p38α MAPK (IC50 = 13 nM); TNFα (IC50 = 50 nM)

发现 BMS-582949 可以阻止细胞中 p38 的激活，如 p38 的磷酸化所示。 p38 磷酸化的丧失证明，BMS-582949 对已被 LPS 激活 p38 的细胞进行处理后，可迅速逆转 p38 激活。 BMS-582949 是一种双作用 p38 激酶抑制剂，可抑制细胞中的 p38 激酶活性和 p38 激活。通过改变被上游激酶磷酸化的激活环的构象，BMS-582949 抑制上游 MKK 对 p38 的磷酸化[2]。这是通过使激活环呈现不易接近的构象来完成的。

在 BALB/c 雌性 LPS 诱导的急性刺激范例中，BMS-582949（5 mg/kg，口服，90 分钟）显着降低 TNFα 的产生 [1]。 BMS-582949（0.3-100 mg/kg，口服，每日一次）25% N-吡咯烷酮、33% Polysolution 400、9% 丙二醇和 33% 水用于 BMS-582949 的静脉 (iv) 溶解技术。 BMS-582949 胃内 (po) 溶解技术中使用的溶剂是 Polyfill 400 [1]。

BMS-582949被发现对Raf的选择性是190倍，对Jnk2（一种参与炎症的MAP激酶）的选择性是450倍。 X 射线晶体学研究进一步证明了 BMS-582949 与 p38R 的结合模式。

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BMS-582949诱导的单核细胞呼吸爆发功能的抑制也以剂量依赖性的方式观察到，但与中性粒细胞呼吸爆发相比，抑制程度较低。经0.5µM和5µM BMS-582949预处理后，PMA刺激的猴子血液呼吸爆发功能与对照组相比差异有统计学意义（p≤0.05）。在这些剂量下，PMA刺激呼吸爆发的中位数百分比抑制值分别为22%和29%。统计推断和中位数百分比抑制值表明，BMS-582949对猴子和大鼠单核细胞呼吸爆发的影响对整体免疫状态的潜在生物学相关性很小；然而，观察到的临床前物种对细菌感染的敏感性是散发的，这一观察结果与猴和大鼠样品中抑制率≥30% 相关。[2]

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BMS-582949对大肠杆菌刺激的单核细胞呼吸爆发作用的IC30值没有计算，因为用于描述百分比抑制与BMS-582949浓度之间关系的线性回归函数的斜率估计与“0”没有显著差异（p≤0.10，大鼠），或者观察到的百分比抑制数据范围不包括30%（猴子）。尽管如此，个别大鼠（0.5µM条件下8只大鼠中有1只，5µM条件下3只，8只大鼠中有3只）显示BMS-582949对大肠杆菌刺激的呼吸爆发bbb的抑制作用大于30%。[2]

BMS-582949 抑制 p38 激酶活性以及 p38 激活。当 p38 磷酸化时，BMS-582949 可抑制细胞中 p38 的激活。 p38 磷酸化的丧失证明，BMS-582949 对已被 LPS 激活 p38 的细胞进行处理后，可迅速逆转 p38 激活。

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最终评估结果表明，BMS-582949以剂量依赖的方式抑制猴子和大鼠中性粒细胞的吞噬(图5)。0.5µM（0.2µg/ml）、5µM（2.1µg/ml）和50µM（21µg/ml）时，大鼠和猴中性粒细胞的吞噬功能显著降低（p≤0.05）。在5µM和50µM时，猴子的中位数抑制率（分别为37%和44%）高于大鼠（分别为16%和27%）。在猴子中，抑制率≥30%的发生率也更高（表3）。抑制率中位数和抑制率≥30%的物种差异反映在大鼠的IC30值（62µM, 25µg/ml）高于猴子（23.2µM, 9.4µg/ml）。无论猴子和大鼠之间的组中位数差异如何，在5µM(2.1µg/ml) BMS-582949下，在猴子和大鼠中观察到中性粒细胞吞噬抑制≥30%的个别发生率（表3），这是感染动物达到的Cmax值的0.1 - 10倍。没有bms - 582949相关影响单核细胞吞噬功能演示了猴子和老鼠(数据未显示)。[2]

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如图6所示，BMS-582949以剂量依赖的方式抑制猴子和大鼠中性粒细胞的呼吸爆发功能。与对照相比，0.5µM（0.2µg BMS-582949/ml）和5µM（2.1µg BMS-582949/ml）浓度下猴子和大鼠细胞的呼吸爆发功能显著降低（p≤0.05）。在0.5µM时，猴子对PMA和大肠杆菌刺激的呼吸爆发的中位数抑制（分别为40%和30%）大于大鼠（分别为39%和25%）。然而，在5µM时，PMA和大肠杆菌刺激的呼吸爆发对大鼠中性粒细胞的抑制（分别为67%和57%）大于猴子细胞（分别为58%和51%）。在大鼠中观察到的与猴子相比，在5µM时所观察到的相对增强的效果可能是由于某些猴子的潜在抑制峰在0.5和5µM之间。在距离评估中，对于一些样品，在扩大浓度范围的上端达到抑制峰后，观察到轻微的下降。IC30值由中位数百分比抑制值计算得出，见表6。PMA刺激呼吸爆发的IC30值与大鼠差异不大，但大肠杆菌刺激呼吸爆发的IC30值明显低于大鼠。然而，如表7所示，在0.5和5µM时，两种物种的呼吸爆发抑制发生率均较高，≥30%

Animal/Disease Models: Acute inflammation model from BALB/c female mice [1]
Doses: 5 mg/kg
Route of Administration: po (oral gavage) Stomach (po), content detection results 90 minutes after LPS injection: TNFα production was diminished by 89% 2 hrs (hrs (hours)) before LPS challenge and 78% at 6 hrs (hrs (hours)).

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Animal/Disease Models: Rat adjuvant arthritis from male Lewis rats (rat AA) Model[1]
Doses: 1, 10, 100 mg/kg one time/day (qd)
Route of Administration: Oral tube feeding (po)
Experimental Results: Paw swelling was diminished in a dose-dependent manner at 10 and 100 mg Efficacy was observed at doses of (po)
Experimental Results: Efficacy in reducing paw swelling was Dramatically improved at doses of 1 and 5 mg/kg. Doses as low as 0.3 mg/kg Dramatically diminished paw swelling.

[1]. Discovery of 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949), a clinical p38α MAP kinase inhibitor for the treatment of inflammatory diseases. J Med Chem. 2010 Sep 23;53(18):6629-39.

BMS-582949 has been investigated for the treatment of psoriasis. This article describes the discovery and characterization of compound 7k (BMS-582949), a highly selective p38α MAP kinase inhibitor currently undergoing a phase II clinical trial for the treatment of rheumatoid arthritis. The key to this discovery is the rational substitution of the N-methoxy group in the previously reported clinical candidate p38α inhibitor 1a with an N-cyclopropyl group. Unlike alkyl and other cycloalkyl groups, the sp² hybridization of the cyclopropyl group can confer better hydrogen bonding properties to the directly substituted amide NH group. Inhibitor 7k showed slightly lower p38α enzyme activity than 1a but had better pharmacokinetic properties, thus showing higher efficacy in both acute mouse inflammation and pseudo-rat AA models. X-ray crystallography confirmed the binding mode of 7k to p38α. [1]

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Functional innate immune assessment, including phagocytosis and respiratory burst, is a frontier area of preclinical animal immunotoxicology evaluation. Although the assessment of phagocytosis and respiratory burst has been reported in clinical and academic studies for over two decades, its widespread application in toxicology and safety programs has only recently gained attention. This article discusses general methods for assessing phagocytosis and respiratory burst in preclinical animals such as mice, rats, dogs, and monkeys, including microplate-based and flow cytometry-based methods. Focusing on methods, this article reviews relevant techniques and describes their application, and presents analytical results for reported phagocytosis and respiratory burst inhibitors (rottlerin, wortmannin, and SB203580). A case study is used to illustrate the rationale for implementation, strategic experimental design, and feasibility of assessing the effects of test substances on phagocytosis and respiratory burst function. This case study investigates the effects of the small molecule p38 kinase inhibitor BMS-582949 on phagocytosis and respiratory burst function in rat and monkey neutrophils and monocytes in vitro and in vitro experiments. Monkeys treated with BMS-582949 during a one-week repeated-dose study were used for in vitro experiments. In vitro and ex vivo results showed that BMS-582949 inhibited phagocytosis and respiratory burst. These findings are consistent with the incidence of opportunistic infections observed in rat and monkey toxicity studies. [2]

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Case Study Summary [2] In vitro assessments of phagocytosis and respiratory burst showed that BMS-582949 inhibited these functions at concentrations similar to the drug exposure concentrations in infected animals in toxicity studies. For example, a concentration of 5 µM BMS-582949 was 0.1–10 times the Cmax value reached in infected animals (Price, Citation 2010). Generally, the inhibition of these functions was greater in monkeys than in rats, which is consistent with the observed severity and incidence of infection in monkeys compared to rats. In addition, ex vivo analysis showed that both phagocytosis and respiratory burst were inhibited at doses that caused infection in monkeys. In both in vitro and ex vivo assessments, the inhibition of respiratory burst was greater than that of phagocytosis, and the inhibition of neutrophils was greater than that of monocytes. In summary, the results of in vitro and ex vivo phagocytosis and respiratory burst assessments support the hypothesis that opportunistic pathogens may exhibit clinically significant infection under the immunomodulatory effects of p38 inhibitors (reduced phagocytosis and respiratory burst).

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Methodological Conclusions [2] The phagocytosis and respiratory burst assessment methods described herein are suitable for assessing the effects of test articles on these important innate immune functions. The technical level and applicability of these methods can be validated using common commercially available immunomodulators. These assessments can be performed in vitro or under ex vivo conditions. For each test article and test species, multiple detection parameters should be assessed to ensure optimal detection conditions. In vitro assessment provides a convenient platform for testing numerous parameters, and these conditions can be translated to ex vivo assessment, as demonstrated in the case study described herein. Flow cytometry-based methods are more suitable for ex vivo assessment than microplate-based methods because flow cytometry can analyze whole blood. Although the effects of test articles can be investigated in survey studies, the 96-well plate format based on plates and flow cytometry facilitates the addition of these functional endpoints to standard toxicology studies with minimal logistical barriers and can be used in preclinical species.

C22H26N6O2

406.48084

406.212

623152-17-0

BMS-582949 hydrochloride;912806-16-7

10409068

Typically exists as solid at room temperature

3.693

110.63

3

5

7

30

627

0

O=C(C1=CN2N=CN=C(NC3=CC(C(NC4CC4)=O)=CC=C3C)C2=C1C)NCCC

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));
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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；
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