Adavosertib (AZD-1775; MK-1775)   中文名称: Wee1 抑制剂 adavosertib

Wee1 (IC50 = 5.2 nM)

体外活性：MK-1775 以 ATP 竞争性方式抑制 Wee1 激酶。与 Wee1 相比，MK-1775 对 Yes 的效力低 2 至 3 倍，IC50 为 14 nM，对其他 7 种激酶的效力低 10 倍，在 1 μM 时抑制 >80%，选择性比人类高 100 倍Myt 1，另一种通过替代位点 (Thr14) 磷酸化来抑制细胞周期蛋白依赖性激酶 1 (CDC2) 的激酶。通过阻断携带突变 p53 的 WiDr 细胞中的 Wee1 活性来消除 DNA 损伤检查点，MK-1775 治疗可抑制 CDC2 在 Tyr15 (CDC2Y15) 的基础磷酸化，EC50 为 49 nM，并抑制吉西他滨、卡铂或顺铂诱导的损伤CDC2 磷酸化和细胞周期停滞呈剂量依赖性，EC50 分别为 82 nM 和 81 nM、180 nM 和 163 nM、以及 159 nM 和 160 nM。 30-100 nM 的 MK-1775 单独处理对 WiDr 和 H1299 细胞没有显着的抗增殖作用，而 300 nM 的 MK-1775 足以抑制 Wee1 > 80%，在 WiDr 细胞中显示出中等但显着的抗增殖作用，抗增殖作用达 34.1% H1299 细胞中为 28.4%。激酶测定：使用重组人Wee1。使用 10 μM ATP、1.0 μCi 的 [γ-33P]ATP 和 2.5 μg 聚（Lys、Tyr）作为底物，在浓度递增的 MK-1775 存在下，在 30°C 下进行激酶反应 30 分钟。掺入基质中的放射性被捕获在 MultiScreen-PH 板上，并在液体闪烁计数器上进行计数。细胞测定：细胞（WiDr、NCI-H1299、TOV21G 和 HeLa）用或不用吉西他滨处理 24 小时，然后用 MK-1775 再处理 24 小时。使用 SpectraMax 通过 WST-8 试剂盒测定细胞活力。使用 Caspase-3/7 Glo 试剂盒测定细胞 caspase-3/7 活性。

MK-1775 单独治疗（约 20 mg/kg）对大鼠 WiDr 异种移植物显示出最小的抗肿瘤作用，第 3 天 T/C 为 69%。单独使用 MK-1775 对裸鼠 HeLa-luc 和 TOV21G-shp53 异种移植物的抗肿瘤功效款式也适中。

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Adavosertib (AZD-1775; MK-1775)增强H1299异种移植物肿瘤对分次放疗的反应[2]
基于Adavosertib (AZD-1775; MK-1775)在p53缺陷型NSCLC细胞系中的显著放射增敏作用（表1），我们确定了这种作用是否延伸到体内情况。我们进行了一系列实验，使用由p53缺陷型NSCLC系之一制成的裸鼠异种移植物肿瘤来检验这个问题，并用MK-1775和外束辐射的组合进行治疗，其中肿瘤生长延迟被用作分析的终点。本研究选择Calu-6细胞系是基于其在体外存活曲线分析中被MK-1775显著放射增敏（图1A和表1）。研究了各种治疗方案，包括测试不同的药物和辐射序列、不同剂量的药物和不同的辐射分级方案。其中许多方案表明，与单独使用辐射相比，药物/辐射组合显著增强了肿瘤生长延迟。当肿瘤每天用1 Gy照射两次，持续5天，并在照射的同一天每天两次给予60 mg/kg时，观察到最大的反应。该实验的结果如图4所示。该治疗方案的增强因子为3.2（p<0.01）。这些结果强调了及时对药物和放射治疗进行测序的重要性，并表明MK-1775的放射增敏作用延伸到体内环境。

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Adavosertib (AZD-1775; MK-1775)治疗导致Wee1激酶受到抑制，其底物Cdc2的抑制性磷酸化减少。与对照组和GEM治疗的肿瘤相比，MK-1775在服用GEM时消除了检查点阻滞以促进有丝分裂进入并促进了肿瘤细胞死亡。MK-1775单一疗法没有诱导肿瘤消退。然而，与p53缺陷型肿瘤的GEM治疗相比，GEM与MK-1775的组合产生了强大的抗肿瘤活性，并显著增强了肿瘤消退反应（4.01倍）。药物治疗期后绘制的肿瘤再生曲线表明，联合治疗的效果比GEM更持久。所有药物均未在p53野生型异种移植物中产生肿瘤消退。 结论：这些结果表明，Adavosertib (AZD-1775; MK-1775)选择性地与GEM协同作用，以实现肿瘤消退，在p53缺陷型胰腺癌症异种移植物中是选择性的[3]。

Wee1 是一种人类重组蛋白。使用 10 μM ATP、1.0 μCi 的 [γ-₃₃P]ATP 和 2.5 μg 聚（Lys、Tyr）作为底物进行激酶反应，所有这些都在增加 MK 的情况下进行-1775 浓度，30°C 30 分钟。混合到基质中的放射性被捕获在 MultiScreen-PH 板上，并使用液体闪烁计数器进行定量。

使用含有 0.4 mol/L NaCl、1 mM EDTA 和 50 mM HEPES (pH 7.9) 的裂解液从细胞沉淀中提取整个蛋白质。然后用蛋白酶抑制剂、1% NP-40 和 10 µL/mL 磷酸酶抑制剂混合物 1 和 2 强化蛋白质。Bio-Rad 蛋白质测定可得出裂解物的蛋白质浓度。在 Immobilon 膜上，使用 12% SDS-PAGE 分离等体积的蛋白质。含有 0.1% 吐温 (TBS-T) 和 5% 脱脂奶粉的缓冲盐水（150 mM，pH 7.4）会阻断膜上的非特异性结合位点。将膜浸泡在含有一抗的 5% 脱脂奶粉中，在 4°C 下过夜，即可鉴定蛋白质信号。随后在已过氧化物酶偶联的相应二抗中孵育 45 分钟。之后，使用 Typhoon 9400 扫描仪对膜进行显影，并使用 ECL 和蛋白质印迹检测试剂增强化学发光。

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细胞周期分析[2]
用200 nmol/LAdavosertib (AZD-1775; MK-1775)处理细胞1小时，以7.5 Gy照射，然后在0、4、8、12、16和24小时后收获。然后用PBS洗涤细胞，并在4°C下在PBS中的70%乙醇中固定过夜。将固定的细胞在缓冲液A（0.5%牛血清白蛋白（BSA）和2%FBS的PBS溶液）中洗涤，然后在冰上的裂解缓冲液（0.1%Triton X-100、0.5%BSA和2%FBS在PBS溶液中）中孵育5分钟。通过离心将细胞造粒，并在缓冲液B（2%BSA和10%FBS在PBS中）中温育。再次，通过离心使细胞造粒，然后在4°C下与缓冲液A中1:50稀释的p-HH3抗体一起孵育过夜。然后在室温下用缓冲液A洗涤细胞，并在缓冲液A中以1:100的稀释度在抗小鼠FITC二抗中孵育1小时。再次用缓冲液B洗涤细胞，通过离心造粒，并在2%BSA、2%吐温-20、5µg/mL碘化丙啶（PI）和2µg/mL RNAse A中在黑暗中孵育一小时，然后立即进行流式细胞术分析。使用Beckman Coulter EPICS-ALTRA和Hyperport系统进行流式细胞术，该系统配备有发射488nm的水冷氩激光器。使用EXPO32软件进行分析。使用525nm带通滤波器测量p-HH3。收集了至少10000个事件进行分析。闸门设置为排除细胞碎片，并测量闸门区域内事件的荧光强度。

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免疫荧光[2]
A549或H1299细胞在放置在35 mm培养皿中的盖玻片上培养，用0.2µg/mL的诺考达唑处理，用1 Gy照射，并用200 nmol/LAdavosertib (AZD-1775; MK-1775)处理，如所示。然后吸出培养基，在PBS中短暂冲洗细胞，然后用2%多聚甲醛固定15分钟。通过在-20°C下用100%甲醇孵育10分钟实现渗透性。在PBS中冲洗三次5分钟后，将细胞在室温下在封闭缓冲液（1X PBS、50µL/mL正常山羊血清和0.3%Triton X-100）中孵育1小时。接下来，在4°C下，将细胞在抗体稀释缓冲液（1X PBS，10 mg/mL牛血清白蛋白，0.3%Triton X-100）中的γ-H2AX一抗中孵育过夜，并轻轻摇晃。用PBS洗涤后，用1:500稀释的适当Alexa Fluor偶联的二抗（山羊抗兔FITC或山羊抗小鼠Alexa Fluor 594）孵育2小时后，观察到一抗。用1:500 4'6-二脒基-2-苯基吲哚二盐酸盐（DAPI）在PBS中复染细胞核，并用Vectashield将盖玻片安装在载玻片上。使用配备CCD相机的徕卡荧光显微镜检查载玻片，并将图像导入Advanced Spot Image分析软件。为了量化γ-H2AX病灶，对50个核进行了评估。通过DAPI染色鉴定微核细胞并定量（200个细胞/盖玻片）。

Inoculation of 1×10⁶ Calu-6 cells in 10 µL results in the production of tumor xenografts in the leg. Tumors with a diameter of 8 mm are treated with radiation and Adavosertib (AZD-1775; MK-1775) for 5 days. Unanesthetized mice are given gamma-rays locally at a dose rate of 5 Gy/min via a small-animal irradiator that consists of two parallel-opposed ¹³⁷Cs sources for their tumor-bearing legs. Tumors are exposed to radiation twice a day, six hours apart. Give adavosertib (MK-1775) by gavage in volumes of 0.1 mL one hour prior to and two hours following the initial daily radiation dosage.

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Tumor xenografts were produced in the leg by im inoculation of 1 × 106 Calu-6 cells in 10 µL. Irradiation and Adavosertib (AZD-1775; MK-1775) treatment were started when tumors reached 8 mm diameter and continued for 5 days. Gamma-rays were delivered locally to the tumor-bearing legs of unanesthetized mice using a small-animal irradiator consisting of two parallel-opposed 137Cs sources, at a dose rate of 5 Gy/min. Tumors were irradiated twice daily separated by 6 h. Adavosertib (AZD-1775; MK-1775) was given by gavage in 0.1 mL volumes 1 h before and 2 h after the first daily radiation dose.[2]
Three mutually orthogonal tumor diameters were measured 2–3 times/week with a Vernier caliper and means calculated for plotting tumor growth delay. Mice were euthanized when tumors grew to 15 mm diameter. Tumor regrowth was expressed as the time in days for tumors in the treated groups to grow from 8 mm to 12 mm diameter minus the time for control tumors to reach the same size (absolute growth delay [AGD]). For treatment with both Adavosertib (AZD-1775; MK-1775) and radiation, normalized growth delay (NGD) was determined as time for tumors in the combined therapy group to grow to 12 mm minus time for tumors treated with drug alone to grow to 12 mm. Radiation enhancement factor (EF) was determined by dividing NGD for MK-1775 plus radiotherapy by the AGD for irradiation plus vehicle. p values for EFs were determined by Student’s t-test comparing NGD for Adavosertib (AZD-1775; MK-1775) plus irrradiation versus AGD for irradiation plus vehicle. [2]

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Nine pancreatic cancer xenografts (six with p53-deficient and 3 with p53-wild type status) were allowed to grow separately on both flanks of athymic mice. When tumors reached a volume of ~200 mm3, mice were individually identified and randomly assigned to treatment groups, with 5–6 mice (8–10 evaluable tumors) in each group: 1) control; 2) Adavosertib (AZD-1775; MK-1775) (30 mg/kg. p.o., once daily for 4 weeks; 3) GEM (100 mg/kg, i.p., twice weekly on days 1 and 4) for 4 weeks; 4) GEM followed 24 h later by Adavosertib (AZD-1775; MK-1775) in the above mentioned dose. Tumor growth was evaluated twice per week by measurement of two perpendicular diameters of tumors with a digital caliper. Individual tumor volumes were calculated as V = a × b2/2, a being the largest diameter, b the smallest. Relative tumor growth index (TGI) on day 28 was calculated using the formula: (mean tumor volume of drug-treated group/mean tumor volume of control group) × 100. Number of tumors that regressed more than 50% of its initial size in each xenograft was noted. Animals were sacrificed 1 h after the last dose of GEM or MK-1775 and tumors were harvested for analysis except three mice each from GEM and combination treatment group, which were kept longer to check tumor re-growth after the treatment. Mice kept for the re-growth study were sacrificed when the tumors reached the size of control tumors in that xenograft.[3]

The geometric mean plasma concentration profiles over 24 h of 250 mg and 200 mg adavosertib in Cycle 1, Day 1 and Day 5 are shown in Fig. 1. Adavosertib was steadily absorbed following the first and fifth QD doses over 5 days. The median tmax was 4.03 and 2.08 h after the first dose and 2.82 and 1.90 h after the fifth dose, in the 250 and 200 mg cohorts, respectively (Table 3). Adavosertib was slowly eliminated and generally similar between the two treatment cohorts; the mean t1/2λz was 7.36 and 7.30 h after the first dose and 10.55 and 8.88 h after the fifth dose, in the 250 and 200 mg cohorts, respectively. The accumulation of adavosertib in plasma following multiple QD doses for 5 days was generally minimal with mean accumulation ratios based on AUC0–24 of 1.63 and 1.73 in the 250 and 200 mg cohorts, respectively.[4]
Systemic exposure to adavosertib increased in a slightly more than dose-proportional manner. A 1.25-fold increase in dose (200 mg to 250 mg) resulted in 1.70- and 1.65-fold increases in the geometric mean of the Cmax and AUC0–24, respectively, after the first dose and 1.38- and 1.62-fold increases in the geometric mean of the Cmax and AUC0–24, respectively, after the fifth dose.[4]

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Introduction: We aimed to assess the safety, pharmacokinetic profile, and antitumor activity of adavosertib monotherapy in Japanese patients with advanced solid tumors. Materials and methods: This was a single-center, open-label, phase I study with two consecutive cohorts (250 mg and 200 mg cohorts). Patients received adavosertib at 250 mg or 200 mg, orally once daily for 5 days on and 2 days off for Weeks 1 and 2 of a 21-day cycle. Results: Dose-limiting toxicities (Grade 3 febrile neutropenia) occurred in 2/6 patients in the 250 mg cohort. None of the three patients in the 200 mg cohort developed dose-limiting toxicities. The most frequent treatment-emergent adverse event was nausea (250 mg: 83.3 %; 200 mg: 100.0 %). Median time to peak drug concentration was 4.03 and 2.08 h after the first dose and 2.82 and 1.90 h after multiple dosing in the 250 and 200 mg cohorts, respectively; respective mean terminal elimination half-lives were 7.36 and 7.30 h (first dose) and 10.55 and 8.88 h (multiple dosing). Systemic exposure increased in a slightly more than dose-proportional manner. No RECIST v1.1 response was observed. Disease control rate was 0 % and 33.3 % in the 250 and 200 mg cohorts, respectively. One patient (33.3 %) in the 200 mg cohort showed a best overall response of stable disease at ≥ 8 weeks; the rest showed progressive disease. Conclusions: Adavosertib 200 mg once daily was well tolerated in this patient population and no safety concerns were raised. Exposure increased in a slightly more than dose-proportional manner and limited antitumor activity was shown.[4]

Safety and tolerability [4]
Overall, 8/9 patients (88.9 %) reported at least one AE, including 5/6 patients (83.3 %) in the 250 mg cohort and all three patients (100.0 %) in the 200 mg cohort. In the overall study population, the most commonly reported TEAEs were nausea (8/9; 88.9 %), followed by decreased appetite, constipation, diarrhea, vomiting, and platelet count decreased (4/9; 44.4 % each) (Table 2). In the 250 mg cohort (n = 6), the most commonly reported TEAEs were nausea (5/6 patients, 83.3 %), followed by vomiting and decreased appetite (4/6 patients, 66.7 % each). In the 200 mg cohort, the most commonly reported TEAEs were nausea (3/3 patients, 100.0 %), followed by diarrhea and hypoalbuminemia (2/3 patients, 66.7 % each). [4]
No AEs leading to discontinuation of treatment or death were reported in the study. Overall, 2/9 patients (22.2 %), both from the 250 mg cohort, reported serious AEs, including Grade 3 febrile neutropenia (2/9 patients, 22.2 %) and Grade 4 platelet count decreased (1/9 patients, 11.1 %). All three serious AEs were assessed by the investigator as possibly related to the study drug and all three were resolved. [4]
Four out of nine patients (44.4 %) reported CTCAE Grade ≥ 3 AEs that were assessed by the investigator as possibly related to the study drug. In the 250 mg cohort, 3/6 patients (50 %) reported CTCAE Grade ≥ 3 AEs that were assessed by the investigator as possibly related to the study drug, including Grade 4 events (neutropenia, white blood cell count decreased, platelet count decreased [serious AE], and neutrophil count decreased in 1/6 patients, 16.7 % each) and Grade 3 events (febrile neutropenia and anemia in 2/6 patients [33.3 %] each and white blood cell count decreased, lymphocyte count decreased, and decreased appetite in 1/6 patients, 16.7 % each). In the 200 mg cohort, 1/3 patients (33.3 %) reported one Grade 3 hypoalbuminemia event that was assessed by the investigator as possibly related to the study drug, while no Grade 4 events were reported. [4]
Grade 3 febrile neutropenia DLTs were reported in 2/6 patients in the 250 mg cohort. No DLTs were reported in the 200 mg cohort. [4]
No clinically meaningful changes in mean values over time were noted for any laboratory parameter, electrocardiogram parameters, or vital signs.

[1]. MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor efficacy of various DNA-damaging agents, including 5-FU. Cancer Biol Ther. 2010 Apr;9(7):514-22.

[2]. MK-1775, a novel Wee1 kinase inhibitor, radiosensitizes p53-defective human tumor cells. Clin Cancer Res. 2011 Sep 1;17(17):5638-48.

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[3]. MK-1775, a potent Wee1 inhibitor, synergizes with NSC 613327 to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts.Clin Cancer Res. 2011 May 1;17(9):2799-806.

[4]. Safety, tolerability, pharmacokinetics, and antitumor activity of adavosertib in Japanese patients with advanced solid tumors: A phase I, open-label study. Cancer Treat Res Commun . 2024:39:100809.

1-[6-(2-hydroxypropan-2-yl)-2-pyridinyl]-6-[4-(4-methyl-1-piperazinyl)anilino]-2-prop-2-enyl-3-pyrazolo[3,4-d]pyrimidinone is a member of piperazines.
MK-1775 has been used in trials studying the treatment of LYMPHOMA, Neoplasms, Ovarian Cancer, Tongue Carcinoma, and Adult Glioblastoma, among others.
Adavosertib is a small molecule inhibitor of the tyrosine kinase WEE1 with potential antineoplastic sensitizing activity. Adavosertib selectively targets and inhibits WEE1, a tyrosine kinase that phosphorylates cyclin-dependent kinase 1 (CDK1, CDC2) to inactivate the CDC2/cyclin B complex. Inhibition of WEE1 activity prevents the phosphorylation of CDC2 and impairs the G2 DNA damage checkpoint. This may lead to apoptosis upon treatment with DNA damaging chemotherapeutic agents. Unlike normal cells, most p53 deficient or mutated human cancers lack the G1 checkpoint as p53 is the key regulator of the G1 checkpoint and these cells rely on the G2 checkpoint for DNA repair to damaged cells. Annulment of the G2 checkpoint may therefore make p53 deficient tumor cells more vulnerable to antineoplastic agents and enhance their cytotoxic effect.

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Drug Indication
Treatment of malignant endometrial neoplasms, Treatment of pancreatic cancer

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Adavosertib is a member of the class of pyrazolopyrimidines that is 1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one substituted by 6-(2-hydroxypropan-2-yl)pyridin-2-yl, prop-1-en-3-yl, [4-(4-methylpiperazin-1-yl)phenyl]amino groups at positions 1, 2, and 6, respectively. It is a potent and selective oral inhibitor of WEE1 kinase. It has a role as an antineoplastic agent and an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor. It is a pyrazolopyrimidine, a member of pyridines, a tertiary alcohol, a secondary amino compound, a N-arylpiperazine and a N-methylpiperazine.
MK-1775 has been used in trials studying the treatment of LYMPHOMA, Neoplasms, Ovarian Cancer, Tongue Carcinoma, and Adult Glioblastoma, among others.

Adavosertib is a small molecule inhibitor of the tyrosine kinase WEE1 with potential antineoplastic sensitizing activity. Adavosertib selectively targets and inhibits WEE1, a tyrosine kinase that phosphorylates cyclin-dependent kinase 1 (CDK1, CDC2) to inactivate the CDC2/cyclin B complex. Inhibition of WEE1 activity prevents the phosphorylation of CDC2 and impairs the G2 DNA damage checkpoint. This may lead to apoptosis upon treatment with DNA damaging chemotherapeutic agents. Unlike normal cells, most p53 deficient or mutated human cancers lack the G1 checkpoint as p53 is the key regulator of the G1 checkpoint and these cells rely on the G2 checkpoint for DNA repair to damaged cells. Annulment of the G2 checkpoint may therefore make p53 deficient tumor cells more vulnerable to antineoplastic agents and enhance their cytotoxic effect.
ADAVOSERTIB is a small molecule drug with a maximum clinical trial phase of II (across all indications) and has 29 investigational indications.

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MK-1775 is a potent and selective small molecule Wee1 inhibitor. Previously we have shown that it abrogated DNA damaged checkpoints induced by gemcitabine, carboplatin, and cisplatin and enhanced the anti-tumor efficacy of these agents selectively in p53-deficient tumor cells. MK-1775 is currently in Phase I clinical trial in combination with these anti-cancer drugs. In this study, the effects of MK-1775 on 5-fluorouracil (5-FU) and other DNA-damaging agents with different modes of action were determined. MK-1775 enhanced the cytotoxic effects of 5-FU in p53-deficient human colon cancer cells. MK-1775 inhibited CDC2 Y15 phosphorylation in cells, abrogated DNA damaged checkpoints induced by 5-FU treatment, and caused premature entry of mitosis determined by induction of Histone H3 phosphorylation. Enhancement by MK-1775 was specific for p53-deficient cells since this compound did not sensitize p53-wild type human colon cancer cells to 5-FU in vitro. In vivo, MK-1775 potentiated the anti-tumor efficacy of 5-FU or its prodrug, capecitabine, at tolerable doses. These enhancements were well correlated with inhibition of CDC2 phosphorylation and induction of Histone H3 phosphorylation in tumors. In addition, MK-1775 also potentiated the cytotoxic effects of pemetrexed, doxorubicin, camptothecin, and mitomycin C in vitro. These studies support the rationale for testing the combination of MK-1775 with various DNA-damaging agents in cancer patients.[1]

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Purpose: Radiotherapy is commonly used to treat a variety of solid tumors. However, improvements in the therapeutic ratio for several disease sites are sorely needed, leading us to assess molecularly targeted therapeutics as radiosensitizers. The aim of this study was to assess the wee1 kinase inhibitor, MK-1775, for its ability to radiosensitize human tumor cells. Experimental design: Human tumor cells derived from lung, breast, and prostate cancers were tested for radiosensitization by MK-1775 using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of MK-1775 to abrogate the radiation-induced G₂ block, thereby allowing cells harboring DNA lesions to prematurely progress into mitosis, was determined using flow cytometry and detection of γ-H2AX foci. The in vivo efficacy of the combination of MK-1775 and radiation was assessed by tumor growth delay experiments using a human lung cancer cell line growing as a xenograft tumor in nude mice. Results: Clonogenic survival analyses indicated that nanomolar concentrations of MK-1775 radiosensitized p53-defective human lung, breast, and prostate cancer cells but not similar lines with wild-type p53. Consistent with its ability to radiosensitize, MK-1775 abrogated the radiation-induced G₂ block in p53-defective cells but not in p53 wild-type lines. MK-1775 also significantly enhanced the antitumor efficacy of radiation in vivo as shown in tumor growth delay studies, again for p53-defective tumors. Conclusions: These results indicate that p53-defective human tumor cells are significantly radiosensitized by the potent and selective wee1 kinase inhibitor, MK-1775, in both the in vitro and in vivo settings. Taken together, our findings strongly support the clinical evaluation of MK-1775 in combination with radiation.[2]

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Purpose: Investigate the efficacy and pharmacodynamic effects of MK-1775, a potent Wee1 inhibitor, in both monotherapy and in combination with gemcitabine (GEM) using a panel of p53-deficient and p53 wild-type human pancreatic cancer xenografts. Experimental design: Nine individual patient-derived pancreatic cancer xenografts (6 with p53-deficient and 3 with p53 wild-type status) from the PancXenoBank collection at Johns Hopkins were treated with MK-1775, GEM, or GEM followed 24 hour later by MK-1775, for 4 weeks. Tumor growth rate/regressions were calculated on day 28. Target modulation was assessed by Western blotting and immunohistochemistry. Results: MK-1775 treatment led to the inhibition of Wee1 kinase and reduced inhibitory phosphorylation of its substrate Cdc2. MK-1775, when dosed with GEM, abrogated the checkpoint arrest to promote mitotic entry and facilitated tumor cell death as compared to control and GEM-treated tumors. MK-1775 monotherapy did not induce tumor regressions. However, the combination of GEM with MK-1775 produced robust antitumor activity and remarkably enhanced tumor regression response (4.01-fold) compared to GEM treatment in p53-deficient tumors. Tumor regrowth curves plotted after the drug treatment period suggest that the effect of the combination therapy is longer-lasting than that of GEM. None of the agents produced tumor regressions in p53 wild-type xenografts. Conclusions: These results indicate that MK-1775 selectively synergizes with GEM to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts. [3]

C27H32N8O2

500.6

500.264

C, 64.78; H, 6.44; N, 22.38; O, 6.39

955365-80-7

24856436

Yellow solid powder

1.3±0.1 g/cm3

723.8±70.0 °C at 760 mmHg

391.5±35.7 °C

0.0±2.5 mmHg at 25°C

1.655

0.5

104.34

2

9

7

37

795

0

O=C1N(CC=C)N(C2C=CC=C(C(C)(C)O)N=2)C2C1=CN=C(NC1C=CC(N3CCN(C)CC3)=CC=1)N=2

BKWJAKQVGHWELA-UHFFFAOYSA-N

InChI=1S/C27H32N8O2/c1-5-13-34-25(36)21-18-28-26(29-19-9-11-20(12-10-19)33-16-14-32(4)15-17-33)31-24(21)35(34)23-8-6-7-22(30-23)27(2,3)37/h5-12,18,37H,1,13-17H2,2-4H3,(H,28,29,31)

1-[6-(2-hydroxypropan-2-yl)pyridin-2-yl]-6-[4-(4-methylpiperazin-1-yl)anilino]-2-prop-2-enylpyrazolo[3,4-d]pyrimidin-3-one

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: ≥ 2.87 mg/mL (5.73 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.16 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澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (4.16 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。

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配方 4 中的溶解度: ≥ 2.08 mg/mL (4.16 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900 μL 玉米油中，混合均匀。

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配方 5 中的溶解度: 2% DMSO +30% PEG 300 +5% Tween+ddH2O: 5 mg/mL

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配方 6 中的溶解度: 5 mg/mL (9.99 mM) in 0.5% Methylcellulose/saline water (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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。

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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。