In vivo feasibility of a peripheral nerve imaging agent

描述（由申请人提供）：“神经成像剂减少前列腺切除术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关键挑战是使神经并发症最小化，所述神经并发症可导致约10%-30%的病例中的阳痿和约10%的病例中的失禁。我们提出了一种新的外周神经成像剂或“神经涂料”的近红外（NIR）染料共轭重组人神经生长因子（rhNGF）（rhNGF-NIR）组成的体内可行性。该提案的目标是显示rhNGF-NIR可以可视化。我们的产品愿景是一种可注射的显像剂，可在手术前给予，以协助外科医生在开始乳房切除术之前。为了显示可行性，我们选择了两种NIR染料，Alexa Fluor 790和Dyomics 831，因为它们的化学性质（1）与LI-COR开发的IRDye（R）800 CW相似（林肯，NE），其安全性在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5.5，提供增加的光的组织穿透和增强的信噪比背景比;（3）与吲哚菁绿色（ICG）不同，可以容易地缀合;和（4）可以使用商业成像系统观察。NGF作为靶向部分的原理。在初步研究中，我们表明，与Alexa Fluor 488（rhNGF-488）结合的NGF在远端的隔室培养物中被吸收并移动到近端。我们选择的靶向部分是rhNGF，因为它是耐受性良好的，非免疫原性的，因为它的双重靶向特性。我们希望在临床研究中使用rhNGF-NIR的最大耐受剂量（MTD）范围内（0.1 μ g/kg-0.3 μ g/kg）。当给出子图（sub-c）时，大约80%的未修饰的rhNGF定位于外周注射部位，被高亲和力TrkA受体吸收，并通过轴突运输移动到背根神经节（DRG），在那里它降解。研究计划。在这项提议中，我们将可靠地合成和表征NGF探针，然后在体外、神经元存活测定和隔室培养中证实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。假说.我们假设，神经生长因子提供了一个安全的和选择性的神经元内靶向部分，可以提供选定的NIR染料，通过外周注射成像周围神经与注射部位之前的脊髓切除术。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，以产生具有一致的蛋白质：探针比例的生物活性探针。目标2.用于（1）神经元存活和（2）隔室培养物中逆行转运的生物测定。神经元对NGF的生存有绝对的需求。我们通过将探针施用于交感神经元的大量培养来确认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NGF。接下来，我们将确认每个rhNGF-NIR被感觉神经元选择性吸收。我们使用隔室培养来证实rhNGF-NIR被远端的TrkA受体内化，然后转运到培养系统的中央室中的细胞体。目标3。确认rhNGF-NIR可在体内可视化并被周围神经选择性吸收。我们将在外周注射微剂量的rhNGF-NIR变体，并在市售成像系统中观察活体动物中的探针。在对照研究中，我们将在两个部位注射rhNGF-NIR sub-c和im，在足垫和上臀部。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）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将拥有开展IND研究的专业知识，包括分布，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.