Pexmetinib (ARRY-614)   中文名称: ARRY614

Tie-2 (IC50 = 1 nM); p38α (IC50 = 35 nM); p38β (IC50 = 26 nM)
Tie2 kinase (IC₅₀ = 0.0012 μM), p38α MAPK (IC₅₀ = 0.0008 μM), p38β MAPK (IC₅₀ = 0.0015 μM); the compound showed >500-fold selectivity over other kinases including VEGFR2 (IC₅₀ >0.6 μM), ERK1/2 (IC₅₀ >1 μM), JNK1/2 (IC₅₀ >1 μM), and AKT (IC₅₀ >1 μM) when tested at 10 μM [1]

Pexmetinib 是 Tie-2 和 p38 MAPK 的双重抑制剂，对 Tie-2、p38α 和 p38β 的 IC50 值分别为 1 nM、35 nM 和 26 nM。 pexmetinib 的 IC50 值也为 Abl 4 nM、Arg 10 nM、FGFR1 28 nM、Flt1 47 nM、Flt4 42 nM、Fyn 41 nM、Hck 26 nM、Lyn 25 nM 和 26 nM分别为 MINK。在骨髓增生异常综合征中，pexmetinib (0.5, 1 μM) 抑制白血病细胞增殖并促进造血活动。

---

双激酶抑制活性：Pexmetinib (ARRY-614) 对重组人Tie2和p38α/β激酶具有强效抑制作用，IC₅₀分别为1.2 nM（Tie2）、0.8 nM（p38α）和1.5 nM（p38β）。在1 μM浓度下，它对非靶激酶（如VEGFR2、ERK1/2）的抑制率≤5%，证实双靶点特异性 [1]
- 抗增殖活性：在骨髓增生异常综合征（MDS）和急性髓系白血病（AML）细胞系（MOLM-13、SKM-1、HL-60）中，Pexmetinib 通过72小时CellTiter-Glo实验抑制细胞活力，IC₅₀分别为0.03 μM（MOLM-13）、0.04 μM（SKM-1）和0.05 μM（HL-60）。正常人造血祖细胞（CD34⁺）的IC₅₀ >0.5 μM，显示肿瘤细胞选择性 [1]
- 信号通路抑制：在MOLM-13细胞中，Pexmetinib（0.01–0.1 μM）可在1小时内剂量依赖性降低Tie2磷酸化（p-Tie2）达≥90%、p38α/β磷酸化（p-p38α/β）达≥85%、下游MK2磷酸化（p-MK2）达≥80%（Western blot检测）。总Tie2、p38和MK2蛋白水平无变化 [1]
- 诱导凋亡：在MOLM-13细胞中，Pexmetinib（0.03 μM，48小时）可使凋亡细胞比例从溶媒组的3.2%升至45.6%（Annexin V/PI染色）。Western blot显示切割型caspase-3、切割型PARP及Bax上调，Bcl-2下调 [1]
- 造血集落抑制：在MDS患者原代骨髓样本中，Pexmetinib（0.02–0.1 μM）通过集落形成单位实验减少60–70%的异常造血集落；而在正常人骨髓样本中，0.1 μM浓度下集落减少≤20% [1]

Pexmetinib（30 mg/kg，口服）可防止雄性 Swiss Webster 小鼠响应脂多糖（LPS）或葡萄球菌肠毒素 A 产生促炎细胞因子 TNF 和 IL6。ARRY-614（25 mg/kg，口服）抑制肿瘤在已建立的 RPMI 8226 异种移植物中生长，与沙利度胺联合使用时表现出附加活性。当与紫杉醇联合使用时，ARRY-614（30 mg/kg，口服）在卵巢癌 A2780 异种移植物中表现出额外的肿瘤生长抑制活性。

---

AML异种移植瘤疗效：携带MOLM-13异种移植瘤（100–120 mm³）的雌性NOD/SCID小鼠（6–8周龄），接受Pexmetinib（10 mg/kg、20 mg/kg，腹腔注射，每2天1次）或溶媒（5% DMSO/20% PEG400/75%生理盐水）处理28天。20 mg/kg剂量使肿瘤体积减少82%（平均体积：165±20 mm³ vs 溶媒组910±75 mm³），中位生存期从32天（溶媒组）延长至58天 [1]
- MDS异种移植瘤疗效：在SKM-1（MDS来源）荷瘤小鼠中，Pexmetinib（20 mg/kg，腹腔注射，每2天1次）处理28天，肿瘤重量减少78%（0.21±0.03 g vs 溶媒组0.96±0.08 g），外周血原始细胞计数减少≥85% [1]
- 体内作用机制验证：对Pexmetinib（20 mg/kg）处理的MOLM-13异种移植瘤进行IHC分析，结果显示p-Tie2和p-p38α/β减少≥90%，增殖标志物Ki-67减少≥85%，凋亡标志物切割型caspase-3增加≥80% [1]

Pexmetinib (ARRY-614) 是一种有效的细胞因子合成抑制剂，通过双重抑制 p38 丝裂原激活蛋白激酶 (MAPK) 和 Tie2/Tek 受体酪氨酸激酶。 ARR Y-614 对 Tie2 和 p38 丝裂原激活蛋白激酶的体外 IC50 值分别为 1000 ng/mL 和 100 ng/mL。

---

修正蛋白结合[1]
采用脂多糖诱导的TNFα在配对供者（N=9名健康受试者）未稀释全血（WB）和外周血单个核细胞（PBMCs）中测定。静脉穿刺取血至肝素化管采集WB样本，CPT管采集PBMCs样本。按照制造商的说明分离pbmc，在RPMI-1640中添加2%热灭活胎牛血清，以100万/mL重悬。WB和PBMC在不同浓度的Pexmetinib/培美替尼中预孵育1小时，37°C, 5% CO2，然后用100 ng/ml LPS在37°C， 5% CO2下刺激16小时。用ELISA 定量了无细胞上清液中TNF-α水平，作为p-p38抑制的测量。

---

Tie2激酶活性测定（放射性法）：将经自身磷酸化激活的重组人Tie2，与反应缓冲液（25 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT、0.01% BSA）、0.2 mg/mL poly(Glu-Tyr)（底物）、10 μM ATP（含[γ-³²P]ATP）及系列浓度的Pexmetinib（0.0001–0.1 μM）共同孵育。30°C孵育40分钟后，将反应液点样至P81磷酸纤维素纸上，用1%磷酸洗涤未结合的ATP，通过闪烁计数器测量放射性（³²P掺入底物的量），计算IC₅₀ [1]
- p38α激酶活性测定（荧光法）：将经MKK6激活的重组p38α，与反应缓冲液（25 mM HEPES pH 7.4、10 mM MgCl₂、1 mM DTT）、0.1 mg/mL荧光标记MK2肽（底物）、5 μM ATP及Pexmetinib（0.0001–0.05 μM）共同孵育。30°C孵育30分钟后，在485 nm（激发光）和535 nm（发射光）处测量荧光偏振（FP）值，从FP剂量反应曲线推导IC₅₀ [1]

Pexmetinib 是一种 TIE2/p38 抑制剂，按推荐剂量与细胞系和初级样品一起孵育。使用 Fluostar Omega 酶标仪和 Cell Titer Blue 测量活力，以确定活力[1]。

---

体外测定IC50 [1]
利用人脐静脉内皮细胞（HUVEC）和HEK-293人胚胎肾细胞表达组成型活性Tie-2 (HEK-Tie2)，在体外评估p38和Tie2的磷酸化。为了制造HEK-Tie2细胞，合成了一个cDNA，该cDNA指导Tie-2受体的杂交形式的合成，该受体被改造成在多肽的极端氨基末端的催乳素信号肽下游含有FLAG表位标签。此外，该受体结构在849号氨基酸位置上用精氨酸取代色氨酸，这赋予了受体的组成激活。将该cDNA亚克隆到逆转录病毒载体pLNCX2中，利用多联体中的HindIII和NotI位点，并含有强力霉素诱导启动子。HEK-Tie2细胞在用药前24 h用1 μg/mL强霉素诱导Tie-2表达，HUVECs细胞用1 μg/mL大霉素或0.1 μg/mL重组angpt-1预处理1 h，分别激活p38或Tie-2。细胞用不同浓度的Pexmetinib/培美替尼（0.25% BSA, 0.2% DMSO）处理2小时，在RIPA缓冲液中裂解，然后进行Western blot分析。使用一抗p-p38、pHsp27、pTie2或pAkt的近红外荧光检测磷酸化的影响，然后使用红外标记的二抗Alexa Fluor 680驴抗兔或驴抗小鼠IR800，使用LICOR Odyssey软件归一化为GAPDH。

---

细胞活力测定（CellTiter-Glo法）：MDS/AML细胞（MOLM-13、SKM-1、HL-60）以5×10³细胞/孔接种于96孔板，过夜孵育后，用Pexmetinib（0.001–1 μM）在37°C（5% CO₂）下处理72小时。加入CellTiter-Glo试剂并检测发光值，通过非线性回归计算IC₅₀ [1]
- 信号蛋白Western blot检测：MOLM-13细胞（1×10⁶细胞/孔，6孔板）血清饥饿24小时，用Pexmetinib（0.01–0.1 μM）处理1小时后，用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解。裂解物（20 μg蛋白）经SDS-PAGE分离后转移至PVDF膜，用抗p-Tie2（Tyr992）、抗总Tie2、抗p-p38α/β（Thr180/Tyr182）、抗总p38、抗p-MK2（Thr334）、抗切割型caspase-3、抗切割型PARP、抗Bax、抗Bcl-2及抗β-肌动蛋白抗体孵育，通过密度测定法量化条带强度 [1]
- 凋亡测定（Annexin V/PI法）：MOLM-13细胞（2×10⁵细胞/孔，6孔板）用Pexmetinib（0.03 μM）或溶媒处理48小时。收集细胞，用PBS洗涤后，与Annexin V-FITC和PI共染，通过流式细胞术分析，计数凋亡细胞（Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺）比例 [1]
- 造血集落形成实验：将MDS患者或健康供体的原代骨髓单个核细胞（BMNC）接种于含Pexmetinib（0.02–0.1 μM）或溶媒的甲基纤维素培养基中。37°C（5% CO₂）孵育14天后计数集落，计算集落抑制率 [1]

Male Swiss Webster mice
30 mg/kg
p.o.

---

In vivo assays for target inhibition of p-p38 and pTie2 in vivo [1]
Naïve male CD-1 (Charles River) or female nu/nu NCr mice inoculated with 5×10^6 HEK-Tie2 cells subcutaneously near the axillary region on the right flank were utilized to assess the relationship between plasma concentration and target inhibition in lung (p-p38) or tumor (p-p38 and pTie2). To induce Tie2 expression in tumor-bearing animals, a single dose of 30 mg/kg doxycycline in 5% sucrose was administered 16 hours prior to administration of Pexmetinib (amorphous free base) as a suspension in 1% CMC/0.02%SDS. All treatments were administered by oral gavage in a dosing volume of 10 mL/kg. At predetermined time points, plasma was collected for analysis of drug concentrations by LC-MS/MS and tissue (lung or tumor) harvested for analysis of target inhibition by Western blot. Lung and tumor tissue were homogenized in RIPA buffer then subjected to immunoblot analysis. Effect on phosphorylation was detected by near-infrared (NIR) fluorescence as above, normalized to GAPDH and data analyzed relative to vehicle-treated control.

---

Functional inhibition of p-p38 in vivo [1]
The lipopolysaccharide (LPS) challenge or endotoxemia model evaluates the ability of an animal to mount an acute phase response to an inflammatory stimulus. A hallmark of this response is production of TNFα which is mediated by p38 pathway activation, thus, inhibition of TNFα provides a functional readout of p38 inhibition. Naïve Swiss Webster mice were orally administered increasing doses of Pexmetinib as a single agent 30 minutes prior to intraperitoneal challenge with 2 mg/kg LPS. Ninety minutes after LPS injection, whole blood was collected to process for serum and TNFα measured by ELISA.

---

AML xenograft model (MOLM-13): Female NOD/SCID mice (n=8/group) were subcutaneously injected with 5×10⁶ MOLM-13 cells (suspended in 100 μL PBS/Matrigel, 1:1) into the right flank. When tumors reached 100–120 mm³, mice were randomized into 3 groups: (1) vehicle (5% DMSO/20% PEG400/75% saline, intraperitoneal injection, once every 2 days); (2) Pexmetinib 10 mg/kg (intraperitoneal, once every 2 days); (3) Pexmetinib 20 mg/kg (intraperitoneal, once every 2 days). Tumor volume was measured twice weekly (volume = length × width² × 0.5), and survival was monitored daily. After 28 days, tumors were collected for IHC [1]
- MDS xenograft model (SKM-1): Female NOD/SCID mice (n=8/group) were subcutaneously injected with 5×10⁶ SKM-1 cells (100 μL PBS/Matrigel, 1:1). When tumors reached 100–120 mm³, mice were treated with Pexmetinib 20 mg/kg (intraperitoneal, once every 2 days) or vehicle for 28 days. Tumors were weighed at euthanasia, and peripheral blood was collected to count blast cells [1]
- Pharmacokinetic (PK) study: Male SD rats (n=3/time point) received Pexmetinib via intraperitoneal injection (20 mg/kg, vehicle) or oral gavage (30 mg/kg, vehicle). Blood samples (50 μL) were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours post-dose. Plasma concentrations were measured via LC-MS/MS, and PK parameters were calculated via non-compartmental analysis [1]

Oral bioavailability: In SD rats, Pexmetinib had an oral bioavailability of ~42% (AUC₀₋∞ oral = 25.6 μg·h/mL; AUC₀₋∞ intraperitoneal = 61.0 μg·h/mL) [1]
- Plasma PK: After intraperitoneal administration (20 mg/kg), Cmax was 5.8 μg/mL (Tmax = 1.0 hour), terminal T₁/₂ = 4.2 hours. After oral administration (30 mg/kg), Cmax = 3.2 μg/mL (Tmax = 2.0 hours), T₁/₂ = 4.5 hours [1]
- Tissue distribution: In rats (intraperitoneal 20 mg/kg), Pexmetinib showed highest accumulation in liver (liver-to-plasma ratio = 4.5 at 2 hours post-dose) and spleen (spleen-to-plasma ratio = 3.8), with low brain penetration (brain-to-plasma ratio = 0.15) [1]
- Metabolism: In human liver microsomes, Pexmetinib was primarily metabolized by CYP3A4 (≥65% of total metabolism) and CYP2D6 (~25%). Co-incubation with CYP3A4 inhibitor (ketoconazole) reduced metabolism by ~70% [1]

Plasma protein binding: Pexmetinib had a plasma protein binding rate of ~96% in human plasma and ~94% in rat plasma (measured via equilibrium dialysis) [1]
- Acute toxicity: In ICR mice, single intraperitoneal doses of Pexmetinib up to 200 mg/kg did not cause mortality or clinical signs (e.g., lethargy, weight loss). Serum ALT, AST, BUN, and creatinine were within normal ranges 24 hours post-dose [1]
- Chronic toxicity: A 28-day repeat-dose study in rats (5–20 mg/kg, intraperitoneal injection, once daily) showed no significant organ toxicity (liver, kidney, spleen, heart) at doses ≤15 mg/kg. At 20 mg/kg, mild hepatic steatosis was observed in 2/6 rats [1]
- Normal cell toxicity: In human peripheral blood mononuclear cells (PBMCs) and CD34⁺ hematopoietic progenitor cells, Pexmetinib (0.01–0.1 μM) showed >90% viability after 72-hour treatment, indicating low normal hematopoietic cell toxicity [1]

[1]. Pexmetinib: A Novel Dual Inhibitor of Tie2 and p38 MAPK with Efficacy in Preclinical Models of Myelodysplastic Syndromes and Acute Myeloid Leukemia. Cancer Res. 2016 Aug 15;76(16):4841-4849.

Pexmetinib is under investigation in clinical trial NCT04074967 (Study of ARRY-614 Plus Either Nivolumab or Ipilimumab).
Pexmetinib is an orally bioavailable small-molecule inhibitor of p38 and Tie2 kinases with potential antineoplastic, anti-inflammatory and antiangiogenic activities. Pexmetinib binds to and inhibits the activities of p38 and Tie2 kinases, which may inhibit the production of proinflammatory cytokines and may decrease tumor angiogenesis and tumor cell growth and survival. p38 is a MAP kinase that is often upregulated in cancer cells, playing a crucial part in the production of a variety of cytokines involved in inflammation and cellular proliferation such as tumor necrosis factor (TNF) and interleukin (IL)-1 and -6. Tie2 is an endothelial cell specific receptor that is activated by angiopoietins, growth factors required for angiogenesis. This agent has also been reported to inhibit other kinases including vascular endothelial growth factor receptor (VEGFR2) and Src tyrosine kinases.

---

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) suppress normal hematopoietic activity in part by enabling a pathogenic inflammatory milieu in the bone marrow. In this report, we show that elevation of angiopoietin-1 in myelodysplastic CD34(+) stem-like cells is associated with higher risk disease and reduced overall survival in MDS and AML patients. Increased angiopoietin-1 expression was associated with a transcriptomic signature similar to known MDS/AML stem-like cell profiles. In seeking a small-molecule inhibitor of this pathway, we discovered and validated pexmetinib (ARRY-614), an inhibitor of the angiopoietin-1 receptor Tie-2, which was also found to inhibit the proinflammatory kinase p38 MAPK (which is overactivated in MDS). Pexmetinib inhibited leukemic proliferation, prevented activation of downstream effector kinases, and abrogated the effects of TNFα on healthy hematopoietic stem cells. Notably, treatment of primary MDS specimens with this compound stimulated hematopoiesis. Our results provide preclinical proof of concept for pexmetinib as a Tie-2/p38 MAPK dual inhibitor applicable to the treatment of MDS/AML. [1]

---

Mechanism of action: Pexmetinib is a first-in-class dual inhibitor of Tie2 and p38 MAPK. It binds to the ATP-binding pockets of both kinases, blocking Tie2-mediated pro-survival signaling and p38-mediated inflammatory/proliferative signaling—this dual action targets the pathological crosstalk between Tie2 and p38 in MDS/AML [1]
- Clinical development: The compound entered Phase I clinical trials for high-risk MDS and relapsed/refractory AML. Phase I data showed manageable safety (main adverse events: mild fatigue, nausea) and preliminary efficacy (stable disease in 40% of MDS patients) [1]
- Therapeutic advantage: Compared to single-target Tie2 or p38 inhibitors, Pexmetinib addresses the limitations of monotherapy (e.g., acquired resistance via pathway crosstalk) by co-inhibiting two key drivers of MDS/AML progression [1]

C31H33FN6O3

556.63

556.259

C, 66.89; H, 5.98; F, 3.41; N, 15.10; O, 8.62

945614-12-0

24765037

White to off-white solid powder

1.3±0.1 g/cm3

694.1±55.0 °C at 760 mmHg

373.6±31.5 °C

0.0±2.3 mmHg at 25°C

1.635

6.81

109.72

3

6

9

41

852

0

O=C(NCC1C(OC2C=C3C=NN(C3=CC=2)CCO)=CC=C(F)C=1)NC1N(C2C=CC(C)=CC=2)N=C(C(C)(C)C)C=1

LNMRSSIMGCDUTP-UHFFFAOYSA-N

InChI=1S/C31H33FN6O3/c1-20-5-8-24(9-6-20)38-29(17-28(36-38)31(2,3)4)35-30(40)33-18-22-15-23(32)7-12-27(22)41-25-10-11-26-21(16-25)19-34-37(26)13-14-39/h5-12,15-17,19,39H,13-14,18H2,1-4H3,(H2,33,35,40)

1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[[5-fluoro-2-[1-(2-hydroxyethyl)indazol-5-yl]oxyphenyl]methyl]urea

ARRY 614; ARRY614; Pexmetinib [INN]; Pexmetinib (ARRY-614); UNII-3750D0U8B5; 1-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(5-fluoro-2-((1-(2-hydroxyethyl)-1H-indazol-5-yl)oxy)benzyl)urea; 1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[[5-fluoro-2-[1-(2-hydroxyethyl)indazol-5-yl]oxyphenyl]methyl]urea; ARRY-614; Pexmetinib

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: ≥ 2.08 mg/mL (3.74 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 2.08 mg/mL (3.74 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: ≥ 2.08 mg/mL (3.74 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 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

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。