p38α MAPK (IC50 = 13 nM); TNFα (IC50 = 50 nM)
p38α MAP kinase (IC50 = 0.3 nM); p38β MAP kinase (IC50 = 6.1 nM); p38γ MAP kinase (IC50 = 1200 nM); p38δ MAP kinase (IC50 = 190 nM) [1]

BMS-582949 显示 p38α IC50 为 13 nM，细胞 TNFα IC50 为 50 nM[1]。 BMS-582949 是一种中度弱的 CYP3A4 抑制剂，BMS-582949 对 p38α 的选择性超过 57 种激酶，包括丝氨酸激酶、非受体酪氨酸激酶、受体酪氨酸激酶以及 p38γ 和 δ 亚型[1 ]。

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用BMS-582949处理可抑制LPS刺激的人外周血单个核细胞（PBMCs）中TNFα的产生，IC50为1.8 nM [1]
BMS-582949可抑制LPS诱导的人PBMCs中IL-6的分泌，IC50为2.4 nM [1]
该化合物可抑制LPS刺激的人PBMCs中IL-1β的产生，IC50为2.1 nM [1]
在LPS刺激的THP-1细胞中，BMS-582949可减少TNFα的释放，IC50为3.2 nM [1]
Western blot检测显示，该化合物可阻断茴香霉素处理的HeLa细胞中p38α MAP激酶的磷酸化 [1]

BMS-582949（5-100 mg/kg，口服）是有效的，尽管在大鼠佐剂关节炎模型和急性炎症小鼠模型中的效力稍低[1]。

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BMS-582949 以 4.4 mL/min/kg 的速率从小鼠体内清除。口服剂量 10 mg/kg 时，BMS-582949 的小鼠 AUC0-8 h 为 75.5 μM•h。在小鼠和大鼠中，BMS-582949 的口服生物利用度值分别为 90% 和 60%[1]。

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为了进一步评估BMS-582949对吞噬和呼吸爆发的潜在影响，我们在食蟹猴体内进行了包括离体功能评估的研究。简而言之，4个剂量组，每组6名男性，每天口服1、10或75 mg/kg的BMS-582949或 vehicle (5% [w/v] propylene glycol, 5% [w/v] polyethylene glycol 300, 5% [w/v] glycerin, 1.5% Methocel E5, 9% [w/v] 1N hydrochloric acid, and 1.5% [w/v] anhydrous alcohol in reverse osmosis water),给药7天。在一项为期一年的猴子重复剂量毒性研究中，观察到10和75 mg/kg/天的感染。在研究第4天、第5天和第8-12天（试验可逆性），用肝素钠管采集两次血液（≈2 ml），用于吞噬和呼吸爆发分析。在给药阶段，在药物的近似Cmax（给药后2-4小时）采集血液。用于吞噬和呼吸爆发评估的方法是上述基于流式细胞术的方法，分别使用PhagoTest®和PhagoBurst®测试试剂盒（Orpegen Pharma）。所用的测定条件与先前讨论的体外确定性评估相同(表2和表5；猴)，不经BMS-582949预处理。采血后90分钟内开始刺激血液。然后使用BD FACScalibur流式细胞仪进行分析。使用Cell Quest Pro软件基于正向和侧向散射对中性粒细胞和单核细胞进行门控。根据FL1（488或绿色）通道的荧光强度，分别对两种细胞类型进行数据汇编，并使用重复测量方差分析（ANOVA）程序来评估对照和BMS-582949处理组之间的差异。 [2]
BMS-582949每日剂量4次（10和75 mg/kg）后，中性粒细胞的吞噬能力显著（p≤0.05）下降（与对照组相比，抑制率分别为54%和56%）（图8）。在每日剂量5次后，1 mg BMS-582949/kg的吞噬能力也显著下降（与对照组相比，抑制率为36%）。末次给药后24小时（研究第8天），小鼠吞噬能力无明显下降。然而，10和75 mg BMS-582949/kg在末次给药后24小时均存在数值差异，并在末次给药后48小时完全恢复。单核细胞吞噬活性在任何点均未见明显下降。［2］

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小鼠口服BMS-582949，剂量分别为1、3和10 mg/kg，在LPS攻击后90分钟，可分别抑制35%、68%和89%的LPS诱导的TNFα产生 [1]
在小鼠胶原诱导关节炎（CIA）模型中，从免疫后第21天至第35天，每日口服BMS-582949（3、10、30 mg/kg）可显著减轻爪肿胀，与溶媒对照组相比，30 mg/kg剂量组的爪体积减少65% [1]
在大鼠佐剂诱导关节炎（AIA）模型中，口服BMS-582949（10、30 mg/kg），每日两次，持续14天，可分别减少42%和68%的爪肿胀，并通过减轻炎症和骨侵蚀改善关节组织病理学 [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]

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采用重组p38α、p38β、p38γ和p38δ MAP激酶检测BMS-582949的抑制活性。实验在含有ATP、MgCl2和特异性肽底物的缓冲液中进行。将酶、底物、ATP和测试化合物在37°C下孵育指定时间后终止反应，采用闪烁邻近分析法（SPA）检测磷酸化底物的量，进而计算IC50值 [1]

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%

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从健康供体中分离人PBMCs，在存在或不存在系列稀释的BMS-582949的条件下，用LPS（1 μg/mL）刺激。在37°C、5% CO2环境中孵育24小时后，收集上清液，采用酶联免疫吸附试验（ELISA）检测TNFα、IL-6和IL-1β的水平，以确定IC50值 [1]
THP-1细胞在适宜培养基中培养，并用LPS（1 μg/mL）和不同浓度的BMS-582949共同刺激。孵育24小时后，通过ELISA定量上清液中TNFα的释放量 [1]
HeLa细胞接种到培养板中，过夜贴壁。先用BMS-582949预处理细胞1小时，再用茴香霉素（1 μM）刺激30分钟。制备细胞裂解液，采用特异性抗体通过Western blot检测磷酸化p38 MAP激酶的水平 [1]

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.

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Monkeys were administered vehicle or BMS-582949 at 1, 10, and 75 mg/kg for 7 days and peripheral blood neutrophils were analyzed for phagocytosis on study days 4, 5, 8, and 9 (x-axis). Monkeys were administered vehicle or BMS-582949 at 1, 10, and 75 mg/kg for 7 days and peripheral blood neutrophils were analyzed for respiratory burst function on study days 4, 5, 8, 9, 10, and 12 (PMA stimulation only day 12; x-axis). [2]

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For LPS-induced TNFα production in mice: Male CD-1 mice were fasted overnight before oral administration of BMS-582949 (1, 3, 10 mg/kg) or vehicle. One hour later, mice were injected intraperitoneally with LPS (10 μg/mouse). Blood samples were collected 90 minutes after LPS injection, and plasma TNFα levels were measured by ELISA [1]
For mouse CIA model: DBA/1 mice were immunized with bovine type II collagen emulsified in complete Freund's adjuvant on day 0 and boosted on day 21. From day 21 to day 35, mice were dosed orally with BMS-582949 (3, 10, 30 mg/kg) or vehicle once daily. Paw volume was measured every 3 days using a plethysmometer, and joint histopathology was evaluated at the end of the study [1]
For rat AIA model: Male Lewis rats were immunized with Mycobacterium tuberculosis emulsified in mineral oil via intradermal injection on day 0. From day 14 to day 28, rats were dosed orally with BMS-582949 (10, 30 mg/kg) or vehicle twice daily. Paw swelling was measured using a plethysmometer, and joint tissues were collected for histopathological analysis [1]

The clearance rate of BMS-582949 in mice was 4.4 mL/min/kg. The AUC0-8 h of BMS-582949 administered orally to mice at a dose of 10 mg/kg was 75.5 μM•h. The oral bioavailability of BMS-582949 in mice and rats was 90% and 60%, respectively [1].

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After a single oral dose of 10 mg/kg in mice, the oral bioavailability of BMS-582949 was 45%[1]
In mice, after intravenous injection of 5 mg/kg, the plasma half-life (t1/2) of the compound was 2.8 hours[1]
In rats, the oral bioavailability after a 10 mg/kg dose was 38%, and the plasma t1/2 was 3.2 hours[1]
BMS-582949 showed good tissue penetration, and 2 hours after oral administration, the drug concentrations in the liver, spleen, and joints of rats were 2.3 times, 1.8 times, and 1.2 times the plasma concentrations, respectively[1]

In a 14-day repeated-dose toxicity study in rats, oral administration of BMS-582949 at doses up to 30 mg/kg twice daily did not cause significant changes in body weight, hematological parameters, or clinical chemical indicators (including liver and kidney function indicators) [1]. BMS-582949 has a plasma protein binding rate of 92% in human plasma, 90% in mouse plasma, and 88% in rat plasma [1].

[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.

[2]. Methods: Implementation of in vitro and ex vivo phagocytosis and respiratory burst function assessments in safety testing. J Immunotoxicol . 2013 Jan-Mar;10(1):106-17.

BMS-582949 has been investigated for the treatment of psoriasis.
This article describes the discovery and characterization of 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 sp(2) hybridization of the cyclopropyl group can confer better hydrogen bonding properties to the directly substituted amide NH. Inhibitor 7k is slightly less effective than 1a in p38α enzyme activity assays but has superior pharmacokinetic properties and is therefore more effective in both acute mouse inflammation models 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, 5 µM of 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. Furthermore, ex vivo analysis showed that 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 cause clinically significant infections 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 substances on these important innate immune functions. Commonly available immunomodulators can be used to validate the technical level and applicability of these methods. These assessments can be performed in vitro or ex vivo. For each test substance and test species, multiple detection parameters should be assessed to ensure optimal detection conditions. Where feasible, in vitro assessment provides a convenient platform for testing multiple parameters, and these conditions can be translated to ex vivo assessment, as shown 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. While the effects of test substances can be studied in investigational studies, the 96-well plate format based on microplate and flow cytometry methods facilitates the addition of these functional endpoints in standard toxicology studies, minimizes logistical barriers, and can be applied to preclinical animal models. BMS-582949 is a potent and selective p38α MAP kinase inhibitor for the treatment of inflammatory diseases [1]. This compound has entered clinical trials for the treatment of rheumatoid arthritis and other inflammatory diseases [1].

C₂₂H₂₇CLN₆O₂

442.94

442.188

C, 59.66; H, 6.14; Cl, 8.00; N, 18.97; O, 7.22

912806-16-7

BMS-582949;623152-17-0

11848302

White to off-white solid powder

4.495

110.63

4

5

7

31

627

0

Cl.O=C(C1C=C(NC2C3N(C=C(C=3C)C(NCCC)=O)N=CN=2)C(C)=CC=1)NC1CC1

BIYQUPNVBIOJIY-UHFFFAOYSA-N

InChI=1S/C22H26N6O2.ClH/c1-4-9-23-22(30)17-11-28-19(14(17)3)20(24-12-25-28)27-18-10-15(6-5-13(18)2)21(29)26-16-7-8-16;/h5-6,10-12,16H,4,7-9H2,1-3H3,(H,23,30)(H,26,29)(H,24,25,27);1H

4-[5-(cyclopropylcarbamoyl)-2-methylanilino]-5-methyl-N-propylpyrrolo[2,1-f][1,2,4]triazine-6-carboxamide;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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 (5.64 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 玉米油中，混合均匀。

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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);
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