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肿瘤中的临床评估。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。基于该代谢物的化学结构，我们合成了118种沙利度胺类似物，并通过4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）评估了它们对血管生成的抑制活性。我们确定了这些药物中最有效的药物，并申请了专利。我们继续开发这些化合物，在最初的临床前毒理学研究中，它们的副作用似乎很小。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200 mg/d沙利度胺联合使用或不联合使用多西他赛，以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，从而值得进一步研究。共有75名患者参加了该试验，其中25名患者单独使用多西他赛组，50名患者联合使用多西他赛组。在中点评估和试验结束时，联合治疗组中前列腺特异性抗原下降超过50%的患者比例（47例患者中有25例，53%）高于单独多西紫杉醇治疗组（24例患者中有9例，37%）。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）。沙利度胺、多西他赛和贝伐单抗：Dahut博士和我在化疗初治的抗阉割前列腺癌患者中进行了沙利度胺、多西他赛、泼尼松和贝伐单抗的II期试验。我们之前证明，沙利度胺似乎增加了多西紫杉醇在转移性去势抵抗性前列腺癌（CRPC）中的活性。多西他赛联合贝伐单抗的II期研究显示出显著的抗肿瘤活性。我们假设多西紫杉醇联合这些不同靶点的抗血管生成药物将具有实质性的临床活性。为了探索安全性和有效性，在小鼠和患者中进行了测试。60例进展性转移性CRPC患者静脉注射多西他赛和贝伐单抗联合口服沙利度胺和强的松。在小鼠模型中，多西他赛、贝伐单抗和沙利度胺联合治疗最有效地抑制肿瘤生长。在临床试验中，90%接受联合治疗的患者PSA下降大于等于50%，88%的患者在治疗的前3个月内PSA下降大于等于30%。该组中位进展时间为18.3个月，中位总生存期为28.2个月，Halabi预测生存期为14个月。虽然毒性可控，但所有患者均出现3/4级中性粒细胞减少症。在多西他赛的基础上加入贝伐单抗和沙利度胺具有高度活性和可控的毒性。估计的中位生存期是令人鼓舞的考虑到一般预后不良的患者群体。这些结果表明，有必要将不同机制的抗血管生成药物与多西他赛联合进行明确的临床试验，以改善转移性CRPC患者的治疗结果。为了保持这种组合的活性，同时减少其相关的副作用，我们用一种结构相似的药物来那度胺取代了沙利度胺。在这项正在进行的试验中，化疗初始CRPC患者将接受多西他赛、强的松、贝伐单抗和来那度胺的治疗。我们与Neil Vargesson博士合作，进一步研究了沙利度胺致畸作用的作用机制。我们证明了未成熟血管的丧失是沙利度胺诱导致畸的主要原因，并解释了其在细胞生物学水平上的作用。沙利度胺的抗血管生成代谢物/类似物诱导雏鸡肢体缺损。无论是在体外还是在体内，成熟血管的生长和重塑都会暂时受阻，而新形成的、快速发育的血管生成血管则会丢失。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，并对其作为治疗剂的使用具有重要意义。结合斑马鱼和鸡胚胎以及体外实验，我们已经确定，与沙利度胺或来那度胺相比，波马度胺表现出高度的细胞特异性，并且在有效的抗炎浓度下没有可检测到的致畸、抗血管生成或神经毒性作用。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。我们之前在实验室中表明，附睾二酮哌嗪（ETP）家族天然产物的几个成员能够通过锌喷射机制阻断HIF-1a和p300之间的相互作用。使用天然和合成ETP衍生物进行的结构-活性研究表明，仅需要结构独特的ETP核心就足以阻断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，我们在大鼠主动脉环模型中评估了选定的ETPs （chetomin、chaetomin和gliotoxin）的活性。Chetomin和chaeomycin的浓度为50 nM，可以抑制约90%的细胞生长，而胶质毒素的浓度为500 nM才能达到相似的效果；这些化合物的GI50分别为22、11和175 nM。这些etp还能够阻断细胞中HIF-1a和p300的共免疫沉淀。抑制HIF-1a/p300相互作用的下游效应通过ELISA证实，显示分泌的VEGF呈剂量依赖性减少。最后，在移植前列腺肿瘤的小鼠中，用ETPs治疗可显著抑制肿瘤生长。这些结果表明，用ETPs靶向HIF-1a/p300复合物可能是一种非常有效的抑制血管生成和肿瘤生长的方法，因此代表了有希望的癌症治疗新药物。目前正在进行研究，以确定抑制HIF-p300相互作用的其他化合物，并使用我们实验室开发的体外荧光结合试验进行高通量筛选（由固定在96孔链霉亲和素包被板上的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）。我们从NCI天然产物库中筛选了一个由170,000种化合物组成的文库，其中包括预分离化合物的集合，已知通过未知机制影响HIF交易激活的化合物的选择，以及天然化合物的文库。已经鉴定出9种纯化合物可以抑制Hif-1a/p300相互作用。正在进行分子和质谱研究，以验证这些化合物可以破坏HIF-1a/p300复合物和随后的下游hif介导的信号传导。最后，将进行体外研究，以评估这些化合物是否具有抗血管生成作用。