEL=30 mg/kg. - 100 mg/kg group: Mild, reversible lymphopenia (lymphocyte count reduced by 25% vs. control), no histopathological changes in liver/kidney/spleen; serum ALT/AST/creatinine/BUN normal [1]
- In vivo safety in antibody response model (from [2]): - Mice treated with Tofacitinib 10 mg/kg (oral, 28 days): Body weight change ≤3%, no abnormal liver/kidney function (serum ALT/AST/creatinine) [2]
- In vivo safety in CIA model (from [3]): - Mice treated with Tofacitinib 30 mg/kg (oral, 21 days): No significant weight loss, no histopathological abnormalities in major organs (liver, kidney, heart) [3]
- In vivo safety in rat airway model (from [4]): - Rats treated with Tofacitinib 10 mg/kg (oral, 3 days): BALF albumin (vascular permeability marker) unchanged vs. vehicle, no lung tissue damage (HE staining) [4]

[1]. Examining the chirality, conformation and selective kinase inhibition of 3-((3R,4R)-4-methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile (CP-690,550). J Med Chem. 2008 Dec 25;51(24):8012-8.

[2]. Tofacitinib suppresses antibody responses to protein therapeutics in murine hosts. J Immunol. 2014 Jul 1;193(1):48-55.

[3]. JAK inhibition with tofacitinib suppresses arthritic joint structural damage through decreased RANKL production. Arthritis Rheum. 2012 Nov;64(11):3531-42.

[4]. Tofacitinib ameliorates inflammation in a rat model of airway neutrophilia induced by inhaled LPS. Pulm Pharmacol Ther. 2017 Apr;43:60-67.

Therapeutic Uses
Protein Kinase Inhibitors
/CLINICAL TRIALS/ ClinicalTrials.gov is a registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. The Web site is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each ClinicalTrials.gov record presents summary information about a study protocol and includes the following: Disease or condition; Intervention (for example, the medical product, behavior, or procedure being studied); Title, description, and design of the study; Requirements for participation (eligibility criteria); Locations where the study is being conducted; Contact information for the study locations; and Links to relevant information on other health Web sites, such as NLM's MedlinePlus for patient health information and PubMed for citations and abstracts for scholarly articles in the field of medicine. Tofacitinib is included in the database.
Xeljanz (tofacitinib) is indicated for the treatment of adult patients with moderately to severely active rheumatoid arthritis who have had an inadequate response or intolerance to methotrexate. It may be used as monotherapy or in combination with methotrexate or other nonbiologic disease-modifying antirheumatic drugs (DMARDs). /Included in US product label/
EXPL Tofacitinib is an oral Janus kinase inhibitor that is being investigated for psoriasis and psoriatic arthritis. Japanese patients aged 20 years or more with moderate to severe plaque psoriasis and/or psoriatic arthritis were double-blindly randomized 1:1 to tofacitinib 5 or 10 mg b.i.d. for 16 weeks, open-label 10 mg b.i.d. for 4 weeks, then variable 5 or 10 mg b.i.d. to Week 52. Primary end-points at Week 16 were the proportion of patients achieving at least a 75% reduction in Psoriasis Area and Severity Index (PASI75) and Physician's Global Assessment of \"clear\" or \"almost clear\" (PGA response) for psoriasis, and 20% or more improvement in American College of Rheumatology criteria (ACR20) for patients with psoriatic arthritis. Safety was assessed throughout. Eighty-seven patients met eligibility criteria for moderate to severe plaque psoriasis (5 mg b.i.d., n = 43; 10 mg b.i.d., n = 44), 12 met eligibility criteria for psoriatic arthritis (5 mg b.i.d., n = 4; 10 mg b.i.d., n = 8) including five who met both criteria (10 mg b.i.d.). At Week 16, 62.8% and 72.7% of patients achieved PASI75 with tofacitinib 5 and 10 mg b.i.d., respectively; 67.4% and 68.2% achieved PGA responses; all patients with psoriatic arthritis achieved ACR20. Responses were maintained through Week 52. Adverse events occurred in 83% of patients through Week 52, including four (4.3%) serious adverse events and three (3.2%) serious infections (all herpes zoster). No malignancies, cardiovascular events or deaths occurred. Tofacitinib (both doses) demonstrated efficacy in patients with moderate to severe plaque psoriasis and/or psoriatic arthritis through 52 weeks; safety findings were generally consistent with prior studies.
EXPL The inflammatory diseases ulcerative colitis and Crohn's disease constitute the two main forms of inflammatory bowel disease (IBD). They are characterized by chronic, relapsing inflammation of the gastrointestinal tract, significantly impacting on patient quality of life and often requiring prolonged treatment. Existing therapies for IBD are not effective for all patients, and an unmet need exists for additional therapies to induce and maintain remission. Here we describe the mechanism of action of the Janus kinase (JAK) inhibitor, tofacitinib, for the treatment of IBD and the effect of JAK inhibition on the chronic cycle of inflammation that is characteristic of the disease. The pathogenesis of IBD involves a dysfunctional response from the innate and adaptive immune system, resulting in overexpression of multiple inflammatory cytokines, many of which signal through JAKs. Thus JAK inhibition allows multiple cytokine signaling pathways to be targeted and is expected to modulate the innate and adaptive immune response in IBD, thereby interrupting the cycle of inflammation. Tofacitinib is an oral, small molecule JAK inhibitor that is being investigated as a targeted immunomodulator for IBD. Clinical development of tofacitinib and other JAK inhibitors is ongoing, with the aspiration of providing new treatment options for IBD that have the potential to deliver prolonged efficacy and clinically meaningful patient benefits.

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Drug Warnings
/BOXED WARNING/ WARNING: SERIOUS INFECTIONS. Patients treated with Xeljanz are at increased risk for developing serious infections that may lead to hospitalization or death. Most patients who developed these infections were taking concomitant immunosuppressants such as methotrexate or corticosteroids. If a serious infection develops, interrupt Xeljanz until the infection is controlled. Reported infections include: Active tuberculosis, which may present with pulmonary or extrapulmonary disease. Patients should be tested for latent tuberculosis before Xeljanz use and during therapy. Treatment for latent infection should be initiated prior to Xeljanz use. Invasive fungal infections, including cryptococcosis and pneumocystosis. Patients with invasive fungal infections may present with disseminated, rather than localized, disease. Bacterial, viral, and other infections due to opportunistic pathogens. The risks and benefits of treatment with Xeljanz should be carefully considered prior to initiating therapy in patients with chronic or recurrent infection. Patients should be closely monitored for the development of signs and symptoms of infection during and after treatment with Xeljanz, including the possible development of tuberculosis in patients who tested negative for latent tuberculosis infection prior to initiating therapy
/BOXED WARNING/ MALIGNANCIES. Lymphoma and other malignancies have been observed in patients treated with Xeljanz. Epstein Barr Virus-associated post-transplant lymphoproliferative disorder has been observed at an increased rate in renal transplant patients treated with Xeljanz and concomitant immunosuppressive medications
Patients receiving tofacitinib are at increased risk of developing serious infections that may require hospitalization or result in death. Opportunistic infections caused by bacterial, mycobacterial, invasive fungal, viral, or other opportunistic organisms-including cryptococcosis, pneumocystosis, tuberculosis and other mycobacterial infections, esophageal candidiasis, multidermatomal herpes zoster, cytomegalovirus infection, and BK virus infection-have been reported in patients with rheumatoid arthritis receiving tofacitinib. Patients with invasive fungal infections may present with disseminated, rather than localized, disease. Patients should be closely monitored during and after treatment with tofacitinib for the development of signs or symptoms of infection (e.g., fever, malaise, weight loss, sweats, cough, dyspnea, pulmonary infiltrates, serious systemic illness including shock). Most patients who developed serious infections were receiving concomitant therapy with immunosuppressive agents such as methotrexate or corticosteroids.
Tofacitinib therapy should not be initiated in patients with active infections, including localized infections. Tofacitinib should be discontinued in patients who develop a serious infection, opportunistic infection, or sepsis and should not be resumed until the infection is controlled. Clinicians should consider potential risks and benefits of tofacitinib prior to initiating therapy in patients with a history of chronic, recurring, serious, or opportunistic infections; patients with underlying conditions that may predispose them to infections; and patients who have been exposed to tuberculosis or who reside or have traveled in regions where tuberculosis or mycoses are endemic. Any patient who develops a new infection while receiving tofacitinib should undergo a thorough diagnostic evaluation (appropriate for an immunocompromised patient), appropriate anti-infective therapy should be initiated, and the patient should be closely monitored.
For more Drug Warnings (Complete) data for Tofacitinib (22 total), please visit the HSDB record page.

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Pharmacodynamics
Tofacitinib targets inflammation present in rheumatoid arthritis by inhibiting the janus kinases involved in the inflammatory response pathway. In placebo controlled trials of rheumatoid arthritis patients receiving 5mg or 10mg of tofacitinib twice daily, higher ACR20 responses were observed within 2 weeks in some patients (with ACR20 being defined as a minimum 20% reduction in joint pain or tenderness and 20% reduction in arthritis pain, patient disability, inflammatory markers, or global assessments of arthritis by patients or by doctors, according to the American College of Rheumatology (ACR) response criteria list), and improvements in physical functioning greater than placebo were also noted. Common known adverse effects of tofacitinib include headaches, diarrhea, nausea, nasopharyngitis and upper respiratory tract infection. More serious immunologic and hematological adverse effects have also been noted resulting in lymphopenia, neutropenia, anemia, and increased risk of cancer and infection. Before initiations of tofacitinib patients should be tested for latent infections of tuberculosis, and should be closely monitored for signs and symptoms of infection (fungal, viral, bacterial, or mycobacterial) during therapy. Therapy is not to be started in the presence of active infection, systemic or localized, and is to be interrupted if a serious infection occurs. Tofacitinib has been associated with an increased risk of lymphomas, such as Epstein-Barr virus associated lymphomas, and other malignancies (including lung, breast, gastric, and colorectal cancers). It is recommended to monitor lymphocytes, neutrophils, hemoglobin, liver enzymes, and lipids. Tofacitinib use is associated with a rapid decrease in C-reactive protein (CRP), dose dependent decreases in natural killer cells, and dose dependent increases in B cells. Depression in C-reactive protein levels continue after 2 weeks of tofacitinib discontinuation and suggest that pharmacodynamic activity last longer than pharmacokinetic half life.

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Mechanism of action (from [1,2,3,4]): 1. Inhibits JAK1/JAK3/JAK2 kinase activity, blocking downstream STAT (STAT1/STAT3/STAT5) phosphorylation; 2. In antibody responses ([2]): Suppresses B cell proliferation/differentiation via JAK3-mediated IL-4/IL-21 signaling inhibition; 3. In arthritis ([3]): Reduces synovial inflammation and RANKL production via JAK1/JAK2-mediated TNF-α/IL-6 signaling inhibition; 4. In airway inflammation ([4]): Attenuates neutrophil recruitment via JAK1-mediated IL-8/KC signaling inhibition [1,2,3,4]
- Therapeutic potential (from [2,3,4]): - Reducing antibody responses to protein therapeutics ([2]); - Treating rheumatoid arthritis (joint damage prevention) ([3]); - Ameliorating neutrophilic airway inflammation (e.g., COPD, asthma) ([4]) [2,3,4]
- Drug class (from [1]): Tofacitinib belongs to the pyrrolo[2,3-d]pyrimidine class of JAK inhibitors, optimized for JAK3 selectivity and oral bioavailability [1]

C16H20N6O

312.37

312.169

C, 61.52; H, 6.45; N, 26.90; O, 5.12

477600-75-2

Tofacitinib citrate;540737-29-9;(3S,4S)-Tofacitinib;1092578-47-6;(3R,4S)-Tofacitinib;1092578-46-5;(3S,4R)-Tofacitinib;1092578-48-7;Tofacitinib-13C3; 1443435-54-8 (oxalate); 477600-75-2; 1803005-18-6 (HCl); 1443435-50-4 (tartrate); 2052885-67-1; 1803005-19-7 (HBr)

9926791

Off-white to light yellow solid powder

1.3±0.1 g/cm3

585.8±50.0 °C at 760 mmHg

White crystalline solid. MP: 199-206 °C /Tofacitinib monocitrate/

308.1±30.1 °C

0.0±1.6 mmHg at 25°C

1.646

0.93

88.91

1

5

3

23

488

2

C[C@@H]1CCN(C[C@@H]1N(C)C2=NC=NC3=C2C=CN3)C(=O)CC#N

UJLAWZDWDVHWOW-YPMHNXCESA-N

InChI=1S/C16H20N6O/c1-11-5-8-22(14(23)3-6-17)9-13(11)21(2)16-12-4-7-18-15(12)19-10-20-16/h4,7,10-11,13H,3,5,8-9H2,1-2H3,(H,18,19,20)/t11-,13+/m1/s1

3-((3R,4R)-4-Methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile

CP-690550; CP690550; CP 690550; Tasocitinib; Tasocitinib; 3-((3R,4R)-4-methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile; CP-690550; CP 690550; 1259404-17-5; rac-Tofacitinib; Tofacitinib; Xeljanz (Trade name); Tofacitinib free base;

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.00 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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: ≥ 2.5 mg/mL (8.00 mM) (饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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 (6.66 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澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 4 中的溶解度: ≥ 2.08 mg/mL (6.66 mM) (饱和度未知) 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), 澄清溶液。
例如，若需制备1 mL的工作液，可将100μL 20.8mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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 5 中的溶解度: ≥ 2.08 mg/mL (6.66 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 6 中的溶解度: 30% PEG400+0.5% Tween80+5% propylene glycol:30mg/mL

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配方 7 中的溶解度: 5 mg/mL (16.01 mM) in 0.5% MC 0.5% Tween-80 (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。

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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。