Olaparib (AZD2281; KU0059436)   中文名称: 奥拉帕尼

PARP-2 ( IC50 = 1 nM ); PARP-1 ( IC50 = 5 nM ); tankyrase-1 ( IC50 = 1.5 μM ); Autophagy; Mitophagy

体外活性：奥拉帕尼可对抗 BRCA1 或 BRCA2 突变。奥拉帕尼对坦科聚合酶 1 不敏感 (IC50 >1 μM)。浓度为 30-100 nM 的奥拉帕尼可以消除 SW620 细胞中的 PARP-1 活性。与 BRCA1 和 BRCA2 丰富的细胞系（Hs578T、MDA-MB-231 和 T47D）相比，奥拉帕尼对 BRCA1 缺陷细胞系（MDA-MB-463 和 HCC1937）高度敏感。由于PARP抑制作用抑制了碱基切除修复，奥拉帕尼对KB2P细胞高度敏感，这可能导致DNA复制过程中单链断裂转化为双链断裂，从而激活BRCA2依赖性重组途径。激酶测定：向第 1 至第 10 列中添加 1 μL 奥拉帕尼（在 DMSO 中），并且仅将 1 μL DMSO 添加至阳性 (POS) 和阴性 (NEG) 对照孔（分别为第 11 列和第 12 列）预处理的 FlashPlate。 PARP-1 在缓冲液中按 1:40 稀释（缓冲液 B：10% 甘油 (v/v)、25 mM HEPES、12.5 mM MgCl2、50 mM KCl、1 mM DTT、0.01% NP-40 (v/v)、 pH 7.6）和 40 μL 添加到所有 96 个孔中（测定中的最终 PARP-1 浓度约为 1 ng/μL）。将板密封并在室温下摇动 15 分钟。随后，将 10 μL 阳性反应混合物（每孔 0.2 ng/μL 双链寡核苷酸 [M3/M4] DNA、5 μM NAD+ 最终测定浓度和每孔 0.075 μCi 3H-NAD+）添加到适当的反应孔中。孔（第 1-11 列）。将不含 DNA 寡核苷酸的阴性反应混合物添加到第 12 列（使用平均阴性对照值作为背景）。将板重新密封并在室温下再摇动 60 分钟以使反应继续进行。然后，向每孔中加入 50 μL 冰冷的乙酸 (30%) 以终止反应，并将板密封并在室温下再摇动 60 分钟。然后使用 TopCount 读板器以每分钟计数 (CPM) 确定与 FlashPlate 结合的氚化信号。细胞测定：奥拉帕尼的细胞毒性通过克隆形成测定来测量。使用前将奥拉帕尼溶解在 DMSO 中并用培养基稀释。将细胞（乳腺癌细胞系，包括 SW620 结肠细胞、A2780 卵巢细胞、HCC1937、Hs578T、MDA-MB-231、MDA-MB-436 和 T47D）接种在六孔板中并贴壁过夜。然后添加不同浓度的奥拉帕尼并将细胞孵育7-14天。之后对存活的菌落进行计数以计算IC50。

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体外细胞效力[1]
在许多体外模型中评估了47/奥拉帕尼的细胞效力。初步研究表明，47是一种有效的药物，可以增强烷化剂MMS对细胞的杀伤作用。在SW620细胞上建立MMS的生长抑制曲线后，以nM浓度添加PARP-1抑制剂可剂量依赖性地提高MMS的有效性（图2a）。增强水平在100nM浓度附近趋于平稳，这表明47与MMS联合使用的最大细胞活性在这个范围内。我们通过使用标准杆数形成测定法直接测量细胞中的PARP-1抑制活性来证实这一点，其中47以类似的浓度范围应用于SW620细胞裂解物，并确定PARP-1抑制的IC50约为6nM，PARP-1活性的总消融浓度为30-100nM（图2b）。

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结果：遗传、转录和功能分析证实，成功分离出BRCA2缺陷型和BRCA2富集型小鼠乳腺肿瘤细胞系。用11种不同的抗癌药物或γ射线照射治疗这些细胞系表明，新型特异性PARP抑制剂Olaparib/AZD2281对BRCA2缺陷型乳腺肿瘤细胞和BRCA2富集型乳腺癌细胞的生长抑制作用最强。最后，药物组合研究表明，AZD2281和顺铂对BRCA2缺陷细胞具有协同细胞毒性，但对BRCA2-熟练对照细胞没有协同细胞毒性。
结论：我们成功建立了第一套BRCA2缺陷乳腺肿瘤细胞系，这是对现有BRCA突变乳腺癌症临床前模型的重要补充。这些细胞对PARP抑制剂奥拉帕尼单独或与顺铂联合使用的AZD2281的高度敏感性为AZD2281作为针对BRCA缺陷型癌症的新型靶向治疗提供了强有力的支持。

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Olaparib选择性靶向ATM突变淋巴细胞，包括增殖的原代CLL细胞。对Olaparib的敏感性是由ATM活性的缺失和细胞增殖介导的。ATM功能障碍细胞的奥拉帕尼敏感性与DNA损伤的积累有关，并且与凋亡无关。研究人员调查了5个ATM突变淋巴母细胞系（LCL）、一个ATM突变MCL细胞系、一个ATM-敲除PGA-CLL细胞系和9个ATM缺陷原代CLL对聚ADP核糖聚合酶抑制剂olaparib（AZD2281）的体外敏感性，并观察到与ATM野生型对应物相比的差异杀伤。ATM的药理学抑制和ATM敲除证实了这种作用是ATM依赖性的，并且是通过有丝分裂突变介导的，与凋亡无关。

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奥拉帕尼使ATM突变细胞对常规细胞毒性药物敏感[4]
最后，为了解决PARP抑制提高标准CLL治疗和其他化疗药物效果的能力，我们测试了olaparib使ATM突变细胞对嘌呤类似物氟达拉滨、烷化剂4HC和苯达莫司汀、组蛋白脱乙酰酶抑制剂VPA和IR敏感的能力（补充表2；图6A）。当单独用这些药物处理时，Granta-519细胞对苯达莫司汀和4HC具有抗性，但对VPA敏感。奥拉帕尼预处理能够显著地使细胞对所有测试的药物敏感（图6A）。奥拉帕尼与VPA之间观察到最大的协同作用，而奥拉帕尼和IR仅显示出适度的协同作用（补充表2）。4HC和奥拉帕尼的效果在很大程度上是相加的（补充表2），尽管在0.1µM的4HC剂量下观察到适度的协同作用（图6A）。最后，奥拉帕尼对氟达拉滨和苯达莫司汀细胞毒性的影响通常是累加的，尽管在某些剂量的氟达拉滨下可以检测到协同活性（补充表2）。Western blot分析表明，当Granta-519细胞暴露于4HC、VPA或IR与1µM olaparib联合使用时，PARP1和胱天蛋白酶3和7的切割增强（图6B），表明在olaparib存在的情况下，药物诱导的凋亡增加。

奥拉帕尼（10 mg/kg，口服）与替莫唑胺联合使用可显着抑制 SW620 异种移植物中的肿瘤生长。奥拉帕尼对 Brca1-/-;p53-/- 乳腺肿瘤显示出良好的反应（每天 50 mg/kg ip），而对 HR 缺陷的 Ecad-/-;p53-/- 乳腺肿瘤没有反应。奥拉帕尼甚至在荷瘤小鼠中没有表现出剂量限制性毒性。奥拉帕尼已用于治疗 BRCA 突变肿瘤，例如卵巢癌、乳腺癌和前列腺癌。此外，Olaparib 对 ATM（共济失调毛细血管扩张突变）缺陷型肿瘤细胞表现出选择性抑制作用，这表明它是治疗 ATM 突变淋巴肿瘤的潜在药物。

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体内疗效[1]
根据我们的体外细胞数据，在SW620肿瘤模型中评估了化合物47/Olaparib增强甲基化化疗药物TMZ抗肿瘤活性的能力。 携带SW620异种移植物肿瘤的动物用化合物47/Olaparib（10mg/kg，po）联合TMZ（50mg/kg，po）治疗，每天一次，连续5天，之后让肿瘤生长出来。与单独使用TMZ组相比，TMZ加化合物47组合对肿瘤体积有相当大的抑制作用（平均值以相对肿瘤体积（RTV）给出，图4）。这相当于在TMZ治疗和联合治疗之间的整个研究末期，肿瘤生长抑制率超过80%。TMZ和联合治疗达到2×RTV的时间分别为44天和70天（潜伏期增加59%）。更高的RTV值，如4×RTV（两倍），无法进行比较评估，因为在研究期间，用联合治疗的肿瘤没有达到这种大小，这清楚地表明化合物47对TMZ活性的显著增强（图4）。TMZ耐受良好，第7天（给药结束后3天）最大平均体重减轻6%，一周内完全恢复。同样，当联合使用时，PARP抑制剂不会加剧TMZ的全身毒性，在第6天最大平均体重减轻9%，体重在3天内完全恢复，没有死亡（体重减轻>20%），这表明在这种给药方案下，联合治疗具有良好的耐受性。

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为了在现实的体内环境中评估PARP1的抑制作用，我们在BRCA1相关乳腺癌症的基因工程小鼠模型（GEMM）中测试了PARP抑制剂OlaparibAZD2281。用AZD2281治疗荷瘤小鼠抑制了肿瘤生长，没有毒性迹象，导致存活率显著提高。在该模型中，AZD2281的长期治疗确实导致了耐药性的发展，这是由编码P-糖蛋白外排泵的Abcb1a/b基因的上调引起的。这种对AZD2281的耐药性可以通过联合使用P-糖蛋白抑制剂tariquidar来逆转。AZD2281与顺铂或卡铂的联合使用增加了无复发和总体生存率，表明AZD2281增强了这些DNA损伤剂的作用。我们的研究结果证明了AZD2281对BRCA1缺乏型癌症的体内疗效，并说明了癌症的GEMM如何用于新疗法的临床前评估和测试克服或规避治疗耐药性的方法。[3]

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ATM突变淋巴肿瘤细胞在体内对Olaparib敏感[4]
为了研究奥拉帕尼的体内影响，我们建立了ATM突变MCL细胞系Granta-519的小鼠异种移植物模型。为了确定在不同动物队列中开始奥拉帕尼治疗之前是否已经发生了肿瘤细胞系的浸润和植入，在治疗开始当天（静脉注射细胞后14天或皮下注射细胞后5天）分析了每个队列中的3只代表性小鼠。注射后5天（皮下注射）和14天（静脉注射），通过FACS分析观察到骨髓和脾脏中存在至少占所有细胞1%的肿瘤细胞，这被认为是植入的（图5A）。此外，使用免疫细胞化学和抗人CD5、Pax-5和Ki-67抗体，我们证实在治疗开始前的两个时间点，脾脏和骨髓中都有增殖的人B淋巴肿瘤细胞的显著浸润（图5A）

随后，在静脉注射细胞5周后和奥拉帕尼治疗14天后，比较了23只NOD/SCID Granta-519细胞注射小鼠淋巴器官中的肿瘤负荷程度。然而，在实验早期，7只小鼠死于与移植物无关的原因，留下16只小鼠，这些小鼠要么接受奥拉帕尼（n=8）治疗，要么仅接受赋形剂（=8）治疗。通过FACS分析对人CD45染色百分比的分析（图5B）显示，与仅接受载体的小鼠相比，接受奥拉帕尼治疗的小鼠骨髓中Granta-519细胞的百分比显著降低，脾脏中的肿瘤细胞载量呈下降趋势（图5B）。 接下来，我们评估了奥拉帕尼对小鼠局部注射ATM突变Granta-519细胞产生的皮下肿瘤生长的影响，发现奥拉帕尼治疗与肿瘤大小减小之间存在显著的正相关关系（图5C）。最后，与单独使用载体相比，奥拉帕尼治疗显著提高了植入Granta-519细胞的小鼠的总体存活率（图5D）。

该测定评估了奥拉帕尼抑制 PARP-1 酶活性的能力。测量 PARP-2 活性抑制的另一种方法涉及使用 PARP-2 特异性抗体在白壁 96 孔板中结合重组 PARP-2 蛋白。添加 ³H-NAD⁺ DNA 后测量 PARP-2 活性。洗涤后添加闪烁剂以量化 ³H 掺入的核糖基化。创建了针对 tankyrase-1 的 AlphaScreen 测定，包括在 384 孔 ProxiPlate 测定中与生物素化 NAD⁺ 一起孵育 HIS 标记的重组 TANK-1 蛋白。通过添加 α 珠子结合 HIS 和生物素标签来产生邻近信号；该信号的丢失与 TANK-1 活性抑制直接相关。每个实验至少进行三次重复。

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体外分离酶测定[1]
该测定测定了受试化合物抑制PARP-1酶活性的能力。所采用的方法与报道一致。我们通过使用PARP-1测定的变体来测量PARP-2活性抑制，其中PARP-2蛋白（重组）在96孔白壁板中被PARP-2特异性抗体结合。在添加3H-NAD+DNA后测量PARP-2活性。洗涤后，加入闪烁剂以测量3H掺入的核糖基化。对于tankyrase-1，开发了一种AlphaScreen检测方法，其中HIS标记的重组TANK-1蛋白在384孔ProxiPlate检测中与生物素化的NAD+一起孵育。加入α珠以结合HIS和生物素标签，从而产生接近信号，而TANK-1活性的抑制与该信号的损失成正比。所有实验至少重复三次。

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体外PARP活性测定[1]
SW620全细胞蛋白提取物是通过在提取缓冲液（1×PBS，1%NP-40，蛋白酶抑制剂混合物，200μM DTT）中在4°C下孵育10分钟制备的。PARP活性通过体外激活后标准杆数形成量的定量来确定。PARP激活反应通过在30°C下使用65 ng SW620全细胞提取物在反应混合物（50 mM Tris pH 8，4 mM MgCl2，200μM DTT，200μM NAD+，20 ng/μL DNA）中进行5分钟。然后通过使用中尺度发现测定平台对每个反应中形成的标准杆数量进行定量。数据是通过三次重复实验计算得出的，即PARP活性相对于载体对照±SE和IC50的平均百分比，这些百分比是使用XL-FIT 4软件计算的。

增强因子或 PF50 值是通过将对照生长中使用的烷化剂甲磺酸甲酯 (MMS) 的 IC50 除以 MMS 加 PARP 抑制剂的 IC50 来确定的。使用 HeLa B 细胞以固定 200 nM 浓度对 Olaparib 进行 MMS 筛选测试。在结直肠细胞系 SW620 上测试的奥拉帕尼浓度为 1、3、10、100 和 300 nM。磺胺罗丹明 B (SRB) 测定用于测量细胞生长。

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体外细胞PF50测定[1]
PF50值是增强因子，其计算方法为使用烷基化剂甲基甲烷磺酸盐（MMS）的对照生长的IC50除以MMS与PARP抑制剂组合的IC50。使用HeLa B细胞，在固定的200 nM浓度下测试测试化合物，以用MMS进行筛选。对于化合物47/Olaparib在SW620结直肠细胞系上的测试（图2），使用的浓度为1、3、10、100和300 nM。通过使用磺基罗丹明B（SRB）测定来评估细胞生长。

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细胞系和培养[1]
SW620结肠癌、A2780卵巢癌、HCC1937、Hs578T、MDA-MB-231、MDA-MB-436和T47D乳腺癌症细胞系从ATCC或ECACC库获得。所有细胞系均在添加了10%v/v FBS、100μg/mL青霉素和100μg/mL链霉素的RPMI1640培养基中单层生长。

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细胞系细胞毒性试验[1]
KU-0059436对癌症细胞系的细胞存活的影响通过使用克隆发生测定来测定，如前所述。简而言之，将细胞接种在六孔板中，并放置过夜。将载体对照（DMSO）或浓度增加的KU-0059436（高达4μM）加入细胞中，根据细胞类型，将混合物放置7-14天，然后计数存活的菌落。通过XL-FIT 4软件计算三个重复孔的数据，即细胞存活率相对于载体对照±SE和IC50的平均百分比。

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47/奥拉帕尼增强MMS细胞毒性通过使用硫罗丹明B细胞生长试验确定[1]
将SW620细胞接种在96孔板中，并放置过夜。在添加浓度递增的MMS之前，细胞与载体对照（DMSO）或单一浓度的KU-0059436（1、3、10、30、100或300 nM）预孵育1小时。细胞在每种药物组合存在下孵育4天，然后通过SRB测定定量细胞生长。从三个重复孔计算数据，作为相对于仅KU-0059436孔的细胞生长平均百分比，并使用XL-FIT 4软件计算±SE和IC50。当仅以低于300 nM的浓度使用KU-0059436时，SW620细胞显示出<24%的生长抑制（>76%的细胞生长）（数据未显示）。

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实验设计：我们建立并彻底表征了一组来自独立BRCA2缺陷小鼠乳腺肿瘤和BRCA2精通控制肿瘤的克隆细胞系。随后，我们评估了这些细胞系对传统细胞毒性药物和新型PARP抑制剂AZD2281的敏感性。最后，进行了体外联合研究，以研究/OlaparibAZD2281和顺铂之间的相互作用。[2]

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CD40L/IL-4[4]
诱导原代CLL细胞增殖 从外周血获得的原代CLL白血病细胞通常在细胞周期的间隙1/间隙0（G1/G0）处停滞。为了刺激和维持这些细胞的增殖，我们比较了5种不同的促有丝分裂刺激（见图3），发现CD40L/IL-4培养系统是最有效和可重复的。简而言之，在以3×105个细胞/孔的剂量照射（50 Gy）表达CD40L的小鼠成纤维细胞后，将1-1.5×106个原代CLL细胞与10 ng/mL IL-4接种到每个孔中，总体积为2 mL RPMI，含有10%胎牛血清，并在37°C下孵育3-4天。此时，启动了存活检测。由于溴脱氧尿苷（BrdU）染色显示CLL增殖在培养中只能持续7-11天（补充图3），因此开始循环3-4天的原代CLL细胞然后用0-10µM的Olaparib再处理7天。因此，为了保持一致性，在所有存活试验中，所有细胞类型仅暴露于奥拉帕尼7天。

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细胞存活试验[4]
在三次重复实验中，将淋巴细胞悬浮液暴露于浓度越来越高的Olaparib中长达7天，并使用血细胞计数器计数3次；然后计算存活分数。在使用单剂量奥拉帕尼的实验中，无论细胞类型如何，都使用3µM，因为这在曲线的第二部分产生了超出初始急剧减少的存活反应，并确保了正常和ATM缺陷细胞之间的最大差异。它还反映了临床上可达到的最大剂量，使该剂量的细胞效应在临床上具有重要意义。

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联合奥拉帕尼/细胞毒性治疗[4]
以1×105个细胞/mL的浓度在200µL的体积中接种三份Granta-519细胞，用Olaparib（剂量范围0-10µM）预处理2天。随后，将递增剂量的4-羟基环磷酰胺（4HC；0-0.25µM；NIOMECH）、氟达拉滨（0-0.5µM）、丙戊酸（VPA；0-10mM）、苯达莫司汀（0-12.5µM）和IR（0-5 Gy）加入含奥拉帕尼的培养物中，再持续5天。这一时间框架使奥拉帕尼治疗的持续时间（共7天）保持一致，并在计算细胞存活率之前，为常规药物的细胞毒性作用提供了足够的时间。根据制造商的说明，使用CellTiter Glo发光细胞存活率测定法测量细胞存活率。使用Wallac Victor2 1420多标签计数器对发光进行定量。使用Windows软件的Calcusyn 2.1版确定协同作用。

Mice: Four treatment groups (n = 5) are randomly assigned to mice with tumors measuring 220-250 mm³: Vehicle control (10% DMSO in PBS/10% 2-hydroxy-propyl-β-cyclodextrin daily for 5 days by oral gavage), Olaparib (50 mg/kg daily for 5 days by oral gavage), 10 Gy fractionated radiotherapy (2 Gy daily for 5 days), and Olaparib and 10 Gy (5×2 Gy) fractionated radiotherapy (with olaparib given 30 min prior to each daily 2 Gy dose of radiation) are the options available. Measurements of tumor volume are made every day until they reach 1000 mm³. For each group of tumors, the number of days needed for each tumor to quadruple in size from the beginning of the treatment is calculated (relative tumor volume×4; RTV4).

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Mouse Xenografts [1]
Tagged mice were inoculated sc with 5 × 106 cells in 0.1 mL of PBS to the right flank. Tumors were measured thrice weekly, and tumor volumes were estimated from the formula [length/2] × [width2]. For xenograft chemopotentiation studies, established tumors in each animal were individually normalized to their size at the start of the experiment, and the data were calculated as the change in tumor volume relative to the day 0 volume by the use of the relative tumor volume (RTV) formula, RTV = TVx/TV0, where TVx is the tumor volume on any day and TV0 is the tumor volume at the initiation of dosing (i.e., day 0). In the tumor growth curves, the mean represents a full complement of animals in the treatment groups; below this threshold, we ceased plotting.

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Dosing Regimen [1]
When the mean tumor volume reached 100 mm3, tumor-bearing mice were randomized into treatment groups with eight animals in each group being dosed orally once daily for 5 consecutive days (po, q.d., ×5); for the combination therapies, the PARP inhibitor was administered 45 min before TMZ. Compound 47/Olaparib was formulated in solution, and TMZ was formulated as a homogeneous suspension in corn oil; both dosing solutions were freshly made each day. Mice in the no-treatment group received both vehicles on a mg/kg basis. Mice were weighed daily during the dosing phase to calculate the day’s dose and any signs of body weight loss accurately (20% weight loss led to the animal being euthanized). Statistical analyses were calculated on the data only when a full complement of animals was present in the treatment groups (i.e., day 13 for the vehicle and 47 monotherapy groups and day 45 for the TMZ and combination comparisons). From the Jonckheere−Terpstra trend test, we concluded that at day 45 there was a statistically significant effect of increasing the dose of 47 (10 mg/kg, data shown only in Figure 3) when used in combination with 50 mg/kg TMZ as compared with that of TMZ alone (p < 0.0001). This was confirmed by the use of Wilcoxon rank-sum tests, which compared the combination treatment groups versus TMZ alone to demonstrate statistically significant differences between TMZ monotherapy and TMZ in combination with 47 at 10 mg/kg (p = 0.007) at day 45.

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Pharmacokinetics [1]
We carried out pharmacokinetics determinations in mouse, rat, and dog. All doses of the compounds were given as a single dose either intravenously (iv) or orally (po; see dose level, as described in Tables 6 and 7). For the iv studies, the compounds were formulated in a mixture of 10% DMSO/10% cyclodextrin in PBS. For the oral studies, the compounds were also predominantly formulated as a solution in 10% DMSO/10% cyclodextrin in PBS except for compound 47, where a suspension in methylcellulose/PBS was used for oral dosing in dogs.

Absorption, Distribution and Excretion
Following oral administration, olaparib is rapidly absorbed. After administration of a single 300 mg dose of olaparib, the mean (CV%) Cmax was 5.4 μg/mL (32%) and AUC was 39.2 μg x h/mL (44%). The steady state Cmax and AUC following a dose of 300 mg twice daily was 7.6 μg/mL (35%) and 49.2 μg x h/mL (44%), respectively. Tmax is 1.5 hours. A high-fat and high-calorie meal may delay Tmax, but does not significantly alter the extent of olaparib absorption.
Following a single dose of radiolabeled olaparib, 86% of the dosed radioactivity was recovered within a seven-day collection period, mostly in the form of metabolites. About 44% of the drug was excreted via the urine and 42% of the dose was excreted via the feces. Following an oral dose of radiolabeled olaparib to female patients, the unchanged drug accounted for 15% and 6% of the radioactivity in urine and feces, respectively.
The mean (± standard deviation) apparent volume of distribution of olaparib is 158 ± 136 L following a single 300 mg dose.
Following a single oral dose in patients with cancer, the mean apparent plasma clearance was 4.55 L/h.

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Metabolism / Metabolites
Olaparib is metabolized by cytochrome P450 (CYP) 3A4/5 _in vitro_. Following an oral dose of radiolabeled olaparib to female patients, unchanged olaparib accounted for 70% of the circulating radioactivity in plasma. Olaparib undergoes oxidation reactions as well as subsequent glucuronide or sulfate conjugation. In humans, olaparib can also undergo hydrolysis, hydroxylation, and dehydrogenation. While up to 37 metabolites of olaparib were detected in plasma, urine, and feces, the majority of metabolites represent less than 1% of the total administered dose and they have not been fully characterized. The major circulating metabolites are a ring-opened piperazin-3-ol moiety and two mono-oxygenated metabolites. The pharmacodynamic activity of the metabolites is unknown.

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Biological Half-Life
Following a single oral dose in patients with cancer, the mean terminal half-life was 6.10 hours.

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Pharmacokinetic Profiles [1]
To establish whether one of these compounds (46 or 47/Olaparib) had the right PK profile to warrant its selection as an orally bioavailable candidate for clinical use, we initially evaluated both compounds for oral bioavailability and pharmacokinetic stability in rats (Table 7). Despite these compounds being structurally comparable and profiling very similarly in terms of their rat iv PK values, cyclopropyl compound 47/Olaparib showed a significantly greater oral exposure than did compound 46, which confirms the mouse data. Accordingly, compound 47 was advanced to further pharmacokinetic analysis in dogs. Table 7 highlights the PK profile of 47 in dog following its dosing iv at 2.5 mg/kg and po at 10 mg/kg in a methylcellulose vehicle as a suspension formulation. The data show that this compound retains an excellent level of bioavailability that is similar to that seen in rat but has a lower relative clearance that equates to approximately 16% hepatic blood flow (68% in rat). As such, this pharmacokinetic profile provided therapeutically relevant levels of PARP inhibitor over at least 4 h in this species. Cyclopropylamide analog 47 was shown to possess good exposure together with attributes of potency and physicochemistry such that this compound was advanced to further analysis of its cellular potency and in vivo efficacy to identify it as a potential clinical candidate.

Hepatotoxicity
In large clinical trials of olaparib, abnormalities in routine liver tests were uncommon with serum aminotransferase elevations occurring in 4% of patients and values above 5 times the upper limit of normal (ULN) in 1% or less. In trials of olaparib in patients with various advanced solid tumors there were no reports of hepatitis with jaundice or liver failure. Subsequent to its approval and more widescale use, there have been no published reports of clinically apparent liver injury attributed to olaparib.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of olaparib during breastfeeding. Because olaparib is 82% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during olaparib therapy and for one month after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
The protein binding of olaparib is approximately 82% _in vitro_. In solutions of purified proteins, the olaparib fraction bound to albumin was approximately 56% and the fraction bound to alpha-1 acid glycoprotein was 29%.

[1]. J Med Chem . 2008 Oct 23;51(20):6581-91.

[2]. Clin Cancer Res . 2008 Jun 15;14(12):3916-25.

[3]. Proc Natl Acad Sci U S A . 2008 Nov 4;105(44):17079-84.

[3]. Blood . 2010 Nov 25;116(22):4578-87.

Olaparib is a member of the class of N-acylpiperazines obtained by formal condensation of the carboxy group of 2-fluoro-5-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoic acid with the free amino group of N-(cyclpropylcarbonyl)piperazine; used to treat advanced ovarian cancer. It has a role as an antineoplastic agent, an EC 2.4.2.30 (NAD(+) ADP-ribosyltransferase) inhibitor and an apoptosis inducer. It is a N-acylpiperazine, a member of cyclopropanes, a member of monofluorobenzenes and a member of phthalazines.
Olaparib is a selective and potent inhibitor of poly (ADP-ribose) polymerase (PARP) enzymes, PARP1 and PARP2. PARP inhibitors represent a novel class of anti-cancer therapy and they work by taking advantage of a defect in DNA repair in cancer cells with BRCA mutations and inducing cell death. Olaparib is used to treat a number of BRCA-associated tumours, including ovarian cancer, breast cancer, pancreatic cancer, and prostate cancer. It was first approved by the FDA and EU in December 2014, and by Health Canada in April 2016.
Olaparib is a Poly(ADP-Ribose) Polymerase Inhibitor. The mechanism of action of olaparib is as a Poly(ADP-Ribose) Polymerase Inhibitor.
Olaparib is a small molecule inhibitor of poly ADP-ribose polymerase and is used as an antineoplastic agent in the therapy of refractory and advanced ovarian carcinoma. Olaparib therapy is associated with a low rate of transient elevations in serum aminotransferase during therapy and has not been linked to instances of clinically apparent liver injury.
Olaparib is a small molecule inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) with potential chemosensitizing, radiosensitizing, and antineoplastic activities. Olaparib selectively binds to and inhibits PARP, inhibiting PARP-mediated repair of single strand DNA breaks; PARP inhibition may enhance the cytotoxicity of DNA-damaging agents and may reverse tumor cell chemoresistance and radioresistance. PARP catalyzes post-translational ADP-ribosylation of nuclear proteins and can be activated by single-stranded DNA breaks.

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Drug Indication
**Ovarian cancer** Olaparib is indicated for the maintenance treatment of adults with deleterious or suspected deleterious germline or somatic BRCA-mutated advanced epithelial ovarian, fallopian tube or primary peritoneal cancer who are in complete or partial response to first-line platinum-based chemotherapy. Olaparib is indicated in combination with [bevacizumab] for the maintenance treatment of adults with advanced epithelial ovarian, fallopian tube or primary peritoneal cancer who are in complete or partial response to first-line platinum-based chemotherapy and whose cancer is associated with homologous recombination deficiency (HRD)-positive status defined by either: a deleterious or suspected deleterious BRCA mutation, and/or genomic instability. Olaparib is indicated for the maintenance treatment of adult patients with recurrent epithelial ovarian, fallopian tube or primary peritoneal cancer, who are in complete or partial response to platinum-based chemotherapy. **Breast cancer** Olaparib is indicated for the adjuvant treatment of adult patients with deleterious or suspected deleterious g_BRCA_m human epidermal growth factor receptor 2 (HER2)-negative high risk early breast cancer who have been treated with neoadjuvant or adjuvant chemotherapy. Olaparib is indicated for the treatment of adult patients with deleterious or suspected deleterious g_BRCA_m, HER2-negative metastatic breast cancer, who have been treated with chemotherapy in the neoadjuvant, adjuvant, or metastatic setting. Patients with hormone receptor (HR) positive breast cancer should have been treated with a prior endocrine therapy or be considered inappropriate for endocrine therapy. **Pancreatic cancer** Olaparib is indicated for the maintenance treatment of adult patients with deleterious or suspected deleterious gBRCAm metastatic pancreatic adenocarcinoma whose disease has not progressed on at least 16 weeks of a first-line platinum-based chemotherapy regimen. **Prostate cancer** Olaparib is indicated for the treatment of adult patients with deleterious or suspected deleterious germline or somatic homologous recombination repair (HRR) gene-mutated metastatic castration-resistant prostate cancer (mCRPC) who have progressed following prior treatment with a hormone agent, such as [enzalutamide] or [abiraterone]. It is also indicated in combination with [abiraterone] and [prednisone] or [prednisolone] for the treatment of adult patients with deleterious or suspected deleterious BRCA-mutated (BRCAm) metastatic castration-resistant prostate cancer (mCRPC).
Ovarian cancer Lynparza is indicated as monotherapy for the: maintenance treatment of adult patients with advanced (FIGO stages III and IV) BRCA1/2-mutated (germline and/or somatic) high-grade epithelial ovarian, fallopian tube or primary peritoneal cancer who are in response (complete or partial) following completion of first-line platinum-based chemotherapy. maintenance treatment of adult patients with platinum sensitive relapsed high grade epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in response (complete or partial) to platinum based chemotherapy. Lynparza in combination with bevacizumab is indicated for the: maintenance treatment of adult patients with advanced (FIGO stages III and IV) high-grade epithelial ovarian, fallopian tube or primary peritoneal cancer who are in response (complete or partial) following completion of first-line platinum-based chemotherapy in combination with bevacizumab and whose cancer is associated with homologous recombination deficiency (HRD) positive status defined by either a BRCA1/2 mutation and/or genomic instability (see section 5. 1). Breast cancer Lynparza is indicated as: monotherapy or in combination with endocrine therapy for the adjuvant treatment of adult patients with germline BRCA1/2-mutations who have HER2-negative, high risk early breast cancer previously treated with neoadjuvant or adjuvant chemotherapy (see sections 4. 2 and 5. 1). monotherapy for the treatment of adult patients with germline BRCA1/2-mutations, who have HER2 negative locally advanced or metastatic breast cancer . Patients should have previously been treated with an anthracycline and a taxane in the (neo)adjuvant or metastatic setting unless patients were not suitable for these treatments (see section 5. 1). Patients with hormone receptor (HR)-positive breast cancer should also have progressed on or after prior endocrine therapy, or be considered unsuitable for endocrine therapy. Adenocarcinoma of the pancreasLynparza is indicated as: monotherapy for the maintenance treatment of adult patients with germline BRCA1/2-mutations who have metastatic adenocarcinoma of the pancreas and have not progressed after a minimum of 16 weeks of platinum treatment within a first-line chemotherapy regimen. Prostate cancer Lynparza is indicated as: monotherapy for the treatment of adult patients with metastatic castration-resistant prostate cancer (mCRPC) and BRCA1/2-mutations (germline and/or somatic) who have progressed following prior therapy that included a new hormonal agent. in combination with abiraterone and prednisone or prednisolone for the treatment of adult patients with mCRPC in whom chemotherapy is not clinically indicated (see section 5. 1).
Treatment of all conditions included in the category of malignant neoplasms (except central nervous system tumours, haematopoietic and lymphoid tissue neoplasms)

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Mechanism of Action
Poly(ADP-ribose) polymerases (PARPs) are multifunctional enzymes comprising 17 members. They are involved in essential cellular functions, such as DNA transcription and DNA repair. PARPs recognize and repair cellular DNA damage, such as single-strand breaks (SSBs) and double-strand breaks (DSBs). Different DNA repair pathways exist to repair these DNA damages, including the base excision repair (BER) pathway for SSBs and BRCA-dependent homologous recombination for DSBs. Olaparib is a PARP inhibitor: while it acts on PARP1, PARP2, and PARP3, olaparib is a more selective competitive inhibitor of NAD⁺ at the catalytic site of PARP1 and PARP2. Inhibition of the BER pathway by olaparib leads to the accumulation of unrepaired SSBs, which leads to the formation of DSBs, which is the most toxic form of DNA damage. While BRCA-dependent homologous recombination can repair DSBs in normal cells, this repair pathway is defective in cells with BRCA1/2 mutations, such as certain tumour cells. Inhibition of PARP in cancer cells with BRCA mutations leads to genomic instability and apoptotic cell death. This end result is also referred to as synthetic lethality, a phenomenon where the combination of two defects - inhibition of PARP activity and loss of DSB repair by HR - that are otherwise benign when alone, lead to detrimental results. _In vitro_ studies have shown that olaparib-induced cytotoxicity may involve inhibition of PARP enzymatic activity and increased formation of PARP-DNA complexes, resulting in DNA damage and cancer cell death.

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Poly(ADP-ribose) polymerase activation is an immediate cellular response to metabolic-, chemical-, or ionizing radiation-induced DNA damage and represents a new target for cancer therapy. In this article, we disclose a novel series of substituted 4-benzyl-2 H-phthalazin-1-ones that possess high inhibitory enzyme and cellular potency for both PARP-1 and PARP-2. Optimized compounds from the series also demonstrate good pharmacokinetic profiles, oral bioavailability, and activity in vivo in an SW620 colorectal cancer xenograft model. 4-[3-(4-Cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2 H-phthalazin-1-one (KU-0059436, AZD2281) 47 is a single digit nanomolar inhibitor of both PARP-1 and PARP-2 that shows standalone activity against BRCA1-deficient breast cancer cell lines. Compound 47 is currently undergoing clinical development for the treatment of BRCA1- and BRCA2-defective cancers.[1]

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The Ataxia Telangiectasia Mutated (ATM) gene is frequently inactivated in lymphoid malignancies such as chronic lymphocytic leukemia (CLL), T-prolymphocytic leukemia (T-PLL), and mantle cell lymphoma (MCL) and is associated with defective apoptosis in response to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double strand break repair. Poly (ADP-ribose) polymerase (PARP) inhibition that imposes the requirement for DNA double strand break repair should selectively sensitize ATM-deficient tumor cells to killing. We investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis. A nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft model of an ATM mutant MCL cell line demonstrated significantly reduced tumor load and an increased survival of animals after olaparib treatment in vivo. Addition of olaparib sensitized ATM null tumor cells to DNA-damaging agents. We suggest that olaparib would be an appropriate agent for treating refractory ATM mutant lymphoid tumors.[4]

C24H23FN4O3

434.46

434.175

C, 66.35; H, 5.34; F, 4.37; N, 12.90; O, 11.05

763113-22-0

23725625

White solid powder

1.4±0.1 g/cm3

1.702

1.9

86.37

1

5

4

32

790

0

FC1C([H])=C([H])C(C([H])([H])C2C3=C([H])C([H])=C([H])C([H])=C3C(N([H])N=2)=O)=C([H])C=1C(N1C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C(C1([H])C([H])([H])C1([H])[H])=O)=O

FDLYAMZZIXQODN-UHFFFAOYSA-N

InChI=1S/C24H23FN4O3/c25-20-8-5-15(14-21-17-3-1-2-4-18(17)22(30)27-26-21)13-19(20)24(32)29-11-9-28(10-12-29)23(31)16-6-7-16/h1-5,8,13,16H,6-7,9-12,14H2,(H,27,30)

4-[[3-[4-(cyclopropanecarbonyl)piperazine-1-carbonyl]-4-fluorophenyl]methyl]-2H-phthalazin-1-one

AZD2281; Ku-0059436; AZD2281; 763113-22-0; Lynparza; KU-0059,436; 1-(Cyclopropylcarbonyl)-4-[5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoyl]piperazine; AZD-2281; AZD 2281; KU59436; KU-59436; KU 59436; KU0059436; KU-0059436; KU 0059436; Olaparib; trade name Lynparza

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: 10 mg/mL (23.02 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 100.0 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。

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配方 2 中的溶解度: ≥ 5 mg/mL (11.51 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 3 中的溶解度: ≥ 5 mg/mL (11.51 mM) (饱和度未知) in 5% DMSO + 95% (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。
*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: ≥ 2.5 mg/mL (5.75 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100μL 25.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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 5 中的溶解度: ≥ 2.5 mg/mL (5.75 mM) (饱和度未知) in 10% DMF 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。
*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 6 中的溶解度: ≥ 2.5 mg/mL (5.75 mM) (饱和度未知) in 10% DMF 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。
*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 7 中的溶解度: ≥ 2.5 mg/mL (5.75 mM) (饱和度未知) in 10% DMF 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。

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配方 8 中的溶解度: ≥ 2.08 mg/mL (4.79 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，要配制1 mL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，混匀。

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配方 9 中的溶解度: ≥ 0.5 mg/mL (1.15 mM) (饱和度未知) in 1% DMSO 99% 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 10 中的溶解度: 20 mg/mL (46.03 mM) in 0.5% CMC-Na/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;
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