Brain Tumor Animal Therapeutics Core

在Fine博士的领导下，NOB实验室与制药公司和学术机构以及NCI发展治疗项目合作，进行了许多新型抗胶质瘤药物的临床前和临床开发。开发管道的第一步是通过ABTC筛选代理。ABTC提供专业的服务，在体外和体内筛选这些药物，使用标准的皮下和立体定向颅内模型。自2005年以来，大量抗胶质瘤药物被筛选出来。其中，25种新药显示出足够的前景，值得通过ABTC进行延长评估。这些扩展的研究包括基于立体定向的颅内模型，观察各种剂量和给药计划，以及新药与其他药物的联合试验。此外，ABTC为NOB内外的其他研究人员提供实验和技术支持，以评估新开发的治疗方法。例如，干细胞因子（SCF）在胶质瘤血管生成中的作用；Notch-1在胶质瘤细胞存活和增殖中的作用安定素在恶性胶质瘤对DNA烷基化剂体内抵抗中的作用。系统和神经毒性（行为）也通过常规动物筛选进行监测。此外，一些较新的给药技术，包括颈动脉内给药、有或没有选择性或总体血脑屏障破坏的给药、对流给药等，已经在ABTC的动物模型中进行了评估。例如，ABTC与NINDS的SNB合作，并与私营部门合作，使用对流增强给药（CED）直接将各种遗传载体注入携带人类胶质瘤异种移植物的免疫缺陷动物的大脑。许多新型的抗肿瘤治疗药物将具有细胞抑制而不是细胞毒性。由于标准反应标准是基于细胞毒性反应的确定，因此在小型早期临床试验中评估这些药物中哪些在人体中具有生物活性是一项挑战。评估真正的细胞抑制剂活性的唯一真正有效的临床参数是患者生存期或肿瘤无进展生存期。然而，这些并不是筛选小型早期临床试验药物活性的有用参数。因此，如果生物活性的替代标记物可以被识别，人们可以利用这些作为筛选很少或没有临床活性的药物的早期终点。为此，ABTC正积极致力于开发可用于临床试验的药物抗肿瘤活性替代标志物，其中包括三个主要领域：1)成像；2)基因表达谱；3)蛋白质组学/血清标志物。例如，在与NOB、NINDS和临床中心实验成像科学项目的研究人员的合作中，无创磁共振成像已被用于对体内磁性标记的内皮祖细胞进行成像，以直接识别胶质瘤模型中的血管发生。核心为该项目提供了技术支持，该项目涉及MRI跟踪体内阿魏氧化铁-硫酸鱼精蛋白（FE-PRO）复合物标记的内皮祖细胞融入已建立的颅内小鼠胶质瘤血管。ABTC还成功地生成了FDA要求的临床前毒理学数据，用于准备我们的IND临床试验，该试验使用ferrodex标记的内皮祖细胞作为胶质瘤患者血管生成的MRI可追踪标记物。此外，我们还与Robert Innis博士（NIMH）合作，试图将PET扫描应用于监测系统中，以实时成像药物通过血脑屏障的通透性，并跟踪多重耐药（MDR）蛋白抑制剂的使用。这项工作正在扩展到使用ABTC来帮助评估结合外周苯二氮卓受体（PBR）的新型PET配体，PBR在胶质瘤中高度过表达。核心的一项主要工作是利用微阵列技术从特定类型的药物治疗的给定胶质瘤细胞系中产生基因表达谱的RNA。如果能够识别出与抗肿瘤活性相对应的特征模式，那么可以/将设计临床试验，在活检/手术之前立即将这些药物中的一种施用于脑肿瘤患者，以便尝试在临床上识别类似的基因谱。在与NOB实验室和GMDI团队的合作下，在ABTC内测试的所有化合物的所有胶质瘤细胞系和GIC/GSCs中产生了基因表达特征。最后，ABTC储存了用每种新化合物处理过的动物的代表性肿瘤、组织和血清样本，期望新的候选组织和/或基于血清的药物活性、肿瘤活性和/或某些肿瘤生物过程的蛋白质标志物(如：血管新生)。这将是未来验证此类声明的宝贵临床前资源。NOB的一项主要工作是开发人类胶质瘤细胞系，使其在生物学和分子上更接近于模拟原发性人类胶质瘤。ABTC积极参与从NIH手术的每个胶质瘤患者的新鲜手术标本中产生原发性人类胶质瘤细胞系和GIC/GSC细胞系。ABTC工作人员与Fine实验室的癌症干细胞生物学家密切合作，对每种细胞系和动物异种移植物进行生长、繁殖和表征。ABTC使用这些特性良好的细胞系作为两大类药物的筛选；1)最有希望的药物已经通过体外和体内筛选的第一阶段，使用更传统的已建立的胶质瘤细胞系；2)针对标准胶质瘤细胞系生物学不能很好地代表但在GIC/GSCs中复制的途径的药物。这些细胞的核心专业知识，以及不同GIC/GSC系的大量资源，是NCI与制药/生物技术界之间潜在合作伙伴关系的强大吸引力，因为他们越来越认识到标准癌细胞系的局限性，以及癌症干细胞更好地代表人类疾病的前景。最后，鉴于我们每年收到的数百个关于这些有价值的GIC/GSC细胞系的请求，ABTC作为指定的小组发挥着至关重要的作用，该小组负责向NIH内外的研究人员扩展、冻结和分发各种细胞系。在此过程中，ABTC的工作人员花费了大量的时间来教授NIH内外的其他研究人员如何培养GIC/GSCs，以及如何将肿瘤细胞立体定向植入小鼠和大鼠体内。ABTC成功的证据是，我们已经启动了11项临床试验，这是NOB内进行的转化工作的直接结果，所有这些临床试验都在ABTC内进行了临床前动物研究。更重要的是，我们已经通过ABTC临床前筛选项目鉴定了12种化合物，这些化合物已经在NIH进行了临床试验（AZD6918,RO4929097,AZD8005,MLN-518, ZD6474,LY317615，舒尼替尼，CC5013,Talampanel）。ABTC的潜在力量在我们的演示中得到了很好的证明，我们能够接受由我们的一个制药合作者发送给我们进行临床前评估的药物，并生成支持临床试验的临床前数据，从而导致NOB赞助的（两个）I期试验，（两个）II期试验和NOB主持的全球随机注册III期临床试验；同时发现了一种新的药物作用机制（GSK3抑制）。

Under the leadership of Dr Fine,the NOB Lab has collaborated with pharmaceutical companies and academic institutions,and the NCI Developmental Therapeutics Program in the preclinical and clinical development of a number of new anti-glioma agents.The first step in the development pipeline is screening of the agent through the ABTC.The ABTC provides the professional service for screening these agents both in vitro and in vivo using both standard subcutaneous and stereotactic intracranial models.Since 2005,a large number of anti-glioma agents have been screened.Of those,25 new agents showed significant enough promise to warrant extended evaluation through the ABTC.These extended studies involved stereotactic-based intracranial models looking at various dose and administration schedules as well as combination trials of the new drug with other agents.Furthermore,ABTC provides experimental and technical support to other investigators both within and outside of the NOB for evaluating newly developed therapeutics.For example, the role of stem cell factor (SCF) in glioma angiogenesis; Notch-1 in glioma cell survival and proliferation;Stathmin in the resistance of malignant gliomas to DNA alkylating agents in vivo. Systemic as well as neurotoxicity (behavior) is also monitored by routine animal screening.In addition,a number of newer drug delivery technologies including intracarotid administration, delivery with or without selective or gross blood-brain barrier disruption, convection delivery, etc.have been evaluated in animal models within the ABTC.For example, the ABTC in collaboration with the SNB of NINDS and in collaboration with the private sector, has used convection-enhanced drug delivery (CED) to directly administer various genetic vectors into the brains of immundeficient animals harboring human glioma xenografts.Many of the new classes of anti-tumor therapeutics will have cytostatic rather than cytotoxic properties.Evaluating which of these agents will have biologic activity in humans in small,early clinical trials is a challenge since the standard response criteria are based on the determination of cytotoxic responses.The only truly valid clinical parameter available for evaluating the activity of a truly cytostatic agent is patient survival or tumor progression-free survival.These,however,are not useful parameters for screening drug activity in small,early phase clinical trials.Thus,if surrogate markers of biologic activity could be identified,one could utilize these as early endpoints for screening out agents with little or no clinical activity.Toward that end,the ABTC is actively working to develop surrogate markers of drug anti-tumor activity that can be utilized and validated in clinical trials,which includes three major areas:1)Imaging;2)Gene expression profiling;3) Proteinomics/Serum markers. For example, in collaboration with investigators in NOB, NINDS and the Clinical Centers program of experimental imaging science,noninvasive MR imaging has been used to image magnetically labeled endothelial progenitor cells in vivo to directly identify vasculogenesis in a glioma model.The core has provided the technical support for this project which involved the MRI tracking of in vivo Ferumoxides-Protamine Sulfate (FE-PRO) complex-labeled endothelial progenitor cells incorporating into the vasculature of established intracranial mouse gliomas.The ABTC has also successfully generated the preclinical toxicology data required by the FDA for preparation of our IND for the clinical trial of using ferrodex-labeled endothelial progenitor cells as MRI trackable markers of angiogenesis in patients with gliomas.Additionally,we have collaborated with Dr. Robert Innis(NIMH)for attempting to adapt PET scanning into a monitoring system for real time imaging of drug permeability through the BBB and following the administration of inhibitors of the multiple drug resistance (MDR) protein.This work is being extended to use the ABTC tohelp evaluate novel PET ligands that bind to the peripheral benzodiazepine receptors (PBR) which is highly overexpresed in gliomas.A major effort of the core is to generate the RNA for gene expression profiles using microarray technology from given glioma cell lines treated with a specific class of agents.If characteristic patterns could be identified that correspond with anti-tumor activity,then clinical trials can/will be devised to administer one of these agents to patients with brain tumors immediately prior to biopsy/surgery in order to attempt and identify a similar genetic profile clinically.In collaboration with the NOB Lab and the GMDI team,gene expression signatures are being generated in all of glioma cell lines and GIC/GSCs for all compounds tested within the ABTC.Finally,the ABTC stores representative tumor, tissue and serum samples from animals treated with each new compound tested with the expectations that new candidate tissue and/or serum-based protein markers of drug activity, tumor activity and/or some tumor biological process(i.e. angiogenesis)may be found.This will be an invaluable preclinical resource for validating such claims in the future.A major effort of the NOB is to develop human glioma cell lines that more closely model primary human gliomas both biologically and molecularly.The ABTC is actively involved in the generation of primary human glioma cell lines and GIC/GSC lines from fresh surgical specimens for every glioma patient operated on at the NIH.The ABTC staff works closely with the cancer stem cell biologists in the Fine laboratory for the growth, propagation and characterization of each of these cell lines and animal xenografts. The ABTC uses these well-characterized cell lines as screens for two major categories of drugs;1)The most promising of the drugs that have made it through the first levels of in vitro and in vivo screens using the more conventional established glioma cell lines;2)drugs that target pathways that may not be well represented by the biology of standard glioma cell lines but are reproduced in the GIC/GSCs.The cores expertise with these cells, and the large resources of different GIC/GSC lines, are a potent enticement for potential partnerships between NCI and the pharmaceutical/biotechnology community given their growing appreciation of the limitation of standard cancer cell lines and the promise of cancer stem cells for better representing the human disease.Finally, given the hundreds of requests we receive each year for these valuable GIC/GSC lines,the ABTC serves a vital function as the group designated to expand,freeze and distribute various cell lines to investigators both within and outside of the NIH.In doing so,the staff of the ABTC spends a significant amount of time teaching other investigators from within and outside of the NIH how to grow GIC/GSCs and how to perform stereotactic implantation of tumor cells into mice and rats.Evidence of the success of the ABTC is the fact that we have activated 11 clinical trials as a direct result of translational work performed within the NOB,all of which had preclinical animal studies performed within the ABTC.Even more to the point,we have identified 12 compounds solely through the ABTC preclinical screening program that have since been brought forward to clinical trials at the NIH (AZD6918,RO4929097,AZD8005,MLN-518, ZD6474,LY317615,sunitinib,CC5013,Talampanel).The potential power of the ABTC is well documented by our demonstration of being able to take an agent sent to us for preclinical evaluation by one of our pharmaceutical collaborators and generate preclinical data supportive of clinical trials that resulted in NOB sponsored (two) phase I trials,(two)phase II trials and a NOB-chaired phase III worldwide randomized registration clinical trial; all the while discovering a novel mechanism of action of the drug (GSK3 inhibition).