II. Clinical and Laboratory Studies of the Histone Deacetylase Inhibitor Depsipe

我们在临床和实验室研究了组蛋白去乙酰化酶抑制剂抑郁肽。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最初策略感兴趣，但我们已经使用实验室和临床策略，将抑郁肽作为T细胞淋巴瘤的孤儿药使用。我们针对皮肤和外周T细胞淋巴瘤的多机构临床试验迄今已纳入120例患者，分为6个队列。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5年，保持部分缓解。另一名患者在3年多的治疗后仍处于完全缓解状态。根据预先治疗的程度，对皮肤T细胞淋巴瘤患者的反应率在30 - 50%之间，现在称为罗米地辛。Gloucester Pharmaceuticals获得了FDA的快速通道和孤儿药地位，用于开发这种治疗CTCL的药物。一项注册试验已完成，我们的试验反应将包括在NDA包中。我们的数据正在准备提交NDA。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。PTCL的反应也是持久的，Gloucester也为该适应症制定了注册策略。该公司已经启动了一项针对PTCL的注册指导II期临床试验，Richard Piekarz博士将在我们的网站担任首席研究员，我们也将加入该试验。该试验已提交给IRB进行审查。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。这些心电图显示了大多数患者的可逆ST波和T波变化，与心功能异常或肌钙蛋白水平变化无关。此外，校正后QT间期中位数增加14毫秒。这项研究的结论是，没有证据表明暴露于抑郁肽会导致心脏损伤。2006年年度报告中指出的最后一个问题是，在CTEP在全国范围内赞助的临床试验中，在500多名接受治疗的患者中，有5人意外死亡与抑郁肽有关。患者普遍存在猝死易感因素。2006年实施的所有抑郁肽方案现在都排除了已知患有心脏病或已有猝死风险的患者。depsipeptide集中描述心脏的影响的能力,一个有效的代理一种罕见的疾病,是一个很好的例子的关键作用校内的程序可以在药物开发。当人们认识到这些心脏发现也代表了其他组蛋白去乙酰化酶抑制剂所观察到的一类效应时，这种作用就具有更大的意义。该试验有一个重要的翻译组件，已经消耗了我的实验室资源的主要部分。我们已经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。目前正在将这些检测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。其他研究包括在第1、3和5天进行抑郁肽的I期试验，希望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，以翻译Tito Fojos博士实验室的观察结果，即在由沉积肽诱导的基因中，有编码碘化钠同调体和甲状腺球蛋白的基因。这些基因在甲状腺癌细胞中的诱导增强了放射性碘的体外积累。甲状腺癌患者的这种效应可能导致放射性碘摄取增加。I期试验正在进行中，收集样本进行药代动力学和药效学分析。已获得用于组蛋白乙酰化测定和治疗前/治疗后肿瘤样本用于基因表达分析的pbmc。对于甲状腺癌患者（每个剂量水平一名患者，然后在MTD处扩大），允许进行放射性碘成像，如果证实放射性碘积累增加，则随后给予放射性碘剂量。由于只有没有可见放射性碘摄取的甲状腺癌患者被纳入研究，这可能很难实现。如果不能记录放射性碘摄取，我们希望治疗前/治疗后的活检至少能在RT-PCR检测的细胞中显示基因表达水平上Na+/I-同调体的诱导。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。初步研究表明，在这些细胞中存在药物积累缺陷，并且正在寻找其潜在的机制。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。我们对最后一种机制做出了贡献，我们与dr。April Robbins和Dan Sackett，着丝粒的变化如下[摘要被截断为7800个字符]

We have studied the histone deacetylase inhibitor depsipeptide 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 as an orphan drug in T cell lymphoma, using both laboratory and clinical strategies. Our multi-institutional clinical trial for cutaneous and peripheral T cell lymphoma has enrolled 120 patients to date, divided into 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 examples, one patient has received therapy continuously for over 5 years, remaining in a partial remission. Another patient remains in complete remission off of therapy for over 3 years. Depending upon how heavily pretreated the patient population being evaluated, the response rate for depsipeptide, now termed romidepsin, in cutaneous T cell lymphoma ranges from 30 - 50%. Gloucester Pharmaceuticals obtained Fast Track and Orphan Drug status from the FDA for development of this therapy for CTCL. A registration trial has completed accrual, and responses from our trial will be included in the NDA package. Our data are being readied for the NDA submission. 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. Responses with PTCL are also durable and Gloucester has developed a registration strategy for that indication as well. A registration-directed Phase II clinical trial in PTCL has been launched by the company, and with Dr. Richard Piekarz as principal investigator at our site, we will join that trial as well. The trial has been submitted to the IRB for review. Our NCI Phase II trial has 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. These ECGs demonstrate the previously documented reversible ST and T wave changes in the majority of patients, unassociated with any abnormality in cardiac function or change in troponin level. In addition, a median increase in the corrected QT interval of 14 msec. This study concluded that there was no evidence of cardiac damage resulting from depsipeptide exposure. One final concern noted in the 2006 annual report -- in clinical trials sponsored by CTEP across the nation there were 5 unexpected deaths associated with depsipeptide among over 500 patients treated. Generally, the patients had predisposing factors for sudden death. Implemented in 2006, all depsipeptide protocols now exclude patients with known cardiac disease or pre-existing risks for sudden death. Our ability to intensively characterize the cardiac effects of depsipeptide, an effective agent in a rare disease, is an excellent example of the critical role that the intramural program can play in drug development. This role assumes greater significance when one recognizes that these cardiac findings represent a class effect observed with other histone deacetylase inhibitors as well. The trial has a significant translational component that has consumed a major fraction of my laboratory resources. We have 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 are currently being 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. Additional studies include a Phase I trial of depsipeptide on a day 1, 3, and 5 schedule in hopes of achieving a more continuous drug effect. Dr. Richard Piekarz is PI on this study. This study has a focus in thyroid cancer in order to translate the observation made in Dr. Tito Fojos laboratory that among the genes induced by depsipeptide were the genes encoding the sodium iodide symporter and thyroglobulin. Induction of these genes in thyroid cancer cells enhanced the accumulation of radioiodine in vitro. Such an effect in patients with thyroid cancer could lead to increased radioactive iodine uptake. The Phase I trial is ongoing, with samples collected for pharmacokinetic and pharmacodynamic analysis. PBMCs for histone acetylation determination and pre/post treatment tumor samples have been obtained for gene expression analysis. In patients with thyroid cancer (one patient per dose level and then expanding at the MTD), radioiodine imaging is allowed, along with subsequent dosing with radioiodine in the event that increased radioiodine accumulation is demonstrated. Since only patients with thyroid cancer without visible radioiodine uptake are enrolled on the study, this will probably be difficult to achieve. It is our hope that pre/post treatment biopsies will at a minimum show induction of the Na+/I- symporter at the gene expression level, in cells assayed by RT-PCR, if radioiodine uptake cannot be documented. 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. Preliminary studies suggest that there is a drug accumulation defect in these cells and a mechanism underlying that is being sought. 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. We contributed to this last mechanism where we reported, in collaboration with Drs. April Robbins and Dan Sackett, changes in the centromere following [summary truncated at 7800 characters]