Translational Studies of the Histone Deacetylase Inhibitor Romidepsin

我们在临床和实验室研究了组蛋白去乙酰化酶抑制剂罗米地辛和贝利诺他。我们最初对罗米地辛感兴趣是在一期临床试验的背景下，当时我们偶然发现（当时命名的）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该药物出现耐药性的原始策略感兴趣，但我们首先追求将抑郁肽/罗米地辛作为T细胞淋巴瘤的孤儿药，使用实验室和临床策略。我们已经将这项工作扩展到使用羟肟酸衍生物belinostat的实体肿瘤。我们针对皮肤和外周T细胞淋巴瘤（CTCL和PTCL）的多机构临床试验在6个队列的131例患者中完成。发表了详细介绍罗米地辛在CTCL和PTCL中的反应的论文。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7年，另一名PTCL患者在复发前仍处于连续完全缓解状态5年。在我们的NCI试验和Gloucester注册试验中，皮肤T细胞淋巴瘤的主要缓解率为34-35%。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。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。这是对特工安全的确认。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。我们分析了4000多张心电图，并收集了许多辅助心脏安全数据。明年的目标是报告罗米地辛的其他心脏发现，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。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。另外，根据协议，样品被送到了宾夕法尼亚大学的路易斯·肖博士那里。这些样品已通过cDNA阵列进行分析，数据正在准备发表。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。我们已经接近完成了一项新的组蛋白去乙酰化酶抑制剂belinostat的联合临床试验，评估了顺铂和依托泊苷联合48小时的连续输注。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；我们目前正在提炼一种II期剂量。II期剂量将在小细胞肺癌患者人群中进行相同的试验设计。支持临床试验的实验室研究已于今年发表。最后，我们对HDI的敏感性和耐药机制已经感兴趣了一段时间。这使我们产生了具有非pgp介导的罗米地辛抗性的细胞系，我们已经开始询问是否可以确定其他抗性机制。我们继续对人类发展指数的作用机制感兴趣。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。我们已经确定了一种两种药物组合，它已经在淋巴瘤单药治疗中观察到显著提高了活性，我们希望它能转化为实体瘤的活性。支持罗米地辛与实验性药物联合使用方案概念的动物研究正在进行中；我们计划在明年对这一组合进行一期研究。

We have studied the histone deacetylase inhibitors romidepsin and belinostat in both the clinic and in the laboratory. We originally became interested in romidepsin in the context of a Phase I clinical trial, when we made the serendipitous discovery that (then-named) 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 first pursued the use of depsipeptide/romidepsin as an orphan drug in T cell lymphoma, using both laboratory and clinical strategies. We have extended this work into solid tumors with the hydroxamic acid derivative belinostat.Our multi-institutional clinical trial for cutaneous and peripheral T cell lymphoma (CTCL and PTCL) completed accrual at 131 patients in 6 cohorts. Papers detailing responses to romidepsin in both CTCL and PTCL are published. 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 7 years, and another patient with PTCL remained in continuous complete remission for 5 years before relapse occurred. The major response rate in cutaneous T cell lymphoma in both our NCI trial and in the Gloucester registration trial was 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.Our NCI Phase II trial had a major second objective in addition to proving efficacy in the various histologies. That was 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 have analyzed over 4000 ECGs, and collected much ancillary cardiac safety data. A goal in the coming year is to report additional 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. 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 associated 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. I addition, samples were sent as per protocol to Dr. Louise Showe at University of Pennsylvania. These samples have been analyzed by cDNA array and the data are being prepared for publication.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 nearly completed a combination clinical trial with a novel histone deacetylase inhibitor, belinostat, evaluating a 48 hr continuous infusion in combination with cisplatin and etoposide. This trial is based on preclinical evidence of synergy between HDAC inhibitors and chemotherapeutics, when properly scheduled. This was carried out as a Phase I trial in an advanced disease population; we are currently refining a Phase II dose. The Phase II dose will be explored in the same trial design in the small cell lung cancer patient population. Laboratory studies supporting the clinical trial have been published this year.Finally, we have been interested for some time in mechanisms of HDI sensitivity and resistance. This led us to the generation of cell lines with non-Pgp mediated romidepsin 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 the HDIs. 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 have identified a two drug combination that has markedly increased the activity already observed in monotherapy in lymphoma and we hope will translate into activity in solid tumors. Animal studies supporting a protocol concept for the combination of romidepsin with an experimental agent are ongoing; we plan to open a Phase I study of this combination in the coming year.