Syk (IC50 = 4 nM)

PRT318 的 IC50 为 4 nM，是纯 Syk 激酶的强抑制剂。在 PRT318 剂量为 50 nM 时，Syk 激酶被抑制 92%，而其他激酶则保留超过 70%[1]。 PRT318 和 P505-15 成功抑制护士样细胞共培养物中 CLL 细胞的存活以及 B 细胞受体 (BCR) 激活后的存活。它们阻止白血病细胞向组织归巢趋化因子 CXCL12、CXCL13 和基质细胞下方迁移，以及 CLL 细胞依赖 BCR 释放趋化因子 CCL3 和 CCL4。 BCR 触发后，PRT318 和 P505-15 阻断 Syk 和细胞外信号调节激酶的磷酸化[2]。

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在这里，研究人员在模拟CLL与微环境相互作用的检测中，对两种新型、高选择性Syk抑制剂PRT318和P505-15的活性进行了表征。PRT318和P505-15有效地拮抗BCR触发后CLL细胞的存活以及在尿样细胞（NLC）共培养中的存活。此外，它们抑制CLL细胞对BCR依赖性趋化因子CCL3和CCL4的分泌，以及白血病细胞向组织归巢趋化因子CXCL12、CXCL13和基质细胞下方的迁移。PRT318和P505-15在BCR触发后进一步抑制Syk和ERK磷酸化。这些发现表明，选择性Syk抑制剂PRT318与P505-15对CLL存活和组织归巢回路的抑制非常有效，并支持这些药物在CLL、其他B细胞恶性肿瘤和自身免疫性疾病患者中的治疗开发。[2]

当 PRT318 存在时，HIT 免疫复合物完全抑制人类和转基因 HIT 小鼠血小板的聚集。用 PRT318 预处理的小鼠由 HIT IC 引起的肺血栓形成明显减少。接受 PRT318 治疗的小鼠的血栓形成评分显着低于对照组 [1]。

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研究人员测试了酪氨酸激酶Syk的新型选择性抑制剂PRT-060318（PRT318）作为治疗HIT的一种方法。PRT318完全抑制了HIT免疫复合物诱导的人和转基因HIT小鼠血小板的聚集。转基因HIT模型小鼠用KKO（一种小鼠单克隆HIT样抗体）和肝素治疗。实验组口服PRT318，而对照组服用赋形剂。PRT318治疗小鼠的Nadir血小板计数明显高于对照组小鼠。当用一种新的血栓可视化技术进行检查时，用PRT318治疗的小鼠血栓形成显著减少。Syk抑制剂PRT318因此在体内预防了HIT免疫复合物诱导的血小板减少症和血栓形成，证明了其在HIT中的活性。[1]

PRT-060318（PRT318）结构和特异性[1]
通过高通量筛选化学文库，发现了一类新型Syk抑制剂。通过广泛的结构-活性关系研究和合成，优化了属于4-苯胺基-2-（2-氨基乙基氨基）嘧啶-5-甲酰胺类的化合物，以鉴定高效和特异的Syk抑制剂PRT-060318（PRT318），（2-（（1R，2S）-2-氨基环己基氨基）-4-（间甲苯基氨基）嘧啶-5-甲酰胺）（补充图1，可在Blood网站上获得；见在线文章顶部的补充材料链接）。PRT318，也称为P142-76，是嘧啶-5-甲酰胺的衍生物（美国专利号6432963）。[1]
使用激酶分析仪评估PRT318与Syk相互作用的分子特异性。通过测定Y352位Syk的磷酸化来研究PRT318的细胞内特异性，已知Y352位在DHL4 B细胞系（DSMZ）中由src家族酪氨酸激酶（SFTK）在B细胞受体下游磷酸化。在含有10%胎牛血清的RPMI中培养的细胞与PRT318预孵育1小时，然后在37°C下用5μg/mL抗-Ig激活30分钟。通过离心将细胞制成颗粒，并在蛋白酶和磷酸酶抑制剂（完全蛋白酶抑制剂鸡尾酒，PhosSTOP，Roche Diagnostics）存在下裂解。裂解物经过十二烷基硫酸钠聚丙烯酰胺凝胶电泳，并转移到硝化纤维膜上。用兔抗磷酸SYK（Y352）检测印迹。

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血小板中的PRT-060318（PRT318）活性[1]
评估了PRT318在几种不同激动剂存在下对体外血小板聚集的活性。人富血小板血浆（PRP）由签署知情同意书后从健康供体获得的正常人血液制备。在96孔格式的测定中进行了PRP的聚集，以比较PRT318对康夫新和二磷酸腺苷（ADP）的影响。Convulxin是一种糖蛋白VI（GPVI）激动剂，其最终浓度如图所示。ADP来自Chronolog。在96孔格式的测定中测量了人血小板钙通量，以比较康夫新刺激后PRT318与PAR1激动剂TRAP-6的效果。 为了评估PRT-060318（PRT318）在HIT IC刺激后抑制血小板聚集的能力，我们首先在有利于形成超大复合物的条件下制备了肝素-PF4复合物。17简而言之，重组人PF4和肝素以1.5:1的摩尔比混合，并在37°C下孵育1小时。将含有5μg hPF4的超大复合混合物的等分试样（5μL）加入PRP中，在37°C下搅拌下与PRT318（0-3μM）一起孵育15分钟，最终体积为250μL。通过加入KKO（一种单克隆HIT样抗体18，终浓度为80μg/mL）引发聚集。记录了最终的聚合百分比。

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SRA[1]
我们使用血清素释放试验（SRA）研究了PRT-060318（PRT318）在人HIT血清存在的情况下对血小板活化的影响。SRA是确认血小板活化HIT抗体的金标准。简而言之，洗涤后的14C-血清素负载血小板与PRT318在1.0μM下孵育，然后在室温下与0.1或100IU/mL肝素和HIT血清孵育1小时。阳性对照血小板仅用赋形剂处理。通过离心去除血小板，并通过闪烁计数测量上清液中释放的14C-血清素。测定上清液中14C-血清素相对于标记血小板中总14C-血清素的百分比。19阳性结果是在0.1 IU/mL肝素浓度下释放的血清素超过20%，在100 IU/mL的肝素浓度下低于20%。

NLC共培养中CLL细胞的凋亡[2]
通过将CLL患者的PBMC悬浮在含有10%胎牛血清和青霉素-链霉素-谷氨酰胺的完全RPMI培养基中，使其浓度达到107个细胞/mL（共2mL），建立NLC共培养。如前所述，细胞在24孔板中孵育14天（23）。为了评估Syk抑制剂PRT-060318（PRT318）和P505-15是否可以克服NLC的保护作用，CLL细胞在标准化条件下在NLC上或悬浮液中培养，有无PRT318和P505-15。在指定的时间点，收集CLL细胞，并如前所述测定细胞活力。

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酶联免疫吸附试验（ELISA）[2]
为了评估Syk抑制剂PRT-060318 (PRT318)和P505-15在用抗IgM（10mg/mL）或NLC刺激CLL细胞后对CCL3和CCL4分泌的影响，如前所述，在活化的CLL细胞的上清液中测量趋化因子水平。在抗IgM或NLC共培养24小时后，收集上清液，并根据制造商的说明通过定量ELISA检测CCL3、CCL4和CXCL13（仅在NLC共文化中）。

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趋化性测定[2]
为了评估PRT-060318（PRT318）和P505-15对CLL细胞趋化性的影响，我们如上所述对聚碳酸酯Transwell插入物进行了趋化性测定。简而言之，CLL细胞（107/mL）在37°C、5%CO2、10μg/mL抗-IgM的完全RPMI培养基中孵育，该培养基含有或不含有2μM PRT318和P505-15。1小时后，将CLL细胞离心并重新悬浮在含有0.5%牛血清白蛋白的RPMI 1640中，浓度为107个细胞/mL。将100μL细胞悬浮液加入直径为6.5 mm、孔径为2μm的Transwell培养插入物的顶部腔室中。然后将过滤器转移到含有或不含有200 ng/mL CXCL12或1μg/mL CXCL13的培养基的孔中。将腔室在37°C的5%CO2中孵育3小时。孵育后，将下腔室中的细胞悬浮并分成等分，用FACSCalibur以60μL/min的速度计数20秒，一式两份。在相同条件下计数1/20稀释的输入细胞。

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CLL细胞在基质细胞下的迁移（假性脾炎）[2]
为了确定Syk抑制剂PRT-060318（PRT318）和P505-15对CLL细胞在基质细胞下自发迁移的影响，我们如前所述定量了伪脾炎。简而言之，在试验前一天，将小鼠基质细胞（9-15C和TSt-4）以1.8×105个细胞/孔的浓度接种到补充有10%胎牛血清和青霉素-链霉素-谷氨酰胺的RPMI 1640中的胶原蛋白包被的12孔板上。在添加10μg/mL的抗-IgM之前，将浓度为107/mL的CLL细胞在37°C下在添加或不添加2μM PRT318或P505-15的完全培养基中的5%CO2中孵育30分钟。再孵育1小时后，将CLL细胞加入基质细胞层，并在37°C的5%CO2中孵育4小时。通过用RPMI培养基剧烈洗涤3次，去除未迁移到基质细胞层的细胞。

Studies in vivo [1]
C57Bl/6 (Charles River Labs) mice were used for the dose selection study. The transgenic HIT mice used to determine the effect of PRT-060318 (PRT318) on HIT IC-induced thrombocytopenia are homozygous for both FcγRIIA and human PF4 and null for mouse PF4.8,20 [1]
To determine the effect of PRT-060318 (PRT318) on HIT IC-induced thrombocytopenia and thrombosis, HIT model mice were treated with KKO (20 mg/kg body weight, intraperitoneally) on day 0. The mice were divided into sex- and weight-matched experimental and control groups. On days 1 to 7, experimental mice (n = 6) received PRT318 (30 mg/kg body weight) orally via gavage twice a day, whereas control mice (n = 6) received vehicle only (sterile water). Both groups received heparin (1600 U/kg body weight, subcutaneously) once daily. Mice were anesthetized by isoflurane inhalation for injections and blood collections. Blood was collected via the retro-orbital plexus using 50 μL ethylenediaminetetraacetic acid-coated microcapillary tubes (Microcaps, Drummond Scientific). Blood was collected 3 days before the antibody injections for baseline values and then on days 1 through 7 after antibody injections. Platelet counts were measured using a Hemavet Analyzer (model 850, CDC Technologies) and monitored over time to assess thrombocytopenia. Mouse plasma samples, collected 2 hours after PRT318 dosing, were precipitated with acetonitrile, and concentrations of PRT318 were quantified by liquid chromatography/tandem mass spectrometry. [1]
To evaluate the effect of PRT-060318 (PRT318) treatment on thrombosis in vivo, the protocol was modified as follows to take advantage of a novel thrombosis visualization technology. Experimental (n = 3) and control (n = 3) HIT mice were injected with KKO and PRT318 or vehicle only as described in the previous paragraph. For the imaging method, blood was collected in heparin from the retro-orbital plexus of untreated syngeneic donor mice. The donor platelets were washed, pooled, and incubated at 37°C for 10 minutes with Alexa750-labeled anti-GPIX antibody. The labeled platelets were pelleted and resuspended in sterile saline. Both experimental and control mice were infused with 1 × 108 labeled platelets in 100 μL aliquots via the retro-orbital plexus. All mice were then injected with heparin as described in the previous paragraph. Mice were killed 3 hours after heparin treatment, and their organs were perfused with sterile phosphate-buffered saline. Lungs were excised, fixed in 7% paraformaldehyde, and imaged with an Odyssey Imaging System (LI-COR Biosciences). The images were evaluated using Nikon NIS Elements image analysis software Version 3.10 (NIS-Elements, Advanced Research). Thrombi were identified as areas with fluorescence intensity at least 1.5 times above background. The Thrombosis Score represents the number of thrombi multiplied by the average intensity.

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30 mg/kg; i.p.

Mice, rabbit, pig models

[1]. PRT-060318, a novel Syk inhibitor, prevents heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model. Blood. 2011 Feb 17;117(7):2241-6.

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

Our studies document that inhibition of Syk signaling with the orally bio-available PRT318 limits the thrombocytopenic and thrombotic effects of HIT IC in vivo. We focused on the effects of inhibition of Syk in platelets. Similar effects on monocytes and endothelial cells cannot be ruled out yet are probably beneficial with regards to pathology of HIT. It is of note that the antithrombotic effects were readily visualized with a new technique, applicable to other forms of immune thrombocytopenia/thrombosis disorders, (M.S. and W.B., unpublished observations, March 2010). Taken together, these studies clearly demonstrate that the Syk inhibitor PRT318 is an active agent in HIT. These results support the hypothesis that inhibition of FcγRIIA-Syk signaling with PRT318 or related compounds may have beneficial effects in the treatment of human HIT. Future studies will be needed to address planned short-term use, effects on other Syk-expressing cells, and on hemostasis.[1]

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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.[2]

C18H24N6O

340.42

340.201

C, 63.51; H, 7.11; N, 24.69; O, 4.70

1194961-19-7

11493841

Light yellow to yellow solid powder

1.3±0.1 g/cm3

602.1±65.0 °C at 760 mmHg

317.9±34.3 °C

0.0±1.7 mmHg at 25°C

1.672

1.62

118.95

4

6

5

25

447

2

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: 25 mg/mL (73.44 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 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：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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