In vivo feasibility of a peripheral nerve imaging agent

描述（由申请人提供）：“神经显像剂减少前列腺切除术神经系统并发症的可行性”根治性或保留神经的前列腺切除术的一个关键挑战是尽量减少神经系统并发症，这些并发症可导致约10%-30%的阳痿和约10%的尿失禁。我们建议在体内证明一种新的周围神经显像剂或“神经涂料”的可行性，该显像剂由近红外（NIR）染料偶联重组人神经生长因子（rhNGF） （rhNGF-NIR）组成。本提案的目标是表明rhNGF-NIR可以可视化。我们的产品vision是一种可注射显像剂，可以在术前给予，以协助外科医生开始前列腺切除术之前。为了证明可行性，我们选择了两种近红外染料，Alexa Fluor 790和Dyomics 831，因为它们的化学成分(1)与LI-COR （Lincoln， NE）开发的IRDye(R) 800CW相似，其安全性已在FDA药物主文件中确立，即这些试剂也应该是安全的；(2)相对于花青素5.5，提供更高的光穿透组织和增强的信噪比背景；(3)与吲哚菁绿（ICG）不同，可以容易地偶联；(4)可以使用商用成像系统进行观测。NGF作为靶向片段的基本原理。在初步研究中，我们发现与Alexa Fluor 488结合的NGF （rhNGF-488）在远端分离培养中被吸收，并移动到近端。我们选择的靶向部分是rhNGF，因为它具有良好的耐受性，非免疫原性，并且具有双重靶向特性。我们期望在临床研究中单独使用rhNGF的最大耐受剂量（MTD）范围内（0.1 g/kg-0.3 g/kg）使用rhNGF- nir剂量。当皮下注射时（sub-c），大约。80%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神经节（DRG），在那里降解。研究计划。在本提案中，我们将可靠地合成和表征ngf探针，然后在体外、神经元存活试验和区隔培养中确认受体介导的轴突转运靶向感觉神经元(1)。我们将在实时成像研究中探索小鼠体内的可视化。假设。我们假设NGF提供了一种安全和选择性的神经元内靶向片段，可以通过外周注射传递选定的近红外染料，以成像前列腺切除术前与注射部位相关的外周神经。我们的具体目标是：染料共轭物的合成、优化和表征。我们将修改我们专有的rhNGF-488连接系统以合成两种变体（rhNGF-780， rhNGF-DYM或rhNGF-NIR）。我们的目标是：(1)使rhNGF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例的生物活性探针。目标2。(1)神经元存活和(2)在区室培养中逆行运输的生物测定。神经元绝对需要神经生长因子才能存活。我们通过将探针用于交感神经元的大规模培养，证实了rhNGF-NIR变体的生物活性。我们的第一个目标是证明探针含有生物活性的神经生长因子。接下来，我们将确认每个rhNGF-NIR被感觉神经元选择性地吸收。我们使用区隔培养来证实rhNGF-NIRs在远端被TrkA受体内化，然后在培养系统的中心腔室中运输到细胞体。目标3。证实rhNGF-NIR在体内可以显像并被周围神经选择性吸收。我们将在外围注射微剂量的rhNGF-NIR变体，并在市售成像系统中观察活体动物的探针。在对照研究中，我们将在脚垫和上臀部两个部位注射rhNGF-NIR亚c和im。我们的目标不是模拟临床应用或确定剂量，而是证明rhngf - nir(1)可以实时可视化，(2)定位于神经元组织；(3)注射后在合理的时间内降解。如果成功，我们和我们的合作者拥有开展ind研究的专业知识，分布，剂量和毒性研究。

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.