Development of Angiogenesis Inhibitors

血管生成抑制剂的开发

• 批准号: 8763678
• 负责人: William Douglas Figg
• 金额: $ 48.41万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763678
• 关键词: Adverse effects; Affect; Angiogenesis Inhibition; Angiogenesis Inhibitors; Angiogenesis Pathway; Anti-Inflammatory Agents; Anti-inflammatory; Antineoplastic Agents; Aorta; Area; Binding; Biological; Biological Assay; Biological Factors; Blood Vessels; Cancer Patient; Cells; Chemical Structure; Chickens; Clinical; Clinical Drug Development; Clinical Research; Clinical Trials; Clinical Trials Design; Co-Immunoprecipitations; Collaborations; Collection; Combined Modality Therapy; Complex; Defect; Development; Dose; EP300 gene; Embryo; Embryopathy; Endothelial Cells; Enrollment; Enzyme-Linked Immunosorbent Assay; Evaluation; Family; Fluorescence; Fractionation; Genes; Genetic Transcription; Gliotoxin; Human; Hypoxia; In Vitro; Investigation; Isoenzymes; Laboratories; Legal patent; Libraries; Limb structure; Malignant neoplasm of prostate; Mass Spectrum Analysis; Mediating; Metabolic Biotransformation; Metabolism; Modeling; Molecular; Mus; Neoplasm Metastasis; Neutropenia; New Agents; Oral; Patients; Pharmaceutical Preparations; Pharmacology; Phase II Clinical Trials; Play; Preclinical Drug Development; Prednisone; Property; Prostate-Specific Antigen; Prostatic Neoplasms; Proteins; Randomized; Rattus; Recombinants; Regimen; Relative (related person); Reporting; Research; Resistance; Safety; Saphenous Vein; Signal Transduction; Solid Neoplasm; Specificity; Streptavidin; Structure; Testing; Thalidomide; Therapeutic; Therapeutic Agents; Time; Tissues; Toxic effect; Toxicology; Transactivation; Transcription Coactivator; Treatment outcome; Tube; Urologic Diseases; Vascular Endothelial Growth Factors; Zebrafish; Zinc; analog; angiogenesis; arm; base; bevacizumab; cancer cell; cancer site; cancer therapy; castration resistant prostate cancer; chaetocin; chemotherapy; chetomin; design; docetaxel; high throughput screening; human CYP2C19 protein; hypoxia inducible factor 1; improved; in vitro Assay; in vitro Model; in vivo; interest; lenalidomide; member; molecular mass; mouse model; neurotoxic; novel; outcome forecast; patient population; phase 2 study; polypeptide C; pre-clinical; protein protein interaction; repository; research clinical testing; response; small molecule; synthetic peptide; transcription factor; tumor; tumor growth; tumor xenograft

The angiogenic property of thalidomide reported by D'Amato and colleagues has prompted its clinical evaluation in various solid tumors, including prostate cancer. 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 synthesized 118 analogs of thalidomide and evaluated them using 4 in vitro models to assess activity in the inhibition of angiogenesis (rat aorta model, human saphenous vein model, cultured endothelial cells, and tube formation assay). We identified the most potent of these agents and have patented them. We continue to develop these compounds, which appear to 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 to determine whether the combination of thalidomide and docetaxel could produce a sufficiently high clinical response rate to warrant further investigation. A total of 75 patients were enrolled onto this trial, 25 patients in the docetaxel alone arm and 50 patients in the combination arm. Both at the midpoint evaluation and at the conclusion of the trial, the proportion of patients with a greater than 50% decline in Prostate Specific Antigen was higher in the combination arm (25 of 47 patients, 53%) than in the docetaxel alone arm (9 of 24 patients, 37%). 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 Bevacizumab: Dr. Dahut and I conducted a Phase II trial of thalidomide, docetaxel, prednisone and bevacizumab in chemo-naive Castration-Resistant Prostate Cancer patients. We previously demonstrated that thalidomide appears to add to the activity of docetaxel in metastatic Castration-Resistant Prostate Cancer (CRPC). Phase II studies combining docetaxel with bevacizumab have shown substantial anti-tumor activity. We hypothesized that the combination docetaxel plus these antiangiogenic drugs with different targets would have substantial clinical activity. To explore safety and efficacy, this was tested in both mouse and patients. Sixty patients with progressive metastatic CRPC received i.v. docetaxel and bevacizumab plus oral thalidomide and prednisone. In the mouse model, combination therapy of docetaxel, bevacizumab, and thalidomide inhibited tumor growth most effectively. In the clinical trial, 90% of patients receiving the combination therapy had PSA declines of greater than or equal to 50%, and 88% achieved a PSA decline of greater than or equal to 30% within the first 3 mo of treatment. The median time to progression was 18.3 mo and the median overall survival was 28.2 mo in this group with a Halabi predicted survival of 14 mo. While toxicities were manageable, all patients developed grade 3/4 neutropenia. The addition of bevacizumab and thalidomide to docetaxel is highly active with manageable toxicities. The estimated median survival is encouraging given the generally poor prognosis of this patient population. These results suggest that definitive clinical trials combining antiangiogenic agents with different mechanisms with docetaxel are warranted to improve treatment outcomes for patients with metastatic CRPC. To maintain the activity of this combination while reducing its associated side effects, we have replaced thalidomide with a structurally similar drug, lenalidomide. In this ongoing trial, patients with chemo-naive CRPC will be treated with docetaxel, prednisone, bevacizumab, and lenalidomide. In collaboration with Dr. Neil Vargesson, we further investigated the mechanism of action of thalidomide's teratogenic effect. We demonstrated that loss of immature blood vessels is the primary cause of thalidomide-induced teratogenesis and explained its action at the cell biological level. Antiangiogenic but not anti-inflammatory metabolites/analogues of thalidomide induce chick limb defects. Both in vitro and in vivo, outgrowth and remodeling of more mature blood vessels is blocked temporarily, whereas newly formed, rapidly developing, angiogenic vessels are lost. These results explain both the timing and relative tissue specificity of thalidomide embryopathy and have significant implications for its use as a therapeutic agent. Using a combination of zebrafish and chicken embryos together with in vitro assays we have determined that Pomalidomide, displays a high degree of cell specificity, and has no detectable teratogenic, antiangiogenic or neurotoxic effects at potent anti-inflammatory concentrations as compared to thalidomide or lenalidomide. 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. We have previously shown in our laboratory that several members of the epidithiodiketopiperazine (ETP) family of natural products are able to block the interaction between HIF-1a and p300 by a zinc ejection mechanism. Structure-activity studies using both natural and synthetic ETP derivatives reveal that only the structurally unique ETP core is required and sufficient to block the interaction of HIF-1a and p300. To demonstrate that disruption of the HIF-1a/p300 complex has antiangiogenic effects, we evaluated the activity of selected ETPs (chetomin, chaetocin, and gliotoxin) in the rat aortic ring model. Chetomin and chaetocin concentrations of 50 nM inhibited approximately 90% of outgrowth, while 500 nM of gliotoxin was needed to achieve a similar effect; these compounds had GI50 of 22, 11, and 175 nM, respectively. These ETPs were also able to block the co-immunoprecipitation of HIF-1a and p300 in cells. The downstream effect of inhibiting the HIF-1a/p300 interaction was demonstrated by ELISA, which showed a dose-dependent decrease in secreted VEGF. Finally, treatment with ETPs in mice bearing prostate tumor xenografts resulted in significant inhibition of tumor growth. These results suggest that targeting the HIF-1a/p300 complex with the ETPs may be a very effective approach for inhibiting angiogenesis and tumor growth, thus representing promising novel agents for cancer therapy. Studies are currently underway to identify additional compounds that inhibit the HIF-p300 interaction and involve 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 consisting of 170,000 compounds from the NCI Natural Products Repository that include a collection of pre-fractionation compounds, a selection of compounds known to affect HIF transactivation through unknown mechanisms, and a library of natural compounds. Nine pure compounds have been identified that inhibit the Hif-1a/p300 interaction. Molecular and mass spectrometry studies are being conducted to verify that these compounds can disrupt the HIF-1a/p300 complex and subsequent downstream HIF-mediated signaling. Finally, ex vivo studies will be carried out to evaluate if these compounds have anti-angiogenic effects.

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瘤中进行了临床评估。以前，我们表明细胞色素P450 2C19的产物之一是5'-OH- thalidomide的沙利度胺的同工酶生物转化，是该药物的抗血管生成活性的原因。基于该代谢产物的化学结构，我们合成了118个沙利度胺的类似物，并使用4种体外模型对其进行了评估，以评估抑制血管生成的活性（大鼠主动脉模型，人类的静脉模型，培养的内皮细胞和管形成分析）。我们确定了这些代理中最有效的代理商，并为其申请了专利。我们继续开发这些化合物，这些化合物在最初的临床前毒理学研究中似乎具有最小的副作用。使用随机II期试验设计，我们比较了每周有或没有200 mg/d的沙利度胺的每周多西他赛（30 mg/m2），以确定沙利度胺和多西他赛的组合是否可以产生足够高的临床反应率，以保证需要进一步研究。总共有75名患者参加了这项试验，单独使用多西他赛的25例患者，组合组中有50名患者。在中点评估和试验结束时，联合组中前列腺特异性抗原降低50％的患者比例在组合组中（47例患者中有25例，53％）高于单独的多西他赛（24例患者中的9例，37％，37％）。单独的多西他赛组为42.9％，合并组为68.2％。与组合组（p = 0.11），多胺，多西他赛和贝伐单抗的28 mo相比，单独多西他赛组的总体生存率为14 mo：Dahut和I博士对Thalidomide，Docetaxel，prednisone，prednisone，prednisone和bevacizumab进行了II期试验，用于化学疗法的患者。我们先前证明，沙利度胺似乎增加了多西他赛在转移性cast割前列腺癌（CRPC）中的活性。将多西他赛与贝伐单抗结合的II期研究表明，具有实质性的抗肿瘤活性。我们假设多西他赛的组合加上具有不同靶标的抗血管生成药物将具有实质性的临床活性。为了探索安全性和功效，在小鼠和患者中都对此进行了测试。 60例进行性转移性CRPC的患者接受了静脉注射。多西他赛和贝伐单抗加上沙利度胺和泼尼松。在小鼠模型中，多西他赛，贝伐单抗和沙利度胺的联合疗法最有效地抑制了肿瘤的生长。在临床试验中，90％接受联合疗法的患者的PSA下降大于或等于50％，而88％的PSA下降大于或等于30％的PSA治疗。进展的中位时间为18.3 mo，在这一组中，哈拉比预测生存率为14 mo的中值总生存期为28.2 mo。虽然毒性是易于管理的，但所有患者都患有3/4级中性粒细胞减少症。将贝伐单抗和沙利度胺添加到多西他赛的毒性具有高度活跃。鉴于该患者人群的预后通常不佳，估计的中位生存期令人鼓舞。这些结果表明，有必要将抗血管生成剂与不同机制与多西他赛的机制结合起来，以改善转移性CRPC患者的治疗结果。为了在减少其相关副作用的同时维持这种组合的活性，我们用结构相似的药物来纳利度胺代替沙利度胺。在这项正在进行的试验中，接受化学性CRPC的患者将接受多西他赛，泼尼松，贝伐单抗和列纳替米德治疗。与尼尔·瓦尔吉森（Neil Vargesson）博士合作，我们进一步研究了沙利度胺的致畸作用的作用机理。我们证明，未成熟血管的丧失是丘里度胺引起的致畸作用的主要原因，并解释了其在细胞生物学水平上的作用。抗血管生成，而不是沙利度胺的抗炎代谢产物/类似物会诱导雏鸡肢体缺陷。在体外和体内，更成熟的血管的产物和重塑均被暂时阻塞，而新形成，快速发育，血管生成血管丧失。这些结果解释了沙利度胺胚胎病的时间和相对组织特异性，并且对其用作治疗剂具有重要意义。使用斑马鱼和鸡胚胎以及体外测定的结合，我们确定了核酸胺，表现出高度的细胞特异性，并且在有效的抗炎性浓度上没有可检测到的致病性，抗血管生成或神经毒性作用，与Thalidomide相比或LenaliDomide或Lenalalidomidemely进行了有效的抗炎浓度。癌细胞适应低氧微环境的主要机制是通过转录因子缺氧诱导因子1α（HIF-1A）的活性。 HIF-1A在低氧条件下的表达调节在转移，血管生成，癌细胞代谢和抗性中起关键作用的基因。因此，由HIF驱动的转录抑制（通过破坏HIF与P300形成的复合物（一种基本的转录共激活因子）具有癌症治疗的潜力。我们先前在实验室中表明，天然产物的附加二硫代氨基吡嗪（ETP）家族的几个成员能够通过锌弹射机制阻止HIF-1A和P300之间的相互作用。使用天然和合成ETP衍生物的结构活性研究表明，仅需要结构独特的ETP核心，并且足以阻止HIF-1A和p300的相互作用。为了证明HIF-1A/P300复合物的破坏具有抗血管生成作用，我们评估了大鼠主动脉环模型中选定的ETP（Chetomin，chaetocin和Gliotoxin）的活性。 50 nm的切顿蛋白和壳毒素浓度抑制了约90％的生长，而需要500 nm的神经毒素才能达到相似的作用；这些化合物的GI50分别为22、11和175 nm。这些ETP还能够阻止细胞中HIF-1A和P300的共免疫沉淀。 ELISA证明了抑制HIF-1A/p300相互作用的下游效应，ELISA表明分泌的VEGF剂量依赖性降低。最后，用前列腺肿瘤异种移植的小鼠用ETP治疗导致肿瘤生长的显着抑制作用。这些结果表明，用ETP靶向HIF-1A/P300复合物可能是抑制血管生成和肿瘤生长的非常有效的方法，因此代表了有希望的癌症治疗的新型药物。目前正在进行研究以识别抑制HIF-P300相互作用的其他化合物，并使用我们实验室中开发的体外荧光结合测定法（由在96-韦尔链蛋白蛋白中固定在96-韦尔链蛋白培养基上的HIF-1A的C-TAD结构域的生物素化合成肽组成（由生物素化的合成肽组成） P300）。我们筛选了一个由NCI天然产品存储库中的170,000种化合物组成的库，其中包括一系列分级化合物的集合，已知通过未知机制影响HIF反式激活的化合物选择以及自然化合物库。已经鉴定出了抑制HIF-1A/p300相互作用的九种纯化合物。进行了分子和质谱研究，以验证这些化合物会破坏HIF-1A/P300复合物，并随后下游HIF HIF介导的信号传导。最后，将进行离体研究以评估这些化合物是否具有抗血管生成作用。