Optimization of non-peptide probes for the NPFF receptor system

描述（由申请人提供）：本项目根据计划公告（PA）编号：PAR-12-060和标题：体内化学探针发现的验证命中请求（R 01）提交。众所周知，阿片受体与调节疼痛感知密切相关。纵观历史，阿片类药物制剂已被用于有效地管理疼痛，并由于其疗效而继续成为治疗的支柱。然而，这些药物与剂量限制性副作用、耐受性和成瘾性的责任相关。它们仍然是治疗急性和慢性严重疼痛的最广泛的处方药。寻找一种没有副作用的有效镇痛化合物仍然是医疗机构的一个大目标。仅在最近的历史中才认识到存在内源性阿片样物质调节肽，其可用作减轻这些负债的额外治疗手段。这些阿片调节肽（特别是胆囊收缩素和神经肽FF）能够根据剂量和作用部位降低和增强阿片类药物的药理作用。更有趣的是，它们被认为在阿片类药物治疗相关的耐受性和依赖性方面具有作用和可能的责任。神经肽FF（NPFF）是一种内源性抗阿片系统，可阻断阿片类药物引起的痛觉过敏和耐受的发生。在该系统中有两种公认的受体，NPFF 1和NPFF 2。目前，不存在用于NPFF系统的已建立的（亚型选择性或非选择性）小分子、非肽探针。缺乏合适的非肽探针阻碍了对NPFF作用的充分理解，以及该受体系统如何参与阿片类药物的作用以及其他提出的药理学。这是我们的目标 该项目旨在鉴定一种或多种合适的非肽探针，以服务于研究界，从而进一步表征NPFF系统。在我们的项目R 03 DA 029738下产生的我们的先导分子表明，我们有该系统的血管紧张素和拮抗剂，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。目的1 -通过化学合成制备对NPFF 1、NPFF 2具有选择性或对每种受体具有亲和力的组合的新型非肽NPFF 1和NPFF 2配体、激动剂或拮抗剂;目的2 -通过各种体外试验研究高亲和力NPFF配体的理化性质，这些试验将评估其溶解性、稳定性，膜渗透性、蛋白结合、微粒体稳定性和代谢谱;目的3 -在大鼠中进行体内药代动力学，以确定半衰期、生物利用度和脑与血浆的比率。显示出活性的那些化合物将在大鼠中进行完整的PK检查，以最终确定它们作为潜在的体内探针的适合性并了解它们的给药要求（峰值时间、脑：血浆、半衰期、清除率）;目的4 -评估新型NPFF配体的体内痛觉过敏活性。化合物将在温水尾戒断痛觉过敏模型中进行评价，以确认并进一步确定目标2和3中鉴定的那些化合物的药理学特征。总的来说，通过我们的专业知识，通过这个项目，我们将为研究界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，因为目前不存在具有有利的亲和力、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.