Translational Studies of the Histone Deacetylase Inhibitor Romidepsin

我们在临床和实验室中研究了组蛋白去乙酰化酶抑制剂抑郁肽（现在称为罗米地辛）。我们最初对抑郁肽感兴趣是在临床试验策略的背景下，寻求确定能够克服或规避多药耐药的药物。我的实验室确定了组蛋白去乙酰化酶抑制剂抑郁肽作为临床前开发的一种药物和pgp介导的外排的底物。由于抑郁肽被Pgp热切地转运，并且由于它在组蛋白乙酰化改变的基因群中诱导MDR-1，我们计划最终开发与Pgp调节剂联合使用的抑郁肽。然而，在I期研究中，我们偶然发现，抑郁肽对T细胞淋巴瘤亚群非常有效。虽然我们继续对预防对该药物产生耐药性的原始策略感兴趣，但我们已经使用实验室和临床策略，将抑郁肽/罗米地辛作为治疗T细胞淋巴瘤的孤儿药。此外，我们的生物标志物数据并不表明Pgp作为暴露于该化合物后的耐药机制而发展。我们针对皮肤和周围T细胞淋巴瘤（CTCL和PTCL）的多机构临床试验在6个队列的134名患者中完成。队列1，皮肤T细胞淋巴瘤患者少于2个全身化疗方案，已完成，手稿正在准备中。对抑郁肽的反应有时是戏剧性的，并且非常持久。例如，一名患者连续接受治疗超过6年，在治疗的部分缓解中保持了20个月，现在又恢复了CTCL的治疗。另一名CTCL患者在停药后仍处于完全缓解状态超过5年，另一名PTCL患者在复发前仍处于持续完全缓解状态5年。我们为罗米地辛创造了一个有趣的说法，“当你需要它的时候就在那里”，这是基于它对那些在治疗结束后出现疾病进展的患者的作用。在我们的NCI试验和Gloucester注册试验中，皮肤T细胞淋巴瘤的主要反应率为34-35%，现在称为罗米地辛。对于PTCL，我们的有效率为38%。值得注意的是，参与我们超过9个多中心的II期试验的校外研究人员也在这两个亚群患者中获得了持久的反应。这些地点包括纽约曼哈塞特的北岸大学医院；加州杜阿尔特的希望之城国家癌症中心；以及澳大利亚墨尔本的彼得·麦卡勒姆癌症中心。Gloucester Pharmaceuticals获得了FDA的孤儿药资格，用于开发CTCL和PTCL的治疗。NCI CTEP和我们的癌症治疗分支机构（现为肿瘤医学分支机构）在藤泽制药就参与肿瘤开发平台的相对优点进行辩论期间，大力推动了该药物的单独开发。我们的NCI II期试验除了证明在各种组织学中的疗效外，还有一个主要的第二个目标。这是对代理人安全的确认。治疗后心电图异常已被注意到，并且大量的努力已被证明心肌损害与本药的施用缺乏相关。我们在2006年6月的《临床癌症研究》中报道了我们对42例接受抑郁肽治疗的患者的2051张心电图的回顾。明年的目标是继续报道罗米地辛的心脏结果，以帮助确定药物的安全性，以及所有hdi与电解质补充联合使用的参数。这个试验有一个重要的翻译部分，它消耗了我实验室资源的很大一部分。我们开发了一种定量免疫印迹法，用于检测和定量患者样本中的组蛋白乙酰化，主要是外周单个核细胞作为替代。将这些测定结果与药代动力学数据进行比较。我们还通过RT-PCR评估了包括CD25、p21和MDR1在内的基因表达，发现在患者单核细胞中，只有MDR1的表达在沉淀肽后被充分诱导进行常规检测。在开始治疗前和疾病进展时，也对肿瘤样本中的MDR1进行分析。我们的数据表明，外周血单个核细胞中组蛋白乙酰化的24小时时间点与药物动力学参数（包括清除率和曲线下面积）有关。此外，我们的数据表明，这一终点与疾病反应有关。综上所述，这些数据表明，药物暴露可能对罗米地辛和整个组蛋白去乙酰化酶抑制剂类都很重要。其他研究包括罗米地辛在第1、3和5天的I期试验，以获得更持续的药物效果。理查德·皮卡兹博士是这项研究的首席研究员。本研究的重点是甲状腺癌，评估放射性碘摄取，这是在实验模型中观察到的。I期试验已经完成；我们的评估是，剂量和时间表并没有带来最佳的基因表达变化。如何最好地增加放射性碘在甲状腺癌中的积累仍然是一个重要的问题。我们正在进行一项新的组蛋白去乙酰化酶抑制剂的临床试验，评估连续输注belinostat联合顺铂和依托泊苷48小时。该试验是基于临床前证据的HDAC抑制剂和化疗药物之间的协同作用，当适当安排。这是在晚期疾病人群中进行的I期试验，I期确定的II期剂量将在小细胞肺癌患者人群中进行相同的试验。这一患者群体很难积累起来。然而，我们决心完成这项研究，因为我们的实验室研究表明，小细胞肺癌细胞对罗米地辛的敏感性很高。我们刚刚完成了一篇证明顺铂或依托泊苷与罗米地辛之间协同作用的论文。这些研究支持了我们的临床试验。最后，我们对抑郁肽的敏感性和耐药机制已经感兴趣了一段时间。这导致我们产生了具有非pgp介导的抑郁肽抗性的细胞系，我们已经开始询问是否可以确定其他抗性机制。我们对沉积肽的作用机制仍感兴趣。至少有5种机制被引用用于组蛋白去乙酰化酶抑制剂：诱导基因表达、胞质蛋白乙酰化和功能改变、由于Hsp90活性受损导致的胞质蛋白降解增加、血管生成改变和有丝分裂作用。究竟哪个机制是至关重要的将是继续调查的主题。这些研究还得到旨在确定协同药物组合的实验的补充。我们希望确定利用各种药物作用机制的药物组合，并以这种方式将已经在淋巴瘤单药治疗中观察到的活性转化为实体瘤的活性。毫无疑问，在这种情况下需要联合治疗。考虑到在我们的研究和其他研究中观察到的与DNA损伤剂的强体外协同作用，至少应该进行检查罗米地辛和放射治疗联合的研究。罗米地辛与实验性信号转导抑制剂联合的方案概念正在审查中。

We have studied the histone deacetylase inhibitor depsipeptide (now known as romidepsin) in both the clinic and in the laboratory. We originally became interested in depsipeptide in the context of a clinical trial strategy seeking to identify agents that could overcome or circumvent multidrug resistance. My laboratory identified the histone deacetylase inhibitor depsipeptide as an agent in preclinical development and a substrate for Pgp-mediated efflux. Because depsipeptide is avidly transported by Pgp, and because it induces MDR-1 in the constellation of genes altered by histone acetylation, we planned to eventually develop depsipeptide in combination with a Pgp modulator. However, in the Phase I setting, we made the serendipitous discovery that depsipeptide was highly effective in subsets of T cell lymphoma. While we have continued to be interested in our original strategy of preventing the emergence of resistance to this agent, we have pursued the use of depsipeptide/romidepsin as an orphan drug in T cell lymphoma, using both laboratory and clinical strategies. Futhermore, our biomarker data do not suggest that Pgp develops as a mechanism of drug resistance following exposure to this compound. Our multi-institutional clinical trial for cutaneous and peripheral T cell lymphoma (CTCL and PTCL) completed accrual at 134 patients in 6 cohorts. Cohort 1, patients with cutaneous T cell lymphoma with fewer than 2 systemic chemotherapy regimens, is complete and a manuscript is in preparation. The responses to depsipeptide are at times dramatic and have been very durable. As an example, one patient received therapy continuously for over 6 years, remained in a partial remission off of therapy for 20 months and has now resumed therapy for CTCL. Another patient with CTCL remains in complete remission off of therapy for over 5 years, and another patient with PTCL remained in continuous complete remission for 5 years before relapse occurred. We have anecdotally coined the phrase "there when you need it" for romidepsin, based on its activity in retreating patients who demonstrate disease progression while off of therapy. The major response rate for depsipeptide, now termed romidepsin, in cutaneous T cell lymphoma in both our NCI trial and in the Gloucester registration trial is 34-35%. For PTCL, our response rate was 38%. It is important to note that durable responses were also obtained in both subsets of patients by extramural investigators who were participating in our Phase II trial among more than 9 multicenter sites included in the study. These sites included North Shore University Hospital in Manhasset, New York; City of Hope National Cancer Center in Duarte, California; and Peter MacCallum Cancer Center in Melbourne, Australia. Gloucester Pharmaceuticals obtained Orphan Drug status from the FDA for development of this therapy for CTCL and PTCL. NCI CTEP and our Cancer Therapeutics Branch (now Medical Oncology Branch) largely pushed the development of this agent alone during a period in which Fujisawa Pharmaceuticals debated the relative merits of becoming involved in an oncology development platform. Our NCI Phase II trial had a major second objective in addition to proving efficacy in the various histologies. That is confirmation of the safety of the agent. EKG abnormalities have been noted following treatment and a great deal of effort has gone into demonstrating the lack of myocardial damage associated with administration of this agent. We reported in June of 2006 in Clinical Cancer Research, our review of 2,051 ECGs obtained in 42 patients treated with depsipeptide. A goal in the coming year is to continue to report cardiac findings with romidepsin that help establish the safety of the agent, and parameters for using all HDIs in conjunction with electrolyte supplementation. The trial had a significant translational component that consumed a major fraction of my laboratory resources. We developed a quantitative immunoblot assay for detecting and quantitating histone acetylation in patient samples, principally peripheral mononuclear cells as a surrogate. Results from these assays were compared to pharmacokinetic data. We have also evaluated gene expression including CD25, p21, and MDR1 by RT-PCR, finding that only MDR1 expression is induced sufficiently following depsipeptide for routine assay in patient mononuclear cells. MDR1 is also analyzed in tumor samples before therapy is initiated and then at the time of disease progression. Our data suggest that the 24hr timepoint of histone acetylation in peripheal blood mononuclear cells is associated with pharmacokinetic parameters including clearance and area under the curve. In addition, our data suggest that this endpoint is assocated with disease response. Taken together these data suggest that drug exposure may be important for romidepsin and potentially for the entire class of histone deacetylase inhibitors. Additional studies included a Phase I trial of romidepsin on a day 1, 3, and 5 schedule to achieve a more continuous drug effect. Dr. Richard Piekarz was PI on this study. This study had a focus in thyroid cancer, evaluating radioactive iodine uptake, which was observed in experimental models. The Phase I trial was completed; our assessment was that the dose and schedule did not give optimal gene expression changes. The question of how best to increase radioiodine accumulation in thyroid cancer remains an important one. We have a new clinical trial of an histone deacetylase inhibitor ongoing, evaluating 48 hr continuous infusion belinostat in combination with cisplatin and etoposide. This trial is based on preclinical evidence of synergy between HDAC inhibitors and chemotherapeutics, when properly scheduled. This is being carried out as a Phase I trial in an advanced disease population and the Phase II dose established in Phase I will be studied in the same trial in the small cell lung cancer patient population. This patient population has been difficult to accrue. However, we are determined to complete the study - as our laboratory studies suggest exquisite sensitivity of small cell lung cancer cells to romidepsin. We have just completed a publication demonstrating synergy between cisplatin or etoposide and romidepsin. These studies support our clinical trial. Finally, we have been interested for some time in mechanisms of depsipeptide sensitivity and resistance. This led us to the generation of cell lines with non-Pgp mediated depsipeptide resistance and we have begun to ask whether other mechanisms of resistance can be identified. We continue to be interested in the mechanism of action of depsipeptide. At least 5 mechanisms have been cited for histone deacetylase inhibitors: induction of gene expression, acetylation of cytoplasmic proteins and altered function, increased degradation of cytoplasmic proteins due to impaired Hsp90 activity, altered angiogenesis, and mitotic effects. Exactly which mechanism is of critical importance will be the subject of continuing investigation. These studies have also been complemented by experiments aimed at identifying synergistic drug combinations. We hope to identify drug combinations that exploit the various mechanisms of drug action and in this way to translate the activity already observed in monotherapy in lymphoma into activity in solid tumors. Undoubtedly combination therapy will be required in that setting. At a minimum, studies examining the combination of romidepsin and radiation therapy should be conducted, given the strong in vitro synergy observed in our studies with DNA damaging agents and in the studies of others. A protocol concept for the combination of romidepsin with an experimental signal transduction inhibitor is under review.