In vivo feasibility of a peripheral nerve imaging agent

周围神经成像剂的体内可行性

• 批准号: 8590174
• 负责人: STEPHEN B KAHL
• 金额: $ 28.83万
• 依托单位: MANZANITA PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8590174
• 关键词: Affect; Afferent Neurons; Affinity; Animals; Axonal Transport; Binding; Biological; Biological Assay; Characteristics; Chemistry; Clinical; Clinical Research; Controlled Study; Cyclic GMP; Development; Devices; Dimerization; Distal; Dose; Dyes; Excision; Face; Goals; Green S; Hour; Human; Image; Imagery; Impotence; In Vitro; Incontinence; Indocyanine Green; Injectable; Injection of therapeutic agent; Lead; Life; Light; Link; Malignant neoplasm of prostate; Maximum Tolerated Dose; Mediating; Modeling; Mus; Nerve; Nerve Endings; Nerve Growth Factors; Nerve Tissue Proteins; Nerve-Sparing Prostatectomy; Neurologic; Neurons; Neurotrophic Tyrosine Kinase Receptor Type 1; Noise; Operative Surgical Procedures; Outcome; Paint; Penetration; Peripheral; Peripheral Nerves; Pharmaceutical Preparations; Phase; Prostatectomy; Proteins; Protocols documentation; Recombinants; Reconstructive Surgical Procedures; Relative (related person); Research; Safety; Signal Transduction; Site; Skin; Solid Neoplasm; Spinal Ganglia; Surgeon; Surgical incisions; System; Tail; Tetanus Toxin; Time; Tissues; Toxic effect; Variant; Veterans; Vision; aerobic respiration control protein; clinical application; cyanine; improved; in vivo; innovation; neuronal cell body; neuronal survival; novel; preclinical study; public health relevance; receptor; retrograde transport; scale up; uptake

DESCRIPTION (provided by applicant): "Feasibility of a nerve imaging agent to reduce neurological complications of prostatectomies" A key challenge in radical or nerve-sparing prostatectomies is to minimize the neurological complications that can lead to impotence in about 10%-30% of cases and incontinence in about 10% of cases. We propose to show in vivo feasibility of a novel peripheral nerve imaging agent or "nerve paint" comprised of a NearInfrared (NIR) dye conjugated to recombinant human Nerve Growth Factor (rhNGF) (rhNGF-NIR). The goal of this proposal is to show that rhNGF-NIR can be visualized. Our product vision is an injectable imaging agent that may be given pre-operatively to assist surgeons prior to beginning a prostatectomy. To show feasibility we selected two NIR dyes, Alexa Fluor 790 and Dyomics 831 because their chemistries (1) are similar to IRDye(R) 800CW developed by LI-COR (Lincoln, NE) whose safety is established in an FDA drug master file i.e. these agents should also be safe; (2) provide increased tissue penetration of light and an enhanced signal-to-noise background ratio relative to cyanine 5.5; (3) can be readily conjugated unlike indocyanine green (ICG); and (4) can be observed using commercial imaging systems. Rationale for NGF as targeting moiety. In preliminary studies we showed that NGF conjugated to Alexa Fluor 488 (rhNGF-488) was taken up in compartmented cultures at distal ends and moved to proximal ends. Our targeting moiety of choice is rhNGF because it is well-tolerated, non-immunogenic, and because of its dual targeting characteristics. We expect to use doses of rhNGF-NIR within the Maximum Tolerated Dose (MTD) established for rhNGF alone in clinical studies (0.1¿g/kg-0.3¿g/kg). When given sub-cutaneously (sub-c), approx. 80% of unmodified rhNGF localizes at the site of peripheral injection, is absorbed by high- affinity TrkA receptors, and moves via axonal transport to Dorsal Root Ganglia (DRG), where it degrades. Research plan. In this proposal we will reliably synthesize and characterize NGF-probes, then confirm receptor-mediated axonal transport targeted to sensory neurons (1) in vitro, in neuronal survival assays and in compartmented cultures. We will explore visualization in vivo in mouse, in real-time imaging studies. Hypothesis. We hypothesize that NGF offers a safe and selective intraneuronal targeting moiety which can deliver selected NIR dyes via peripheral injection to image peripheral nerves associated with the site of injection prior to a prostatectomy. Our Specific Aims are: Aim 1. Synthesize, optimize, and characterize dye conjugate. We will modify our proprietary linker system for rhNGF-488 to synthesize two variants (rhNGF-780, rhNGF-DYM, or rhNGF-NIR). Our goals are to (1) enable dimerization of rhNGF to optimize uptake and tissue selectivity; and (2) develop analytical protocols to yield bioactive probes with consistent protein:probe ratios. Aim 2. Bioassay for (1) neuronal survival and (2) retrograde transport in compartmented cultures. Neurons have an absolute requirement for NGF to survive. We confirm bioactivity of rhNGF-NIR variants by administering probe to mass culture of sympathetic neurons. Our first goal is to show that probes contain biologically active NGF. Next we will confirm that each rhNGF-NIR is selectively absorbed by sensory neurons. We use compartmented cultures to confirm that rhNGF-NIRs are internalized by TrkA receptors on distal ends, then transported to cell bodies in the central chamber of the culture system. Aim 3. Confirm that rhNGF-NIR can be visualized and is selectively absorbed by peripheral nerves in vivo. We will inject microdoses of rhNGF-NIR variants at the periphery and observe probes in live animals in a commercially available imaging system. In controlled studies we will inject rhNGF-NIR sub-c and im at two sites, in the footpad and in the upper haunch. Our goal here is not to model clinical application or to determine dose, but to show that rhNGF-NIRs (1) can be visualized in real-time, (2) are localized to neuronal tissue; and (3) degrade within a reasonable period after injection. If successful, we and our collaborators have the expertise to carry out IND-enabling studies distribution, dose, and toxicity studies.

描述(申请人提供)：“神经显像剂减少前列腺切除术的神经并发症的可行性”根治性或保留神经的前列腺切除术的一个关键挑战是将神经并发症降至最低，这些并发症可能导致约10%-30%的病例阳萎和约10%的病例大小便失禁。我们建议在体内展示一种新型的周围神经显像剂或由重组人神经生长因子(RhNGF)连接的近红外(NIR)染料(rhNGF-NIR)组成的“神经涂料”的可行性。这项提议的目的是表明重组人神经生长因子-近红外光谱是可视的。我们的产品视觉是一种可注射的显像剂，可以在手术前给予，以在开始前列腺切除术前辅助外科医生。为了证明可行性，我们选择了两种近红外染料，Alexa Fluor 790和DyEconomics 831，因为它们的化学成分(1)类似于Li-COR(林肯，东北)开发的IRDye(R)800CW，其安全性已在FDA的药物主文件中确定，即这些试剂也应该是安全的；(2)相对于花菁5.5，提供更多的组织透过率和增强的信噪背景比；(3)与吲哚青绿(ICG)不同，它们可以很容易地结合在一起；以及(4)可以使用商业成像系统观察到。NGF作为靶向部分的理论基础。在初步研究中，我们发现与Alexa Fluor 488(rhNGF-488)结合的NGF在远端的隔室培养中被吸收并转移到近端。我们选择的靶向部分是重组人神经生长因子，因为它耐受性好，不具免疫原性，而且具有双重靶向特性。我们希望在临床研究中使用的rhNGF-NIR剂量在单独使用rhNGF的最大耐受量(MTD)范围内(0.1？g/kg-0.3？g/kg)。皮下给药时(亚c级)，约80%的未修饰的rhNGF定位于外周注射部位，被高亲和力的TrkA受体吸收，并通过轴突运输到背根节(DRG)，在那里它被降解。研究计划。在这项建议中，我们将可靠地合成和表征NGF探针，然后在体外、神经元存活试验和隔室培养中证实受体介导的轴突运输针对感觉神经元(1)。我们将在小鼠体内探索可视化，在实时成像研究中。假设。我们推测，NGF提供了一种安全和选择性的神经元内靶向部分，它可以通过外周注射将选定的近红外染料输送到前列腺切除术前与注射部位相关的周围神经。我们的具体目标是：目标1.合成、优化和表征染料偶联物。我们将修改我们针对rhNGF-488的专有连接器系统，以合成两个变体(rhNGF-780、rhNGF-DYM或rhNGF-NIR)。我们的目标是(1)实现重组人神经生长因子的二聚化，以优化摄取和组织选择性；(2)开发分析方案，以产生具有一致蛋白质：探针比率的生物活性探针。目的2.隔室培养中神经元存活和逆行运输的生物测定。神经元对NGF的生存有绝对的要求。我们通过向交感神经元的大量培养施加探针来确认rhNGF-NIR突变体的生物活性。我们的第一个目标是证明探针中含有具有生物活性的NGF。接下来，我们将确认每个重组人神经生长因子-近红外光谱都被感觉神经元选择性地吸收。我们使用隔室培养来证实rhNGF-NIRS在远端被TrkA受体内化，然后运输到培养系统中央小室的细胞体。目的3.证实重组人神经生长因子-近红外在体内可被周围神经显示并选择性吸收。我们将在外周注射微量的重组人神经生长因子-近红外变体，并在商业成像系统中观察活体动物的探针。在对照研究中，我们将在足垫和上臀部两个部位注射重组人神经生长因子-近红外光谱亚单位和肌注。我们的目标不是模拟临床应用或确定剂量，而是证明重组人神经生长因子-近红外光谱(1)可以实时可视化，(2)定位于神经元组织，(3)在注射后的合理时间内降解。如果成功，我们和我们的合作者就拥有开展IND研究分布、剂量和毒性研究的专业知识。