JAK3 (IC50 = 0.7 nM); JAK1 (IC50 = 3.9 nM); JAK2 (IC50 = 5 nM); Tyk2 (IC50 = 4.8 nM)

氢溴酸佩菲替尼（0-100 nM；3 天）以浓度依赖性方式抑制 IL-2 驱动的 T 细胞增殖[1]。氢溴酸培菲替尼 (10-1000 nM) 在大鼠全血中的平均 IC50 为 124 nM，在人类细胞中的平均 IC50 为 127 nM，以浓度依赖性方式抑制 IL-2 诱导的 STAT5 磷酸化[1]。

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佩菲替尼的体外活性[1]
pecicitinib的结构如图1所示。佩菲替尼对JAK活性的抑制呈浓度依赖性，IC50值分别为3.9 nM （JAK1）、5.0 nM （JAK2）、0.7 nM （JAK3）和4.8 nM (TYK2)（表1）。在相同的实验条件下，托法替尼的IC50值为3.7 nM （JAK1）、3.1 nM （JAK2）、0.8 nM （JAK3）和16 nM （TYK2）。这两种化合物都表现出对JAK3最有效的抑制活性。对JAK3的选择性比对JAK2的选择性高约7倍，比对托法替尼的选择性高4倍。

在佐剂诱导的关节炎大鼠模型中，氢溴酸培菲替尼（1-30 mg/kg；口服；每天一次，持续 24 天）在预防和治疗剂量方案中均表现出剂量依赖性有效性[1]。 在口服治疗的研究中，培菲替尼对AIA大鼠的足跖肿胀（ED50为2.7 mg/kg）和足跖骨破坏具有预防作用[1]。

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值得注意的是，培菲替尼剂量依赖性地改善了AIA大鼠的足跖肿胀，并抑制了il -2诱导的STAT5磷酸化。更有趣的是，在输液泵实验中，诱导50%爪肿胀抑制的血浆浓度为29.0 ng/mL (88.9 nM)，这似乎与体外全血实验中STAT5磷酸化的IC50一致（124 nM，图3B）。这些发现进一步提示jak1 /3介导的STAT5磷酸化程度可能与AIA的发病机制相关。综上所述，这些数据表明，体外il -2诱导的STAT5磷酸化测定可能是一种有用的药效学标志物，全血测定中STAT5磷酸化的抑制作用可以预测培菲替尼在AIA模型中的作用[1]。 此外，在人全血中观察到il -2诱导的STAT5磷酸化，且培菲替尼浓度依赖性地抑制STAT5磷酸化，其IC50值与大鼠全血相似。这一结果表明，在大鼠和人之间，培菲替尼对STAT5的磷酸化模式不存在种间差异[1]。

激酶分析[1]
人类JAK1、2、3和TYK2激酶结构域是市售的，使用链霉亲和素包被的96孔板进行检测。反应混合物中含有15 mM Tris-HCl （pH 7.5）、0.01%吐温20、2 mM二硫苏糖醇、10 mM MgCl2、250 nM Biotin-Lyn-Substrate-2（用于JAK1、2和3）或Biotin-IRS1-Substrate（用于TYK2）和ATP（最终浓度为200 μM [JAK1]、10 μM [JAK2]、8 μM [JAK3]和4 μM [TYK2]）。将培菲替尼或托法替尼溶解在二甲亚砜中。通过添加激酶结构域启动反应，然后在室温下孵育1小时。使用磷酸酪氨酸特异性ELISA，使用酶标抗磷酸酪氨酸抗体，以生物素- lyn -底物-2或生物素- irs -底物的磷酸化速率来测量激酶活性。在ATP浓度为10 μM的条件下，对培菲替尼进行TYK2激酶检测。

细胞增殖测定[1]
细胞类型：雄性Lewis大鼠的脾细胞
测试浓度： 0 -100 nM
孵育时间： 3 天
实验结果： 以浓度依赖性方式抑制 IL-2 诱导的 T 细胞增殖，IC50 为 10 nM。

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大鼠T细胞的增殖[1]
将雄性Lewis大鼠脾细胞悬浮于添加10%胎牛血清和50 μM 2-巯基乙醇的RPMI1640中，浓度为1.5 × 106个细胞/mL。用刀豆蛋白A在37℃下培养大鼠脾细胞24 h，诱导IL-2受体表达。然后将脾细胞与IL-2和指定浓度的培非替尼或托法替尼在96孔组织培养板中孵育。孵育3天后，将alamarBlue®添加到每个测试孔中，然后孵育4-6小时。在545 nm的激发波长和590 nm的发射波长下测量荧光强度。所有实验都进行了三次，实验分别进行了四次或一次，分别使用了培非替尼或托法替尼。对于每个个体，分别用细胞和培养基单独培养的孔作为空白，用不含JAK抑制剂的IL-2刺激细胞作为对照。为了计算JAK抑制剂的抑制率，空白和对照分别被指定为100%和0%的抑制率。

Animal/Disease Models: Seven-weeks-old female Lewis rats, adjuvant-induced arthritis (AIA) model[1]
Doses: 1, 3, 10, and 30 mg/kg
Route of Administration: Oral administration, one time/day for 24 days
Experimental Results: Dramatically inhibited the increase in paw volume at doses of 1 mg/kg or greater with an ED50 value of 2.7 mg/kg (95% confidence interval: 1.5–4.2 mg/kg). Dramatically decreased the bone destruction score at 10 mg/kg or greater and almost fully ameliorated both paw swelling and bone destruction scores at 30 mg/kg.

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For the oral administration regimen, four of the adjuvant-injected groups received peficitinib (1, 3, 10, and 30 mg/kg) dissolved in 0.5% methylcellulose (MC) once daily. Rats in the normal and control groups received 0.5% MC alone. Two different dosing regimens, prophylactic and therapeutic, were employed using the same set of rats described above. For the prophylactic dosing regimen, rats received peficitinib or 0.5% MC once daily for 24 days starting the day after adjuvant injection. Body weight was measured on days 4, 10, 15, 21, and 25 post-adjuvant injection, and paw volume was measured on days 10, 15, 21, and 25 post-adjuvant injection. For the therapeutic dosing regimen, adjuvant-injected rats were grouped evenly based on body weight and increase in left hind paw volume at day 15. Rats received peficitinib or 0.5% MC from day 15–24, and body weight and paw volume were measured on days 15, 18, 21, and 25 post-adjuvant injection.[1]
In the prophylactic and therapeutic dosing regimens, rats were sacrificed on day 25 after measuring body weight and paw volume, and the left hind paw of each animal was collected. In the intraperitoneal infusion regimen, peficitinib was dissolved in polyethyleneglycol and an equal volume of 500 mM acetic acid at concentrations of 1, 2, and 4 mg/mL, and was administered at day 9 post-adjuvant injection via intraperitoneal infusion using an osmotic infusion pump which was implanted under sterile conditions with isoflurane anesthesia. Dosage levels were calculated as approximately 0.12, 0.24, and 0.48 mg/day per body or 0.75, 1.5, and 3 mg/kg/day per unit body weight. Rats in the normal and control groups underwent pump implantation surgery and vehicle was administered. Body weight and paw volume were measured on days 0, 9, 15, 18, 21 and 24 post-adjuvant injection.[1]

Pharmacokinetic data from a 4-week repeated oral dose study of peficitinib at 3 mg/kg using female SD rats showed a maximum plasma concentration (Cmax) of 367 ng/mL, AUC of 834 ng h/mL, and trough concentration (Ctrough) of 2.9 ng/mL (unpublished observations). Taken together with pharmacokinetic data from the present study, at ED50, the Cmax was estimated as 330 ng/mL, AUC as 751 ng h/mL, and Ctrough as 2.6 ng/mL.[1]
In our examination of the effects of continuous infusion, the plasma levels of peficitinib administered as an intraperitoneal infusion were determined, and the EC50 of paw swelling was estimated as 29.0 ng/mL. AUC was therefore calculated as 696 ng h/mL (29.0 ng/mL × 24 h) – a similar exposure level to that of orally administered peficitinib, even though the transition of plasma level differed between continuous infusion and oral administration. These findings suggest that the efficacy of peficitinib on paw swelling in the AIA model was dependent on AUC rather than Cmax or Ctrough, providing a potentially important insight for the design of a clinical dosing regimen.[1]

[1]. A novel JAK inhibitor, peficitinib, demonstrates potent efficacy in a rat adjuvant-induced arthritis model. J Pharmacol Sci. 2017 Jan;133(1):25-33.

Peficitinib has been used in trials studying the treatment and basic science of Psoriasis, Pharmacodynamics, Drug Interactions, Colitis, Ulcerative, and RHEUMATOID ARTHRITIS, among others.

C18H22N4O2

326.39

326.174

C, 66.24; H, 6.79; N, 17.17; O, 9.80

944118-01-8

Peficitinib hydrobromide;1353219-05-2;Peficitinib hydrochloride;1353219-06-3

57928403

Light yellow to yellow solid powder

1.5±0.1 g/cm3

1.777

3.26

105.02

4

4

3

24

525

2

C1[C@@H]2CC3(C[C@@H](C2NC4=C5C=CNC5=NC=C4C(=O)N)CC1C3)O

DREIJXJRTLTGJC-JQCLMNFQSA-N

InChI=1S/C18H22N4O2/c19-16(23)13-8-21-17-12(1-2-20-17)15(13)22-14-10-3-9-4-11(14)7-18(24,5-9)6-10/h1-2,8-11,14,24H,3-7H2,(H2,19,23)(H2,20,21,22)/t9?,10-,11+,14?,18?

4-[[(1R,3S)-5-hydroxy-2-adamantyl]amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide

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 (7.66 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 25.0 mg/mL澄清DMSO储备液加入到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: ≥ 2.5 mg/mL (7.66 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 100 μL 25.0 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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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。