Optimization of non-peptide probes for the NPFF receptor system

描述(由申请人提供)：本项目提交的计划公告(PA)编号：PAR-12-060和标题：征集确认命中发现体内化学探针(R01)。众所周知，阿片受体与痛觉的调节密切相关。纵观历史，阿片类药物制剂一直被用来有效地控制疼痛，并由于其有效性而继续成为治疗的支柱。然而，这些药物与剂量限制副作用、耐受性和成瘾的易感性有关。尽管如此，它们仍然是治疗急性和慢性剧烈疼痛的最广泛的处方药物。寻找一种有效的、无副作用的止痛化合物，仍然是医疗机构的一大目标。直到最近，人们才意识到内源性阿片调节肽的存在，可以作为一种额外的治疗手段来减轻这些负担。这些阿片调节肽(特别是胆囊收缩素和神经肽FF)能够降低和增强阿片类药物的药理作用，这取决于剂量和作用部位。更有趣的是，他们被提议在阿片类药物治疗相关的耐受和依赖方面发挥作用并可能承担责任。具体地说，神经肽FF(NPFF)被认为是一种内源性抗阿片系统，可阻断耐受和阿片诱导的痛觉过敏的发展。在这个系统中有两个公认的受体，NPFF1和NPFF2。目前，已建立的(亚型选择性或非选择性)小分子、非肽探针用于NPFF系统尚不存在。缺乏合适的非肽探针阻碍了对NPFF作用的充分了解，以及该受体系统如何参与阿片类药物的作用以及其他拟议的药理作用。这就是我们的目标 确定一个或多个合适的非肽探针的项目，以服务于研究界，努力进一步确定NPFF系统的特征。我们在项目R03 DA029738下产生的铅分子表明，对于这个系统，我们既有血管紧张剂，也有拮抗剂，并且已经开始了解选择性问题，但它们需要针对体内方法进行优化。这项工作将通过以下具体目标进行：目的1-通过化学合成制备新型非肽类NPFF1和NPFF2配体、对NPFF1、NPFF2具有选择性的激动剂或拮抗剂，或每个受体的亲和力的组合；目的2-通过各种体外测试来研究高亲和力NPFF1配体的理化性质，以评估其溶解性、稳定性、膜通透性、蛋白质结合、微粒体稳定性和代谢谱；AIM 3-进行大鼠体内药物动力学研究，以确定半衰期、生物利用度和脑血浆比。这些显示活性的化合物将在大鼠身上进行全面的PK检查，以最终确定它们是否适合作为体内潜在的探针，并了解它们的给药要求(峰值时间、脑：血浆、半衰期、清除量)；目的4-评估新型NPFF配体在体内的痛敏活性。化合物将在温水尾部戒断痛觉过敏模型中进行评估，以确认并进一步确定AIMS 2和AIMS 3中确定的那些化合物的药理学特征。总体而言，凭借我们通过该项目的专业知识，我们将为研究社区提供合适的体内非肽探针，以进一步表征NPFF系统。这些将是新的工具，因为目前还不存在具有良好亲和力、活性和体内性质的非肽NPFF配体。

DESCRIPTION (provided by applicant): This project is submitted under Program Announcement (PA) Number: PAR-12-060 and Title: Solicitation of Validated Hits for the Discovery of in vivo Chemical Probes (R01). Opioid receptors are well known to be intimately involved with modulating the perception of pain. Throughout history, opioid preparations have been utilized to effectively manage pain and have continued to be the mainstay of therapy due to their efficacy. However, these drugs are associated with the liabilities of dose-limiting side-effects, tolerance and addiction. Still they are the most widely prescribed medications to treat acute and chronic severe pain. The search for an effective analgesic compound devoid of side-effects, is still a large goal of the medical establishment. Only recently in history was it realizd that endogenous opioid-modulating peptides exist that can be utilized as a means of additional therapy to attenuate these liabilities. These opioid modulating peptides (specifically cholecystokinin and Neuropeptide FF) are able to both reduce and potentiate the pharmacological effects of opioids depending on the dose and site of action. More interestingly, they have been proposed to have a role and possible responsibility for tolerance and dependence associated with opioid therapy. Specifically, Neuropeptide FF (NPFF) has been reported to exist as an endogenous anti-opioid system and block the development of tolerance and opioid-induced hyperalgesia. There are two recognized receptors in this system, NPFF1 and NPFF2. Currently, established (subtype selective or non-selective) small molecule, non-peptide probes for the NPFF system do not exist. This lack of a suitable non-peptide probe has hampered the full understanding of the actions of NPFF and how this receptor system is involved in the actions of opioids as well as other proposed pharmacologies. It is the goal of this project to identify one or more suitable non-peptide probes to serve the research community in an effort to further characterize the NPFF system. Our lead molecules, generated under our project R03 DA029738, show that we have both angonists and antagonists for this system and have begun to understand selectivity issues yet they need to be optimized for in vivo approaches. This work will be carried out through the following specific aims: AIM 1 - To prepare through chemical synthesis, novel non-peptidic NPFF1 and NPFF2 ligands, agonists or antagonists with selectivity for NPFF1, NPFF2, or a combination of affinities at each receptor; AIM 2 - To investigate the physiochemical properties of high affinity NPFF ligands through a variety of in vitro assays that will assess solubility, stability, membrane permeability, protein binding, microsomal stability and metabolic profiling; AIM 3 - To perform in vivo pharmacokinetics in rats to determine half-life, bioavailability and brain to plasma ratios. Those compounds that show activity will be subjected to full PK workup in rats to ultimately determine their suitability as potential in vivo probes and to understand their dosing requirements (peak time, brain:plasma, half-life, clearance); AIM 4 - To evaluate novel NPFF ligands in vivo for hyperalgesic activity. Compounds will be evaluated in a warm-water tail-withdrawal hyperalgesia model to confirm and further determine pharmacological profiles of those compounds identified in Aims 2 and 3. Overall, with our expertise through this project, we will provide the research community with suitable in vivo non-peptide probes to further characterize the NPFF system. These will be new tools as currently, non-peptide NPFF ligands with favorable affinities, activities, and in vivo properties do not exist.