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估。作为我们之前联合多西他赛与两种抗血管生成（贝伐单抗和沙利度胺）药物的II期研究的后续，Bill Dahut博士（GMB， CCR， NCI）和我在mCRPC患者中进行了多西他赛、强的松、贝伐单抗和来那度胺的II期试验。来那度胺替代沙利度胺，因为副作用（疲劳和神经病变）减少。患者也给予广泛的支持措施（生长因子和抗凝），以减轻发病率和治疗限制性毒性。毒性是可控的，最常见的不良事件（ae）是血液学。29名患者（46%）患有4级中性粒细胞减少症，20名（32%）患有3级贫血，7名（11%）患有3级血小板减少症。在61例可评估的患者中，57例（93%）、55例（90%）和33例（54%）的PSA分别下降了30%、50%和90%。在29例可评估的患者中，24例（86%）有证实的放射学部分反应。中位进展时间和总生存期分别为18.2个月和24.6个月。通过适当的支持措施，联合血管生成抑制可以安全地进行，并可能提供临床益处。这些产生假设的数据需要随机试验来证实这些发现。沙利度胺在多种影响免疫调节和血管生成途径的恶性肿瘤中显示出临床活性。开发新的沙利度胺类似物，提高疗效和降低毒性是我们实验室正在进行的研究工作。先前，我们发现细胞色素P450 2C19同工酶生物转化沙利度胺的产物之一，5'- oh -沙利度胺，是该药物抗血管生成活性的原因。基于这种代谢物的化学结构，我们与dr。Nigel Greig （NIA， NIH）和Michael Gutschow合成并表征新型沙利度胺类似物，并使用体外和体内模型评估其活性。在与Neil Vargesson博士的合作下，我们对新的沙利度胺类似物进行了体内筛选，以确定哪些药物具有活性，并描述了一系列具有抗血管生成特性、抗炎特性的化合物，以及一些在斑马鱼和鸡胚胎模型系统中都表现出的化合物。此外，我们最近设计并合成了一类新的化合物，由四氟化沙利度胺类似物（Gu973和Gu998）和四氟苯酰胺（Gu1029和Gu992）组成。所有化合物都能减少大鼠主动脉环中微血管的生长，并在更大程度上抑制huvec，浓度低于先前测试的沙利度胺类似物。这些化合物的抗血管生成特性也在fl1:EGFP斑马鱼胚胎中进行了体内检测，所有化合物都可以抑制新生血管的生长。此外，Gu1029和Gu973降低了mpo:GFP斑马鱼胚胎的抗炎反应。利用人前列腺癌PC3异种移植模型，探讨了化合物的体内抗肿瘤作用。这四种化合物还在鸡胚胎中进行了体内筛选，以研究它们的致畸潜力。本研究确立了这些新型沙利度胺类似物作为一种具有抗癌作用的有前途的免疫调节剂，值得进一步发展以表征其作用机制。血管生成抑制剂现已广泛应用于临床；然而，关于其致畸作用机制的研究相对较少。为了解决这一问题，我们在发育中的斑马鱼和鸡胚胎模型中筛选了多种血管生成抑制剂，以评估其发育缺陷和潜在的致畸作用。我们证实了先前关于舒尼替尼、索拉非尼和TNP-470具有致畸性的报道，并证明阿西替尼、帕唑帕尼、万德替尼和依维莫司在这些模型中也是致畸性的。剂量反应研究发现，药物在体外抑制HUVEC细胞增殖，并在体内靶向胚胎发育中的血管。这进一步证明了在临床环境中使用这些药物存在胎儿毒性的潜在风险，并强调了胚胎血管发育和维持的重要性。我们得出结论，血管生成抑制剂，无论分子靶点，是致畸时暴露于鸡胚胎。癌细胞适应缺氧微环境的主要机制是通过转录因子缺氧诱导因子1 α (HIF-1a)的活性。缺氧条件下HIF-1a的表达调节在转移、血管生成、癌细胞代谢和抵抗中起关键作用的基因。因此，抑制由HIF驱动的转录（通过破坏HIF与p300（一种重要的转录辅激活因子）形成的复合物）具有治疗癌症的潜力。HIF-1a/p300抑制剂的开发一直受到临床前毒性的阻碍；因此，我们的目标是利用我们实验室开发的体外荧光结合试验（由固定在96孔链霉亲素包被板上的HIF-1a C-TAD结构域的生物素化合成肽和含有p300 CH1结构域的重组gst标记蛋白组成），通过高通量筛选鉴定新型HIF-1a/p300抑制剂。我们筛选了170298个天然粗提取物和预分离样品，鉴定了25个活性提取物。其中一种提取自海绵Latrunculia sp.，提供6种吡罗亚胺醌生物碱，鉴定为阳性命中（IC50值：1-35 uM）。荧光素酶测定证实，在HCT 116结肠癌细胞中，discorhabdin B(1)及其二聚体(2)、3-二氢discorhabdin C(3)、makaluvamine F(5)、discorhabdin H(8)、discorhabdin L(9)和discorhabdin W（11）对HIF-1a转录活性有抑制作用（0.1-10 uM, p0.05）。除11外，这些化合物均能降低LNCaP前列腺癌细胞中HIF-1a的转录活性（0.1 ~ 10 μ m, p0.05）。这些影响发生在缺氧条件下的非细胞毒性浓度（50%细胞死亡）。在下游HIF-1a靶水平上，化合物8 （0.5 uM）显著降低LNCaP细胞中VEGF的分泌（p0.05）。吡罗亚胺醌类生物碱是一类新型HIF-1a抑制剂，可阻断HIF-1a与p300之间的蛋白-蛋白相互作用，从而减少hif相关的转录。这些化合物的临床前研究正在进行中。我们继续与Kirk Gustafson博士（分子靶标实验室，CCR， NCI）合作，天然产物筛选也鉴定出海洋海鞘eudisstoma sp.的提取物具有活性。从该提取液中分离到新的杂环生物碱二萜苷A和B，并通过波谱分析确定了它们的结构。我们最近合成了新的七环结构的eudistine C.这些化合物抑制CH1/C-TAD结合，尽管只有在相对较高的浓度。初步的生物学特性表明了在癌症和疟疾方面的潜在应用。基于eudisdidine框架的化合物代表了HIF-1a和p300在癌细胞适应低氧环境中的作用的可能化学探针，并为癌症治疗的发展提供了线索。

The angiogenic property of thalidomide reported by D'Amato and colleagues has prompted its clinical evaluation in various solid tumors, including prostate cancer. As a follow-up to our previous phase II study combining docetaxel with two antiangiogenic (bevacizumab and thalidomide) drugs, Dr. Bill Dahut (GMB, CCR, NCI) and I conducted a phase II trial of docetaxel, prednisone, bevacizumab, and lenalidomide in mCRPC patients. Lenalidomide was substituted for thalidomide in this regimen because of the reduced side effect profile (fatigue and neuropathy). Patients were also given broad supportive measures (growth factor and anticoagulation) in an attempt to mitigate the morbidity and treatment-limiting toxicities. Toxicities were manageable with most common adverse events (AEs) being hematological. Twenty-nine patients (46%) had grade 4 neutropenia, 20 (32%) had grade 3 anaemia and seven (11%) had grade 3 thrombocytopenia. Of 61 evaluable patients, 57 (93%), 55 (90%) and 33 (54%) had PSA declines of 30, 50 and 90%, respectively. Of the 29 evaluable patients, 24 (86%) had a confirmed radiographic partial response. The median times to progression and overall survival were 18.2 and 24.6 months, respectively. With appropriate supportive measures, combination angiogenesis inhibition can be safely administered and potentially provide clinical benefit. These hypothesis-generating data would require randomized trials to confirm the findings. Thalidomide has demonstrated clinical activity in various malignancies affecting immunomodulatory and angiogenesis pathways. The development of novel thalidomide analogs with improved efficacy and decreased toxicity is an ongoing research effort in our laboratory. Previously, we 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 collaborated with Drs. Nigel Greig (NIA, NIH) and Michael Gutschow to synthesize and characterize novel thalidomide analogs and evaluated them using in vitro and in vivo models to assess activity. In collaboration with Dr. Neil Vargesson, we conduct an in vivo screen of a library of new thalidomide analogs to determine which agents demonstrate activity, and describe a cohort of compounds with anti-angiogenic properties, anti-inflammatory properties and some compounds which exhibited both using the in vivo zebrafish and chicken embryo model systems. Additionally, we recently designed and synthesized a new class of compounds, consisting of both tetrafluorinated thalidomide analogs (Gu973 and Gu998) and tetrafluorobenzamides (Gu1029 and Gu992). All compounds were seen to reduce microvessel outgrowth in rat aortic rings as well as inhibit HUVECs to a greater extent, at lower concentrations than previously tested thalidomide analogs. The anti-angiogenic properties of the compounds was also examined in vivo in fli1:EGFP zebrafish embryos, where all compounds were seen to inhibit the extent of outgrowth of newly developing blood vessels. In addition, Gu1029 and Gu973 reduced the anti-inflammatory response in mpo:GFP zebrafish embryos. The compounds anti-tumor effects were also explored in vivo using the human prostate cancer PC3 xenograft model. All four compounds were also screened in vivo in chicken embryos to investigate their teratogenic potential. This study establishes these novel thalidomide analogs as a promising immunomodulatory class with anti-cancer effects that warrant further development to characterize their mechanisms of action. Angiogenesis inhibitors are now widely used in the clinic; however, there are relatively few published studies on the mechanism of their presumed teratogenic effects. To address this issue, we screened a variety of angiogenesis inhibitors in developing zebrafish and chicken embryo models to assess for developmental defects and potential teratogenic effects. Weconfirmed previous reports that sunitinib, sorafenib and TNP-470 are teratogenic and demonstrate that axitinib, pazopanib, vandetanib, and everolimus are also teratogens in these models. A dose response study identified the drugs inhibit HUVEC cell proliferation in vitro, and also target the developing blood vessels of embryos in vivo. This provides further evidence for the potential risk of fetal toxicity when using these drugs in a clinical setting, and emphasizes the importance of the development and maintenance of the vasculature in the embryo. We conclude that angiogenesis inhibitors, regardless of the molecular target, are teratogenic when exposed to chicken embryos. A principal mechanism by which cancer cells adapt to the hypoxic microenvironment is through the activity of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1a). HIF-1a expression under hypoxic conditions regulates genes that play key roles in metastasis, angiogenesis, cancer cell metabolism, and resistance. Therefore, the inhibition of transcription driven by HIF (via disrupting the complex that HIF forms with p300, an essential transcriptional coactivator) has the potential for cancer treatment. Development of HIF-1a/p300 inhibitors has been hampered by preclinical toxicity; therefore, we aimed to identify novel HIF-1a/p300 inhibitors with a high throughput screen using the in vitro fluorescence-binding assay developed in our laboratory (composed of a biotinylated synthetic peptide of the C-TAD domain of HIF-1a immobilized on 96-well streptavidin-coated plates and a recombinant GST-tagged protein containing the CH1 domain of p300). We screened a library of 170,298 crude natural product extracts and prefractionated samples and identified 25 active extracts. One of these extracts, originating from the marine sponge Latrunculia sp., afforded six pyrroloiminoquinone alkaloids that were identified as positive hits (IC50 values: 1-35 uM). Luciferase assays confirmed inhibition of HIF-1a transcriptional activity by discorhabdin B (1) and its dimer (2), 3-dihydrodiscorhabdin C (3), makaluvamine F (5), discorhabdin H (8), discorhabdin L (9), and discorhabdin W (11) in HCT 116 colon cancer cells (0.1-10 uM, p0.05). Except for 11, all of these compounds also reduced HIF-1a transcriptional activity in LNCaP prostate cancer cells (0.1-10 uM, p0.05). These effects occurred at noncytotoxic concentrations (50% cell death) under hypoxic conditions. At the downstream HIF-1a target level, compound 8 (0.5 uM) significantly decreased VEGF secretion in LNCaP cells (p0.05). Pyrroloiminoquinone alkaloids are a novel class of HIF-1a inhibitors, which interrupt the protein-protein interaction between HIF-1a and p300 and consequently reduce HIF-related transcription. Preclinical studies of these compounds are ongoing. Continuing our collaboration with Dr. Kirk Gustafson (Molecular Targets Laboratory, CCR, NCI), the natural product screen also identified an extract of the marine ascidian Eudistoma sp. as active. Novel heterocyclic alkaloids eudistidines A and B were isolated from the extract, and their structures assigned by spectroscopic analyses. We recently synthesized the novel heptacyclic structure of eudistine C. These compounds inhibited CH1/C-TAD binding, albeit only at relatively high concentrations. Initial biological characterization of eudistidine C suggests potential applications in the context of cancer and malaria. Compounds based on the eudistidine framework represent possible chemical probes into the role of HIF-1a and p300 in the adaptation of cancer cells to low oxygen environments and are leads for the development of cancer therapeutics.