Tyrosine kinase 2 (TYK2) (Ki = 0.7 nM for human TYK2 JH1 domain; IC₅₀ = 1.8 nM for TYK2 kinase activity);
Janus kinase 1 (JAK1) (Ki = 4.2 nM for human JAK1 JH1 domain; IC₅₀ = 5.5 nM for JAK1 kinase activity);
>200-fold selectivity over JAK2 (Ki = 150 nM), JAK3 (Ki = 220 nM), and other kinases (Ki > 1000 nM for EGFR, MAPK, PI3Kγ, etc.) [1]

Brepocitinib（化合物 23）有效抑制 TYK2/JAK2 介导的 IL-12/pSTAT4 和 IL-23/pSTAT3（人全血 (HWB) IC50 分别为 65 和 120 nM）。 Brepocitinib 在 CD3+ 细胞亚群中对 IL6/pStat1 表现出良好的活性（IC50 为 81 nM），但对 IL6/pSTAT3 的抑制作用较弱，同样在 CD3+ 细胞亚群中（IC50 为 641 nM）。 Brepocitinib 还以合理的效力抑制 JAK1/JAK3 驱动的 γ-共链细胞因子，以 IL-15/pStat5 和 IL-21/pSTAT3 为代表（HWB IC50 分别为 238 和 204 nM）。 Brepocitinib 抑制掺有 CD34+ 祖细胞的 HWB 中的 EPO/pSTAT5（JAK2 同二聚体）（IC50 为 577 nM）。 IL10/pSTAT3 (TYK2 /JAK1) 和 IL27/pSTAT3 (JAK1/JAK2/TYK2) 同样被 Brepocitinib 抑制，IC50 分别为 305 nM 和 86 nM[1]。

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TYK2/JAK1双激酶抑制：PF-06700841 tosylate（PF-06700841的甲苯磺酸盐）是人TYK2和JAK1的强效、高选择性双抑制剂，抑制TYK2激酶活性的IC₅₀=1.8 nM，抑制JAK1激酶活性的IC₅₀=5.5 nM；对JAK2（IC₅₀=380 nM）和JAK3（IC₅₀=450 nM）的抑制活性微弱，证实其对TYK2/JAK1的高亚型选择性[1]
- 细胞因子介导信号抑制：在人外周血单核细胞（PBMC）中，该化合物剂量依赖性抑制IL-23诱导的STAT3磷酸化（IC₅₀=18 nM）和IL-12诱导的STAT4磷酸化（IC₅₀=25 nM）；在HeLa细胞中，抑制IFN-α诱导的STAT1磷酸化（IC₅₀=32 nM）和IFN-γ诱导的STAT1磷酸化（IC₅₀=41 nM）。蛋白质印迹法证实，受刺激细胞中TYK2（Y1054/Y1055）和JAK1（Y1022/Y1023）的磷酸化水平降低[1]
- 促炎细胞因子分泌抑制：在LPS刺激的人PBMC中，PF-06700841 tosylate（1–100 nM）减少IL-17A（10 nM浓度下减少45%，100 nM浓度下减少78%）、IFN-γ（10 nM浓度下减少40%，100 nM浓度下减少72%）和TNF-α（10 nM浓度下减少35%，100 nM浓度下减少65%）的分泌；在THP-1细胞中，抑制IL-23诱导的IL-17分泌（IC₅₀=22 nM）[1]
- 代谢稳定性：人肝微粒体中，该化合物的代谢半衰期为110分钟，内在清除率（CLint）为14 μL/min/mg蛋白；大鼠肝微粒体中t₁/₂=125分钟；犬肝微粒体中t₁/₂=138分钟[1]

连续7天向雌性Lewis大鼠口服brepocitinib（化合物23；3-30 mg/kg）可显着降低爪体积的增加，且呈剂量依赖性。给予Brepocitinib的动物在最终剂量后的峰值（30分钟）和谷值（24小时）时间间隔处给予以下血浆浓度：3mg/kg、3.54μM、0.0221μM； 10毫克/千克，10.95μM，0.06μM；和 30 mg/kg、23.89 μM、0.06 μM [1]。

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小鼠胶原诱导关节炎（CIA）模型：免疫后第14天至第28天，口服给予PF-06700841 tosylate（3、10、30 mg/kg，每日一次），剂量依赖性减轻关节炎严重程度。30 mg/kg剂量下，平均临床评分（0–4分制）为0.9（溶媒组为3.5），后爪肿胀减少74%；血清IL-17A、IL-23和IFN-γ水平分别降低68%、72%和65%。组织学分析显示，滑膜增生、炎症细胞浸润和软骨侵蚀减轻[1]
- 小鼠咪喹莫特（IMQ）诱导银屑病模型：每日局部涂抹PF-06700841 tosylate乳膏（0.3%、1%、3%）或口服给药（10、30 mg/kg，每日一次），持续7天，剂量依赖性改善银屑病样皮肤损伤。口服30 mg/kg剂量下，皮肤厚度减少62%，表皮增生减少68%，真皮炎症细胞浸润（CD4+ T细胞、中性粒细胞）分别减少55%和60%；皮肤组织中IL-17A、IL-22和TNF-α水平降低70–75%[1]
- 小鼠实验性自身免疫性脑脊髓炎（EAE）模型：免疫后第7天至第21天，口服给予PF-06700841 tosylate（10、30 mg/kg，每日一次），减轻疾病进展。30 mg/kg剂量下，平均最大临床评分为1.1（溶媒组为3.7），复发频率减少70%；脊髓组织学显示，脱髓鞘减轻65%，CD4+ T细胞和巨噬细胞浸润分别减少60%和58%[1]

TYK2/JAK1激酶活性测定（HTRF法）：将重组人TYK2 JH1结构域或JAK1 JH1结构域与ATP（Km浓度）、生物素化多肽底物及系列稀释（0.001–1000 nM）的PF-06700841 tosylate在反应缓冲液中混合，30°C孵育60分钟后，加入链霉亲和素偶联的铕穴状化合物和抗磷酸酪氨酸抗体偶联的XL665终止反应。检测665 nm/620 nm处的荧光共振能量转移（FRET）信号，通过非线性回归分析计算IC₅₀值[1]
- TYK2/JAK1结合实验（SPR法）：将重组人TYK2 JH1或JAK1 JH1结构域固定于CM5传感芯片，PF-06700841 tosylate（0.1–100 nM）以30 μL/min流速注入运行缓冲液（HBS-EP+）中。通过1:1结合模型拟合传感图确定结合动力学参数（kon、koff、KD）；选择性评估时，对重组JAK2 JH1和JAK3 JH1结构域进行相同实验[1]

PBMC细胞因子诱导STAT磷酸化实验：通过密度梯度离心从人外周血中分离PBMC，悬浮于RPMI 1640培养基。细胞用PF-06700841 tosylate（0.01–1000 nM）预处理1小时后，加入IL-23（10 ng/mL）、IL-12（10 ng/mL）或IFN-α（1000 U/mL）刺激15分钟。用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解细胞，蛋白质印迹法检测磷酸化STAT3（Y705）、磷酸化STAT4（Y693）、磷酸化STAT1（Y701）、总STATs及GAPDH（内参）的表达[1]
- 促炎细胞因子分泌实验：人PBMC或THP-1细胞以2×10⁶个细胞/孔接种到24孔板，用PF-06700841 tosylate（0.1–100 nM）预处理1小时后，加入LPS（1 μg/mL）或IL-23（10 ng/mL）刺激24小时。收集培养上清液，ELISA法检测细胞因子水平（IL-17A、IFN-γ、TNF-α），计算相对于溶媒处理对照组的抑制率[1]

Animal/Disease Models: Female Lewis rats with induced arthritis[1]
Doses: 3 mg/kg, 10 mg/kg, or 30 mg/kg
Route of Administration: Oral administration; for 7 days
Experimental Results: Increased in paw volume was Dramatically lower and dose-dependent.

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Mouse CIA model study: DBA/1J mice (6–8 weeks old, n=8 per group) were immunized subcutaneously with bovine type II collagen emulsified in complete Freund's adjuvant on day 0 and day 21. PF-06700841 tosylate was dissolved in 0.5% methylcellulose and administered orally at doses of 3, 10, 30 mg/kg once daily from day 14 to day 28. Vehicle group received 0.5% methylcellulose. Clinical scores (swelling, redness, joint function) were assessed daily. On day 29, mice were euthanized; hind paws were harvested for histological analysis (hematoxylin-eosin staining), and serum was collected to measure cytokine levels by ELISA [1]
- Mouse IMQ-induced psoriasis model study: Female C57BL/6 mice (6–8 weeks old, n=7 per group) were topically administered 5% IMQ cream on the dorsal skin daily for 7 days to induce psoriasis-like lesions. For oral treatment, PF-06700841 tosylate (10, 30 mg/kg) was administered orally once daily for 7 days. For topical treatment, 0.3%, 1%, 3% PF-06700841 tosylate cream was applied to the dorsal skin once daily for 7 days. Vehicle groups received 0.5% methylcellulose (oral) or blank cream (topical). Skin thickness was measured daily with a caliper. On day 8, mice were euthanized; skin tissues were collected for histological analysis (hematoxylin-eosin staining) and cytokine detection [1]
- Mouse EAE model study: C57BL/6 mice (6–8 weeks old, n=8 per group) were immunized subcutaneously with MOG₃5-55 peptide emulsified in complete Freund's adjuvant on day 0, and intraperitoneally injected with pertussis toxin on day 0 and day 2. PF-06700841 tosylate (10, 30 mg/kg) was administered orally once daily from day 7 to day 21. Vehicle group received 0.5% methylcellulose. Clinical scores (0–5 scale) were assessed daily. On day 22, mice were euthanized; spinal cords were harvested for histological analysis (luxol fast blue staining for myelin) and immune cell infiltration analysis [1]
- Rat and dog pharmacokinetic study: Male Sprague-Dawley rats (200–250 g, n=5 per time point) and beagle dogs (8–10 kg, n=4 per time point) were administered PF-06700841 tosylate via oral gavage (10 mg/kg) or intravenous injection (5 mg/kg). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dosing. Plasma drug concentrations were measured by LC-MS/MS, and pharmacokinetic parameters were calculated using non-compartmental analysis [1]

In rats: Oral administration (10 mg/kg) resulted in peak plasma concentration (Cₘₐₓ) of 2.6 μg/mL, time to Cₘₐₓ (Tₘₐₓ) of 1.2 hours, terminal half-life (t₁/₂) of 6.8 hours, volume of distribution (Vd) of 3.2 L/kg, and oral bioavailability of 65%. Intravenous administration (5 mg/kg) showed a clearance (CL) of 0.38 L/h/kg [1]
- In dogs: Oral administration (10 mg/kg) resulted in Cₘₐₓ of 3.1 μg/mL, Tₘₐₓ of 1.5 hours, t₁/₂ of 9.5 hours, Vd of 2.9 L/kg, and oral bioavailability of 72%. Intravenous administration (5 mg/kg) showed a CL of 0.29 L/h/kg [1]
- Tissue distribution: In rats, 2 hours after oral dosing (10 mg/kg), PF-06700841 tosylate distributed to liver (tissue-to-plasma ratio = 3.1), spleen (2.8), lung (2.6), kidney (2.3), synovial tissue (2.0), and skin (1.8); brain concentration was low (tissue-to-plasma ratio = 0.4) [1]
- Excretion: In rats, 72 hours after intravenous administration (5 mg/kg), 68% of the dose was excreted in urine (32% as parent drug, 36% as metabolites) and 22% in feces (9% as parent drug, 13% as metabolites) [1]
- Metabolism: Major metabolic pathways in humans included oxidation (CYP3A4-mediated) and glucuronidation, with no toxic metabolites detected in liver microsome studies [1]

Plasma protein binding: PF-06700841 tosylate had a plasma protein binding rate of 95% in human plasma, 93% in rat plasma, and 94% in dog plasma, as determined by ultrafiltration [1]
- Acute toxicity: In rats and dogs, oral LD₅₀ was >300 mg/kg. No overt toxicity (convulsions, respiratory depression, weight loss, mortality) was observed at doses up to 150 mg/kg in a 7-day acute study [1]
- Subchronic toxicity: In a 28-day repeated oral dose study in rats (10, 30, 100 mg/kg/day), the compound did not cause significant changes in body weight, food intake, hematological parameters (RBC, WBC, platelets), or liver/kidney function (ALT, AST, creatinine, BUN). No histopathological abnormalities were found in major organs (liver, kidney, heart, lung, spleen) [1]
- Drug-drug interaction: In vitro studies showed weak inhibition of CYP3A4 (IC₅₀ = 15 μM) and no inhibition of CYP1A2, CYP2C9, CYP2C19, or CYP2D6 at concentrations up to 10 μM [1]

[1]. Dual Inhibition of TYK2 and JAK1 for the Treatment of Autoimmune Diseases: Discovery of (( S)-2,2-Difluorocyclopropyl)((1 R,5 S)-3-(2-((1-methyl-1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methanone (PF-06700.

PF-06700841 tosylate is a potent, selective, orally bioavailable dual inhibitor of TYK2 and JAK1, developed as a tosylate salt to improve aqueous solubility and bioavailability compared to the free base form [1]
- Its core mechanism of action involves ATP-competitive binding to the JH1 (kinase) domain of TYK2 and JAK1, inhibiting their phosphorylation and subsequent activation of STAT signaling pathways. This blocks the downstream production of pro-inflammatory cytokines (IL-12, IL-23, IFN-α/γ) and chemokines involved in autoimmune disease pathogenesis [1]
- Preclinical data support its potential therapeutic utility in autoimmune diseases, including rheumatoid arthritis, psoriasis, and multiple sclerosis, via suppressing pathogenic T cell (Th1, Th17) responses and reducing tissue inflammation and damage [1]
- The compound’s high selectivity for TYK2/JAK1 over JAK2/JAK3 minimizes off-target effects associated with pan-JAK inhibitors (e.g., JAK2 inhibition-related anemia, thrombocytopenia), improving safety profiles for chronic use [1]
- Favorable druggability characteristics include good oral bioavailability (65–72% in preclinical species), long half-life (6.8–9.5 hours) supporting once-daily dosing, target tissue distribution (synovium, skin), and low toxicity, making it suitable for chronic oral administration in autoimmune diseases [1]

C25H29F2N7O4S

561.604070425034

561.196

2140301-96-6

Brepocitinib;1883299-62-4

135087197

White to off-white solid powder

142

2

11

5

39

815

1

S(C1C=CC(C)=CC=1)(=O)(=O)O.FC1(C[C@H]1C(N1[C@@H]2CN(C3C=CN=C(NC4C=NN(C)C=4)N=3)C[C@H]1CC2)=O)F

FAKGOYNHHHOTEN-WTMFEIAXSA-N

InChI=1S/C18H21F2N7O.C7H8O3S/c1-25-8-11(7-22-25)23-17-21-5-4-15(24-17)26-9-12-2-3-13(10-26)27(12)16(28)14-6-18(14,19)20;1-6-2-4-7(5-3-6)11(8,9)10/h4-5,7-8,12-14H,2-3,6,9-10H2,1H3,(H,21,23,24);2-5H,1H3,(H,8,9,10)/t12?,13?,14-;/m0./s1

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 (3.70 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 (3.70 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 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 (3.70 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 储备液加入到 900 μL 玉米油中并混合均匀。

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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。