Clinical Studies to Circumvent Drug Resistance

1 .药物分布有限导致的耐药尽管近年来我们的实验室已经从Pgp和ABCG2的研究转向了对表观遗传治疗的关注，但我们仍然相信药物分布有限可能导致耐药，并且药物分布有限可能有多种病因，其中之一可能是药物外排泵。这个直截了当的问题无法回答：限制药物分布是否在癌症化疗耐药中起作用？许多测量肿瘤组织中药物浓度的小型试验或标记药物的药效学成像研究表明，药物在癌症中的分布范围很广。我们也观察到肺癌患者对替代药物赛司他比（sestamibi）的不同摄取。没有什么地方的药物分布问题比在大脑中更重要了，在那里药物的摄取受到需要穿过血脑屏障（BBB）的限制。在脑毛细血管内皮的其他调节因子中，穿越血脑屏障需要一个积极的过程和逃避多药转运体ABCG2和ABCB1。我们参与了GRN1005的临床试验，GRN1005是一种新型药物偶联物，由三个紫杉醇分子连接到结合LRP受体的肽。该偶联物结合LRP受体后通过胞吞作用穿过血脑屏障。当细胞内酯酶裂解GRN1005并释放游离紫杉醇时，活性药物被认为在肿瘤细胞中释放（107）。此外，这种缀合物被认为可以逃避多药物外排转运体。GRN1005（现为ANG1005）在I期试验中获得阳性信号后，被Geron Pharmaceuticals授权进行胶质母细胞瘤和乳腺癌的注册研究。我们最初计划参加一个多机构的乳腺癌试验，并进行一个试点试验，包括手术切除乳腺癌或肺癌转移到大脑，研究GNR1005对转移性病变的通路。在研究开始后不久，Geron宣布GRN1005未能达到其首次中期疗效分析——前30名患者中，在550mg /m2的剂量下，0名患者出现应答（而13名患者中有4名（31%）在650mg /m2的剂量下出现客观应答）。审判突然结束，没有进一步的记录；多地点的pi是在事后得知这个决定的。然而，在关闭决定后对数据的审查显示，10例患者的数据缺失，并且在没有GCSF支持的情况下，导致剂量减少到550 mg/m2的过度毒性包括中性粒细胞减少症。Geron将该药物的许可证退还给了最初的开发商Angiochem。鉴于我们对该领域的兴趣，以及我们在几名NCI患者中观察到的药物活性，我们主张并被允许继续开展包括FLT-PET成像在内的NCI子研究。我们目前有一名患者进行了超过8个月的研究，确认GRN1005有主要反应。二世。虽然有多种fda批准的治疗肾细胞癌（RCC）的药物，但它们的有效时间都是有限的。为了开发新型药物，在不直接针对药物外排机制的情况下克服或规避耐药性，我们与NCI药物筛选合作，在体外和异种移植物中鉴定在RCC中具有特定活性的化合物（108,109）。虽然DNA似乎是这些化合物的目标，但它们在信息学评价中是独特的。一种先导化合物DMS612被选为I期药物，首次在NCI（我们作为协调中心）、匹兹堡大学和好时医学中心进行人体研究。我们已经测试了2个方案-第1天，第8天和第15天的方案，累积血小板减少作为剂量限制毒性，但最小的其他毒性。观察到两种反应；我们现在正在研究第二个时间表-每21天第1、2天给药。William Bonner实验室的Christophe Redon进行了γ - h2ax染色，记录了外周血单核细胞和毛囊细胞的DNA损伤，并显示了剂量反应曲线。DNA损伤在拔毛球中比在pbmc中更明显，出现时间更早，数量更多。24 h后，pbmc中3mg /m2的信号约为9mg /m2信号的25%，而毛球中的信号为65%。此外，毛球的损伤在早期就很明显。有趣的是，DNA损伤，正如γ - h2ax的表达所反映的，似乎与骨髓抑制的发生率和严重程度相关，这支持了DNA损伤与作用机制的生物学相关的假设。我们计划在II期环境中研究DMS612，首先在RCC中，使用第1,2天的时间表。我们将检查毛囊、pbmc和肿瘤组织中的γ - h2ax染色。我们认识到开发一种新的RCC药物将是具有挑战性的，因为四种VEGFR酪氨酸激酶抑制剂，两种mTOR抑制剂和贝伐单抗已被FDA批准。我们认为，这些药物具有机制重叠，并且需要具有新作用机制的药物，因为现有的治疗方法无法治愈。此外，新的免疫检查点抑制剂可能会重新引起人们对“免疫原性细胞死亡诱导剂”的兴趣，如细胞毒性治疗（110）。CTEP将支持其II期中心的试验，研究人员已同意参与。更多的阳性数据可能会导致其他肿瘤类型的研究。Yves Pommier发现了一种潜在的选择性生物标志物，他发现编码DNA修复蛋白ATAD5的基因变异可以预测细胞对DMS612的敏感性。我们从参加DMS612 I期研究的患者中收集样本进行ATAD5测序。三世。在与Tito Fojo博士的合作中，我们继续努力表征肿瘤生长动力学，并评估这些生长动力学是否可以用于帮助药物开发。威尔弗雷德·斯坦博士从事这个高产项目，可能会为如何评估药物效果提供一个范式转变。

Project Summary I. Drug Resistance Due to Limited Drug Distribution Although our laboratory has shifted from studies of Pgp and ABCG2 in recent years to a focus on epigenetic therapies, we remain convinced that limited drug distribution likely contributes to drug resistance, and that there are likely diverse etiologies of limited drug distribution, one of which may be drug efflux pumps. This straightforward question cannot be answered: does restricted drug distribution play a role in cancer chemotherapy drug resistance? Numerous small trials measuring drug concentrations in tumor tissue, or pharmacodynamic imaging studies of labeled drug, suggest a wide range of drug distribution in cancer. We too observed variable uptake of a drug surrogate, sestamibi, in lung cancer. Nowhere is the issue of drug distribution more relevant than in the brain, where drug uptake is limited by the need to cross the blood brain barrier (BBB). Crossing the BBB requires an active process and evasion of the multidrug transporters ABCG2 and ABCB1, among other regulators of brain capillary endothelium. GRN1005 in CNS Metastases from Breast Cancer We have participated in a clinical trial of GRN1005, a novel drug conjugate that consists of three molecules of paclitaxel linked to a peptide that binds the LRP receptor. The conjugate was developed to cross the Blood Brain Barrier via transcytosis after binding the LRP receptor. Active drug is thought to be released in tumor cells when intracellular esterases cleave GRN1005 and release the free paclitaxel (107). Additionally, this conjugate is thought to evade the multidrug efflux transporters. GRN1005 (now ANG1005) was licensed by Geron Pharmaceuticals to conduct registration studies in glioblastoma and breast cancer after a positive signal in Phase I testing. We originally planned to participate in both a multi-institutional trial in breast cancer and to conduct a pilot trial that would include surgical resection of breast or lung cancer that had metastasized to the brain, to study the access of GNR1005 to metastatic lesions. Soon after the study opened, Geron announced that GRN1005 had failed to meet its first interim efficacy analysis - 0 responses among the first 30 patients accrued at 550 mg/m2 (whereas 4 of 13 patients (31%) had objective responses at 650 mg/m2). The trial was abruptly closed without further accrual; the multi-site PIs learned of this decision after the fact. However, review of data after the closure decision revealed that data were missing in 10 patients, and that the excessive toxicity that had led to the dose reduction to 550 mg/m2 included neutropenia in the absence of GCSF support. Geron returned the license for the drug to its original developer, Angiochem. Given our interest in this field, and our observation of activity of the agent in several NCI patients, we argued for and were allowed to keep open the NCI sub-study including FLT-PET imaging. We currently have one patient on study for over 8 months with a confirmed major response to GRN1005. II. A Novel Agent for Drug Resistant Cancers While there are multiple FDA-approved agents for renal cell cancer (RCC), all have a limited efficacy duration. To develop novel agents that would overcome or circumvent drug resistance without directly targeting drug efflux mechanisms, we collaborated with the NCI drug screen to identify com-pounds with particular activity in RCC in vitro and in xenografts (108,109). Although DNA appears to be the target for these compounds, they are unique as assessed by informatics evaluation. A lead compound, DMS612, was selected for Phase I, first in human studies carried out at the NCI (we serve as coordinating center), University of Pittsburgh, and Hershey Medical Center. We have tested 2 schedules - a day 1, 8, and 15 schedule that had cumulative thrombocytopenia as dose limiting toxicity, but minimal other toxicity. Two responses were observed; we are now studying a second schedule - day 1,2 administration every 21 days. Christophe Redon in the laboratory of William Bonner performed gamma-H2AX staining, documenting DNA damage in peripheral blood mononuclear cells and hair follicle cells, showing a dose response curve. DNA damage was more obvious in plucked hair bulbs than in PBMCs, appearing earlier and in greater numbers. While in PBMCs the signal at 3 mg/m2 was about 25% of that at 9 mg/m2 after 24 h, in the hair bulbs, it was 65%. In addition, damage was apparent earlier in the hair bulbs. Interestingly, DNA damage, as reflected by expression of gamma-H2AX, appeared to correlate with myelosuppression incidence and severity, supporting a hypothesis that DNA damage is biologically relevant to the mechanisms of action. Going Forward with DMS612 We plan to study DMS612 in the Phase II setting, first in RCC, using the day 1, 2 schedule. We will examine gamma-H2AX staining in hair follicles, PBMCs, and in tumor tissue where accessible. We recognize developing a novel agent in RCC will be challenging, since four VEGFR tyrosine kinase inhibitors, two mTOR inhibitors, and bevacizumab are approved by the FDA. We would argue that those agents have mechanistic overlap, and that agents with new mechanisms of action are needed since no existing therapy is curative. Further, the new immune checkpoint inhibitors may renew interest in "immunogenic cell death inducers" such as cytotoxic therapy (110). CTEP will support the trial in their Phase II centers and investigators have agreed to participate. Additional positive data could lead to studies in other tumor types. A potential selective biomarker was identified by Yves Pommier, who found that variants of a gene encoding a DNA repair protein, ATAD5, predicted cell sensitivity to DMS612. We have gathered samples from patients enrolled on the DMS612 Phase I study to perform ATAD5 sequencing. III. In collaboration with Dr. Tito Fojo, we have continued our efforts to characterize tumor growth kinetics and to evaluate whether those growth kinetics can be used to aid drug development. Dr. Wilfred Stein works on this highly productive project that may offer a paradigm shift for how drug effects can be assessed.