Lck (IC50 = 249 nM); PAK5 (IC50 = 166 nM)

PRT062607 (P505-15) 是一种全新、极其有效、强效的口服 Syk 小分子抑制剂。使用两种不同的纯激酶测定来研究 PRT062607 对其靶激酶 Syk 的功效。根据 FRET 测试，半最大 Syk 抑制需要 6±0.2 nM（平均值±SEM）。放射性酶测定得出的 Syk IC50 为 2.1±0.4 nM（平均值±SEM），表明效力相似。 PRT062607 有效抑制人全血中 Fcε 受体 1 介导的嗜碱性粒细胞脱颗粒 (IC50 0.15 μM) 和 B 细胞抗原受体介导的 B 细胞信号传导和激活（IC50 分别为 0.27 和 0.28 μM）[1]。

当对 CAIA 模型中的小鼠口服PRT062607 (P505-15)  时，与载体对照相比，通过每日炎症评分确定，爪子炎症平均减少了 12%、44% 和 87%。研究结束时，平均血浆浓度（24 小时内的 C 平均值）确定为 0.38、0.95 和 1.47 μM。给予 30 mg/kg PRT062607 的小鼠表现出关节退化显着减少，以至于无法将小鼠与正常小鼠区分开。到研究结束时，大鼠 CIA 模型中的大多数动物（八分之七）（平均炎症评分±SEM=0.63±1.1；与媒介物相比，p<0.0001）由于高剂量的PRT062607（15 毫克/千克，每日两次）[1]。

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在人全血中，PRT062607 (P505-15) 能有效抑制B细胞抗原受体介导的B细胞信号传导和激活（IC50分别为0.27和0.28 μM）和fce1受体介导的嗜碱性粒细胞脱粒（IC50分别为0.15 μM）。给药后，在小鼠体内测量了相似的体外抑制水平（Syk信号IC50 0.32 μM）。在更高的浓度下，syk独立的信号传导和激活不受影响，这表明激酶抑制在细胞系统中的特异性。口服P505-15在两种类风湿关节炎啮齿动物模型中产生剂量依赖性抗炎活性。在特异性抑制Syk活性约67%的浓度下，观察到统计学上显著的疗效。因此，特异性Syk抑制可以模拟Syk基因缺陷来调节免疫功能，为PRT062607 (P505-15) 治疗人类疾病提供了一种治疗策略。[2]

SYK Autophosphorylation/SYK自身磷酸化。[1]
采用PRT062607 (P505-15) 或不用对Ramos细胞进行预孵育30分钟（每实验107个细胞），然后用1 μg/ml抗igm在37℃下刺激30分钟。通过将细胞重悬在含有新鲜蛋白酶和磷酸酶抑制剂的RIPA裂解缓冲液（50 mM Tris 7.4, 1% NP40, 0.5%脱氧胆酸钠，150 mM NaCl, 0.5 mM EDTA）中终止信号传导，并在冰上孵育1小时。离心后，蛋白裂解物用蛋白A/G糖珠预清。裂解液与兔抗syk抗体孵育过夜（4℃），用蛋白A/G sepharose beads沉淀。洗涤珠变性，10% SDS-PAGE溶解蛋白，兔抗syk抗体免疫印迹，剥离，兔抗psyk Y526/526抗体重新印迹。

细胞内磷酸流式细胞术。[1]
Ramos细胞（0.5 × 106）悬浮于200 μl新鲜培养基（RPMI + 10%胎牛血清）中，用载药或PRT062607 (P505-15) 预处理（37℃下30分钟）。细胞不受刺激或以1 μg/ml goat F(ab) ' 2 anti-IgM （Life Technologies）刺激10分钟。从CLL患者（n = 7）外周血中获得的Ficoll纯化（2 × 106）冷冻活CLL细胞，37℃解冻，用10 ml RPMI培养基加10%胎牛血清离心洗涤，以106细胞/ml的浓度在相同培养基中重悬。在0.3 mM H2O2刺激30分钟前，等量（200 μl）细胞用载药或PRT062607 (P505-15) 处理(Reth 2002；Irish et al., 2006) （8.8 M高汤），然后加入6 μg山羊F（ab’）2抗人IgG和抗人IgM 1:1的混合物10分钟。加入60 μl的16%多聚甲醛溶液，室温孵育10分钟，阻断信号传导。将固定的细胞在冷冻的磷酸盐缓冲盐水（PBS）中离心洗涤2次，悬浮在预冷至- 20°C的50%甲醇溶液中，4°C保存过夜。按照制造商的说明，通过在含有1% BSA的PBS中洗涤渗透化细胞两次，然后在含有各种抗体的同一缓冲液中孵育，对细胞进行细胞内磷酸流式细胞术染色。在FACS分析之前，细胞在含有1% BSA的PBS中再次洗涤，其中使用BD Biosciences FACS Calibur收集了至少2000个事件。使用FlowJo软件对数据进行分析。

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细胞凋亡。[1]
采用pe偶联单克隆活性caspase-3抗体凋亡试剂盒检测细胞凋亡。将细胞悬浮在生长培养基（0.5 × 106个细胞/ml）中，并在FACS分析前用指示浓度的PRT062607 (P505-15) 或对照处理72小时。在一些实验中，将SU-DHL6 （0.5 × 106个细胞）与100µl肝素化人全血混合。样品用1µM PRT062607 (P505-15) 处理24小时，然后用抗人CD19抗体表面染色，准备用于FACS分析活性caspase-3。

In Vitro and In Vivo Stimulation with Anti-IgD. [1]
Spleens were harvested from Balb/c mice and separated into a single cell suspension using a single cell strainer. Cells were collected in PBS containing 1% BSA by centrifugation, washed once in the same buffer, and resuspended in RPMI (containing 10% FCS) at 106 cells/ml. Aliquots (190 µl) were then treated with various concentrations of PRT062607 (P505-15)  for 1 hour prior to stimulating overnight in a 37°C tissue culture incubator with 1 µl anti-mouse immunoglobulin D (IgD). Following stimulation, cells were stained with anti-CD80/86 FITC and anti-CD45R/B220 APC for 30 minutes at room temperature, washed once in PBS containing 1% BSA, and resuspend in 300 μl of the same buffer for collection of 2000 CD45-positive events by flow cytometry. Balb/c mice (n = 5 per group) received daily oral BID doses of vehicle (0.5% methylcellulose in water) or PRT062607 (P505-15)  (15 mg/kg) for a total of 5 days. On study day 1, 1 hour after the first oral dose, mice received a single 200-μl subcutaneous injection of control goat serum or anti-IgD serum. On study day 5, mice were anesthetized with SC ketamine cocktail and exsanguinated via cardiac puncture. Spleens were weighed and then sectioned and stained with hematoxylin and eosin for histology.

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Xenograft Studies. [1]
NOD/SCID mice were acclimated in-house at least 3 days prior to use. Ramos cells (3 × 106) were injected subcutaneously into the hind flank area of conscious mice using a 27-gauge needle in an injection volume of less than 0.5 ml. Following injection, mice were randomized into treatment groups (n = 15) and dosed twice daily by oral gavage with vehicle (0.5% methylcellulose in water) or 10, 15, or 20 mg/kg PRT062607 (P505-15) . Body weights were obtained at least once per week, and caliper measurements of tumors were determined twice per week beginning when palpable tumors were formed until the end of the study. Tumor volume was assessed by caliper measurement using a formula [maximum length × width × height × pi/6]. Twice daily dosing of vehicle or PRT062607 (P505-15)  continued until the vehicle or any treatment group exhibited tumors that exceeded 1.5 g in size. At the time of termination (5 weeks post-Ramos inoculation) the mice were anesthetized with a ketamine cocktail. A blood sample via cardiac puncture was obtained for CBC and plasma concentration determination, and then the mice were euthanized via cervical dislocation. Tumors were excised, weighed, and then snap frozen in liquid nitrogen for determination of concentration of PRT062607 (P505-15)  in the tumor tissue. Statistical comparison of tumor weights across groups was performed using a t-test.

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10, 15, or 20 mg/kg; Oral administration

NOD/SCID mice injected with Ramos cells

[1]. Specific inhibition of spleen tyrosine kinase suppresses leukocyte immune function and inflammation in animal models of rheumatoid arthritis. J Pharmacol Exp Ther. 2012 Feb;340(2):350-9.

[2]. The selective SYK inhibitor P505-15 (PRT062607) inhibits B cell signaling and function in vitro and in vivo and augments the activity of fludarabine in chronic lymphocytic leukemia. J Pharmacol Exp Ther. 2013 Feb;344(2):378-87.

[3]. Selective, novel spleen tyrosine kinase (Syk) inhibitors suppress chronic lymphocytic leukemia B-cell activation and migration. Leukemia. 2012 Jul;26(7):1576-83.

Given its preclinical activity and specificity, especially when compared with previously studied SYK inhibitors, P505-15 is an attractive compound for clinical development. Additional studies aimed at defining the biologic characteristics associated with SYK sensitivity are needed. Exploring additional P505-15 combinations including chemotherapy, monoclonal antibodies, or novel combinations of kinase inhibitors targeting multiple branches of signaling pathways, may identify further therapeutic opportunities. A dose finding study using P505-15 in healthy volunteers has been completed and includes single and multiple dosing regimens.[1]

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B-cell receptor (BCR) associated kinases including spleen tyrosine kinase (SYK) contribute to the pathogenesis of B-cell malignancies. SYK is persistently phosphorylated in a subset of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), and SYK inhibition results in abrogation of downstream kinase activity and apoptosis. P505-15 (also known as PRT062607) is a novel, highly selective, and orally bioavailable small molecule SYK inhibitor (SYK IC(50) = 1 nM) with anti-SYK activity that is at least 80-fold greater than its affinity for other kinases. We evaluated the preclinical characteristics of P505-15 in models of NHL and CLL. P505-15 successfully inhibited SYK-mediated B-cell receptor signaling and decreased cell viability in NHL and CLL. Oral dosing in mice prevented BCR-mediated splenomegaly and significantly inhibited NHL tumor growth in a xenograft model. In addition, combination treatment of primary CLL cells with P505-15 plus fludarabine produced synergistic enhancement of activity at nanomolar concentrations. Our findings support the ongoing development of P505-15 as a therapeutic agent for B-cell malignancies. A dose finding study in healthy volunteers has been completed.[2]

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Syk is a protein tyrosine kinase that couples B-cell receptor (BCR) activation with downstream signaling pathways, affecting cell survival and proliferation. Moreover, Syk is involved in BCR-independent functions, such as B-cell migration and adhesion. In chronic lymphocytic leukemia (CLL), Syk becomes activated by external signals from the tissue microenvironment, and was targeted in a first clinical trial with R788 (fostamatinib), a relatively nonspecific Syk inhibitor. Here, we characterize the activity of two novel, highly selective Syk inhibitors, PRT318 and P505-15, in assays that model CLL interactions with the microenvironment. PRT318 and P505-15 effectively antagonize CLL cell survival after BCR triggering and in nurse-like cell-co-cultures. Moreover, they inhibit BCR-dependent secretion of the chemokines CCL3 and CCL4 by CLL cells, and leukemia cell migration toward the tissue homing chemokines CXCL12, CXCL13, and beneath stromal cells. PRT318 and P505-15 furthermore inhibit Syk and extracellular signal-regulated kinase phosphorylation after BCR triggering. These findings demonstrate that the selective Syk inhibitors PRT318 and P505-15 are highly effective for inhibition of CLL survival and tissue homing circuits, and support the therapeutic development of these agents in patients with CLL, other B-cell malignancies and autoimmune disorders.[3]

C19H23N9O.C2H4O2

453.49758

393.202

1370261-96-3

PRT062607 Hydrochloride;1370261-97-4

44462758

White to off-white solid powder

1.5±0.1 g/cm3

707.6±70.0 °C at 760 mmHg

381.7±35.7 °C

0.0±2.3 mmHg at 25°C

1.783

0.74

149.66

4

8

6

29

544

2

NC(C1=CN=C(N[C@H]2[C@@H](N)CCCC2)N=C1NC3=CC=CC(N4N=CC=N4)=C3)=O

TXGKRVFSSHPBAJ-JKSUJKDBSA-N

InChI=1S/C19H23N9O/c20-15-6-1-2-7-16(15)26-19-22-11-14(17(21)29)18(27-19)25-12-4-3-5-13(10-12)28-23-8-9-24-28/h3-5,8-11,15-16H,1-2,6-7,20H2,(H2,21,29)(H2,22,25,26,27)/t15-,16+/m0/s1

2-[[(1R,2S)-2-aminocyclohexyl]amino]-4-[3-(triazol-2-yl)anilino]pyrimidine-5-carboxamide

PRT062607; 1370261-96-3; PRT-062607; 2-[[(1R,2S)-2-aminocyclohexyl]amino]-4-[3-(triazol-2-yl)anilino]pyrimidine-5-carboxamide; K9C42672RH; P505-15; BIIB057; UNII-K9C42672RH;

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。