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]。

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。

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.

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maximum plasma concentration (Cmax) of 367 ng/mL, AUC of 834 ng h/mL, and trough concentration (Ctrough) of 2.9 ng/mL (unpublished observations).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]

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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.

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The Janus kinase (JAK) family of tyrosine kinases is associated with various cytokine receptors. JAK1 and JAK3 play particularly important roles in the immune response, and their inhibition is expected to provide targeted immune modulation. Several oral JAK inhibitors have recently been developed for treating autoimmune diseases, including rheumatoid arthritis (RA). Here, we investigated the pharmacological effects of peficitinib (formerly known as ASP015K), a novel, chemically synthesized JAK inhibitor. We found that peficitinib inhibited JAK1 and JAK3 with 50% inhibitory concentrations of 3.9 and 0.7 nM, respectively. Peficitinib also inhibited IL-2-dependent T cell proliferation in vitro and STAT5 phosphorylation in vitro and ex vivo. Furthermore, peficitinib dose-dependently suppressed bone destruction and paw swelling in an adjuvant-induced arthritis model in rats via prophylactic or therapeutic oral dosing regimens. Peficitinib also showed efficacy in the model by continuous intraperitoneal infusion. Area under the concentration versus time curve (AUC) at 50% inhibition of paw swelling via intraperitoneal infusion was similar to exposure levels of AUC at 50% inhibition via oral administration, implying that AUC might be important for determining the therapeutic efficacy of peficitinib. These data suggest that peficitinib has therapeutic potential for the oral treatment of RA.[1]

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In conclusion, peficitinib is a novel, orally available JAK inhibitor which demonstrates dose-dependent efficacy both in prophylactic and therapeutic dosing regimens in an AIA rat model in which the AUC, not Cmax or Ctrough, appears to underlie therapeutic efficacy. This finding suggests that this compound may have therapeutic potential for the oral treatment of RA, and indeed, peficitinib is currently in clinical development for this purpose. In addition, our findings suggest that the ex vivo IL-2-induced STAT5 phosphorylation assay may be effective as a pharmacodynamic marker to monitor the immune response to peficitinib.[1]

C18H23BRN4O2

407.304823160172

406.1

C, 53.08; H, 5.69; Br, 19.62; N, 13.76; O, 7.86

1353219-05-2

Peficitinib;944118-01-8

67998300

Solid powder

104Ų

5

4

3

25

525

2

ZUVPMAPXNYGJQC-OGTXJUTESA-N

InChI=1S/C18H22N4O2.BrH/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);1H/t9?,10-,11+,14?,18?;

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

Peficitinib hydrobromide; ASP015K hydrobromide; ASP-015K hydrobromide; 1353219-05-2; Peficitinib hydrobromide [USAN]; U55XHZ5X6P; 1H-Pyrrolo(2,3-b)pyridine-5-carboxamide, 4-((anti-5-hydroxytricyclo(3.3.1.13,7)dec-2-yl)amino)-, hydrobromide (1:1); ASP 015K hydrobromide; Peficitinib hydrobromide

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;
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。