Development of Angiogenesis Inhibitors

D'Amato及其同事报道的沙利度胺的血管生成特性促使其在包括前列腺癌在内的各种实体肿瘤中的临床评价。我们实验室之前的研究表明，沙利度胺的细胞色素P450 2C19同工酶生物转化的产物之一，5'- oh -沙利度胺，是该药物抗血管生成活性的原因。基于该代谢物的化学结构，我们合成了118种沙利度胺类似物，并利用四种体外模型（大鼠主动脉模型、人隐静脉模型、内皮细胞培养和血管形成实验）对它们进行了评估，以评估其抑制血管生成的活性。我们已经确定了其中最有效的药物，并申请了专利。我们正在继续开发这些化合物。在最初的临床前毒理学研究中，这些化合物的副作用似乎很小。采用随机II期试验设计，我们比较了每周多西他赛（30 mg/m2）与不服用200 mg/d的沙利度胺。本研究的目的是确定沙利度胺和多西他赛联合使用是否能产生足够高的临床反应率，以保证进一步的研究。所有患者都有转移性AIPC，并在雄激素阻断和抗雄激素停药方面取得进展。该试验共有75名患者入组，其中多西他赛单用组25例，联合用药组50例。两组都选择了两阶段的最小-最大设计，即最小入组患者的最大数量。联合治疗组使用P0=0.25（不良反应率）和P1=0.45（目标反应率），多西他赛单用P0=0.05和P1=0.025，两组分别使用？=0.05和？=0.10。在中点评估和试验结束时，联合组中PSA下降超过50%的患者比例（47例患者中有25例，53%）高于单独多西紫杉醇组（24例患者中有9例，37%）。这些缓解率满足进一步评估的标准（联合用药组）和与先前结果的一致性（单药组）。同样，在多西他赛单用组中，通过CT扫描可测量软组织疾病的11例患者中有3例（27%）出现部分缓解，联合治疗组中20例患者中有7例（35%）出现软组织疾病的部分缓解。没有一个有骨损伤的病人有正常的骨扫描。多西他赛和联合治疗组的中位无进展生存期分别为3.7个月和5.9个月（曲线总差异p=0.32）。多西紫杉醇单独治疗组18个月生存率为42.9%，联合治疗组为68.2%。多西他赛单独治疗组的中位总生存期为14个月，而联合治疗组为28个月（p=0.11）。基于我们发现的沙利度胺、多西他赛和estrumomine联合治疗的阳性数据，我们最近启动了一项estramomstine、多西他赛和Thalidomide联合治疗AIPC的II期研究。这种组合是基于我的实验室所做的临床前工作，在几种前列腺癌异种移植模型中显示了这种组合的协同作用。该研究的主要目的是显示与多西他赛和沙利度胺联合使用相比，PSA下降有统计学上显著的改善。我们也在研究多个次要终点。其中包括研究药物之间可能的药代动力学相互作用，以及患者基因型（CYP2C19）与治疗效果之间的潜在相关性。我们也在寻找治疗前后血液中的循环肿瘤细胞。此外，我们将监测方案的耐受性和生存时间作为终点。我们招募了20例转移性进行性AIPC患者进行II期临床试验，以评估该组合。多西紫杉醇（30mg/m2）每周给药，4周中的3周。沙利度胺剂量为200 mg/天，雌二醇在第1、2、3、8、9、10、15、16、17天给予，每天3次。我们发现该组合的进展空闲时间为7.2个月。90%的患者有？50%的PSA下降，20%的软组织病变患者（n=10）通过CT扫描有部分反应。尽管由于副作用（如疲劳、胃肠道（GI）并发症），沙利度胺和/或雌二醇的剂量多次减少，但该方案耐受性良好。这种联合治疗有24例3级和2例4级并发症。我们还发现总生存率与CYP1B1*3基因型之间有统计学意义的关联（p=0.013）。多西他赛为基础的化疗现在被认为是转移性AIPC的标准方案。在这项研究中，我们能够证明艾莫司汀、多西他赛和沙利度胺在前列腺癌临床前模型中是一种有利的联合治疗方案，并且在AIPC患者中是一种安全、耐受和有效的治疗方案。Dahut博士和我本人正在进行沙利度胺、多西他赛、强的松和贝伐单抗在初次化疗AIPC患者中的二期试验。本研究的主要目的是确定这种组合是否与足够高的PSA反应患者比例相关，值得进一步研究。我们还将研究多个次要终点，类似于上述沙利度胺、多西他赛和雌二醇试验。这项研究的初步结果非常令人鼓舞。1998年，我设计了一项独特的双盲随机III期试验，以确定沙利度胺是否可以提高LHRH激动剂对原发性终末期治疗（XRT或根治性前列腺切除术）后PSA水平升高的AIPC患者的疗效。在LHRH激动剂治疗6个月后，患者接受200mg /d的沙利度胺或安慰剂（A期）。在PSA进展时，LHRH激动剂重新启动6个月。6个月后，患者交叉使用沙利度胺或安慰剂（B期）。280名患者中有154名入组。我们最近对该研究进行了盲法分析，发现沙利度胺治疗在PFS方面具有统计学上的显著优势。低氧诱导因子-1 （HIF-1）是参与细胞适应缺氧的主要转录因子。已经发现了几种小分子可以阻止p300/ hif -1 α的结合。其中之一是Chetomin，它已被证明可以消除p300/CBP与HIF-1？和HIF-2alpha在体外相互作用试验中，通过破坏p300 CH1的结构和功能。结果还表明，Chetomin是一种特异性转录抑制剂，仅抑制与HIF-1alpha在p300上相同表面相互作用的转录因子。这些结果使我们在类似于Chetomin试验的基于荧光的蛋白质结合试验中筛选结构相关的化合物，称为ETPs（附硫碘二氧哌嗪），以检查结构/活性。

The angiogenic property of thalidomide reported by D'Amato and colleagues has prompted its clinical evaluation in various solid tumors including prostate cancer. Our laboratory previously showed that one of the products of cytochrome P450 2C19 isozyme biotransformation of thalidomide, 5'-OH-thalidomide, is responsible for the drug's antiangiogenic activity. Based on the chemical structure of this metabolite, we have synthesized 118 analogs of thalidomide and have evaluated them using four in vitro models to assess activity in the inhibition of angiogenesis (rat aorta model, human saphenous vein model, cultured endothelial cells, and tube formationassay). We have identified the most potent of these and have patented them. We are continuing to develop these compounds. These compounds appear have minimal side effects in initial preclinical toxicology studies. Using a randomized Phase II trial design we compared weekly docetaxel (30 mg/m2) with or without 200 mg/d of thalidomide. The objective of this study was to determine whether the combination of thalidomide and docetaxel could produce a sufficiently high clinical response rate to warrant further investigation. All of the patients had metastatic AIPC and were progressing on androgen blockade, as well as anti-androgen withdrawal. A total of 75 patients were enrolled onto this trial, 25 patients in docetaxel alone arm and 50 patients in the combination arm. A two-stage mini-max design, that is one in which the design sought to minimize the maximum number of patients enrolled, was chosen for both arms. We used P0=0.25 (undesirable response rate) and P1=0.45 (target response rate) for the combination arm, and P0=0.05 and P1=0.025 for the docetaxel-alone arm, with ?=0.05 and ?=0.10 for both arms. Both at the midpoint evaluation and at the conclusion of the trial, the proportion of patients with a more than 50% decline in PSA was higher in the combination arm (25 of 47 patients, 53%) than in the docetaxel alone arm (9 of 24 patients, 37%). These response rates satisfied criteria for further evaluation (combination arm) and for consistency with prior results (single agent arm). Likewise, 3 of 11 patients (27%) in the docetaxel-alone arm with measurable soft tissue disease by CT scan developed a partial response, and 7 of 20 patients (35%) in the combined treatment group had a partial response in soft tissue disease. None of the patients with bony lesions had a normalization of their bone scan. The median progression free survival was 3.7 mo and 5.9 mo in the docetaxel and combined group, respectively (p=0.32 for the overall difference in the curve). The 18 mo survival was 42.9% in the docetaxel alone group and 68.2% in the combined group. The median overall survival in the docetaxel alone group was 14 mo compared with 28 mo for the combination arm (p=0.11) Thalidomide, Docetaxel, and Estrumustine Based on the positive data we found with the combination of thalidomide and docetaxel, we have recently initiated a Phase II study of estramustine, docetaxel and thalidomide in chemo-naive patients with AIPC. This combination was based on preclinical work done in my laboratory showing synergy with this combination in several xenograft models of prostate cancer. The primary objective of the study is to show a statistically significant improvement in PSA decline when compared to the combination of docetaxel and thalidomide. We are also looking at multiple secondary endpoints. These include possible pharmacokinetic interactions among the study agents, and potential correlation between patient genotype (CYP2C19) and efficacy of treatment. We are also looking for circulating tumor cells in blood before and after treatment. Additionally, we will be monitoring the tolerability of the regimen and survival duration as endpoints. We enrolled 20 patients with metastatic progressive AIPC on to a Phase II clinical trial to evaluate this combination. Docetaxel (30mg/m2) was administered every week, 3 out of 4 weeks. The dose of thalidomide was 200 mg/day and estramustine was given three times a day on Day 1, 2, 3, 8, 9, 10, 15, 16 and 17. We found that the progression free time was 7.2 months with this combination. Ninety percent of patients had a ?50% decline in PSA and 20% of those patients with soft tissue lesions (n=10) had a partial response by CT scan. The regimen has been well tolerated, although there have been several dose reductions of thalidomide and/or estramustine due to side effects (e.g., fatigue, gastrointestinal (GI) complications). There were 24 grade 3 and 2 grade 4 complications associated with this combination. We also found a statistically significant association between overall survival and the CYP1B1*3 genotype (p=0.013). Docetaxel-based chemotherapy is now regarded as standard regimen for metastatic AIPC. In this study, we were able to demonstrate that estamustine, docetaxel and thalidomide proved to be advantageous treatment combination in preclinical models of prostate cancer and is a safe, tolerable and active regimen in patients with AIPC. Dr. Dahut and myself are conducting a Phase II trial of thalidomide, docetaxel, prednisone and bevacizumab in chemo-naive AIPC patients. The primary objective of this study is to determine if this combination is associated with a sufficiently high proportion of patients with a PSA response to be worthy of further investigation. We will also be looking at multiple secondary endpoints, similar to those outlined above for the thalidomide, docetaxel and estramustine trial above. The preliminary results from this study are extremely encouraging. Thalidomide in Stage D0 Androgen Independent Prostate Cancer (ADPC) In 1998, I designed a unique double-blinded randommized Phase III trial to determine if thalidomide could improve the efficacy of LHRH agonists in patients with AIPC, that had a rising PSA level after primary definitive therapy (XRT or radical prostatectomy). Following 6 months of LHRH agonist, patients receive either 200 mg/d of thalidomide or placebo (Phase A). At the time of PSA progression, an LHRH agonist is restarted for 6 additional months. After 6 mo, patients are crossed over to either thalidomide or placebo (Phase B). 154 of 280 patients were enrolled. We recently unblinded the study to find a statistically significant advantage in PFS for the the thalidomide treatment. Development of a High Throughput Screening Assay for the Identification of Novel Inhibitors that Target the HIF System Hypoxia Inducible Factor-1 (HIF-1) is a master transcription factor involved in cellular adaptation to hypoxia. Several small molecules have been found to prevent p300/HIF-1alpha binding. One of these is Chetomin, which has been shown to abrogate the normal interaction and binding of p300/CBP with both HIF-1? and HIF-2alpha in an in-vitro interaction assay, by disrupting the structure and function of p300 CH1. The results also indicated that Chetomin was a specific transcriptional inhibitor, only inhibiting transcription factors that interact with the same face on p300 as HIF-1alpha. These results lead us to screen structurally related compounds, known as ETPs (epidithiodioxopiperazines), in a fluorescence based protein binding assay similar to the Chetomin assay, to examine the structure/activity re [summary truncated at 7800 characters]