Intra-cartilage depot delivery of electrically-charged IL-1RA for targeting osteoarthritis-associated inflammation and catabolism in multiple joint tissues

项目总结/摘要 骨关节炎（OA）疼痛仍然是一个棘手的问题，几乎没有治疗方案。中枢作用镇痛药 毒品有使人上瘾的副作用。因此，局部持续镇痛递送是一种有吸引力的策略， 开发新的OA疼痛疗法。然而，由于以下原因，局部关节内（IA）输送仍然不足： 药物从滑膜关节空间快速清除，需要多次注射，这会导致毒性。这进一步 复杂的高密度和高度负电荷的软骨基质，阻止药物到达他们的 目标站点。亲本R 01通过开发软骨靶向和驻留阳离子来应对这一挑战 抗分解代谢和促合成代谢疾病改善OA治疗剂。例如，IL-1的受体拮抗剂 将IL-1 RA与最佳电荷阳离子肽载体（CPC-IL-1 RA）融合，并将其有效地与IL-1 RA融合。 在体外和体内证实了仅用一次剂量就能抑制OA相关的卡他霉素。 在这里，我们将通过评估靶向药物递送对OA疼痛治疗的影响， （i）阳离子融合CPC-IL-1 RA（已经开发作为 亲本R 01任务）和（ii）阳离子CCR 2受体拮抗剂（CCR 2 RA）。我们和其他人已经证明， CCL 2/CCR 2信号传导是促进内侧半月板（DMM）不稳定中持续疼痛的关键 模型，其局部药理学阻断可降低疼痛行为。这些发现表明，CCL 2- 关节中局部的CCR 2信号传导也有助于实验性OA中的疼痛，这突出了需要 开发CCR 2抑制剂如CCR 2受体拮抗剂（CCR 2 RA）的持续局部递送方法， 它是一种小分子。CCR 2 RA将与开发的阳离子软骨靶向纳米结构融合 用于递送小分子的多臂抗生物素蛋白（mAv）。中心假设是一次性IA注射 CPC-IL-1 RA和mAv-CCR 2 RA的联合应用将在4周内在大鼠内侧海马中提供持续的镇痛缓解。 横截（MMT）OA模型。我们拥有一支在OA疼痛和肌肉骨骼关节药物方面具有专业知识的团队 将在以下目标中测试该假设的递送，目标1：合成软骨内储库一个月- 使用可水解酯接头的阳离子mAv的CCR 2 RA的长期持续释放。药物释放速率和 测量缀合物的生物活性并与未修饰的药物进行体外比较。目的2：检验疗效 在大鼠MMT模型中单次IA注射阳离子CPC-IL-1 RA和mAv-CCR 2 RA。雄性和雌性大鼠 将接受生理盐水、CPC-IL-1 RA、未结合的IL-1 RA、mAv-CCR 2 RA或未结合的IL-1 RA的IA注射 CCR2RA。将评估负重不对称和膝关节痛觉过敏;将评价DRG变化 通过IHC和RNAscope;将评估关节组织病理学，包括评估膝关节神经支配变化 和免疫细胞浸润这一具有竞争力的紧急修订将扩大已设计的影响 用于持续OA疼痛抑制超过一个月的阳离子治疗剂， 提供长期疼痛缓解，以及疾病改善（在原始R 01中讨论）。

Project Summary/Abstract Osteoarthritis (OA) pain remains an intractable problem with few treatment options. Centrally acting analgesic drugs carry the risk of addictive side effects. Thus, local sustained analgesic delivery is an attractive strategy for development of novel OA pain therapies. Local intra-articular (IA) delivery, however, remains inadequate due to rapid drug clearance from the synovial joint space requiring multiple injections that cause toxicity. This is further complicated by the dense and highly negatively charged cartilage matrix that prevents drugs from reaching their target sites. The parent R01 addressed this challenge by developing cartilage targeting and residing cationic anti-catabolic and pro-anabolic disease modifying OA therapeutics. For example, a receptor antagonist of IL-1 (IL-1RA) was fused with an optimally charged cationic peptide carrier (CPC-IL-1RA), and its effectiveness in suppressing OA associated catabolism was demonstrated in-vitro as well as in vivo with only a one-time dose. Here we will broaden the impact of our work in targeted drug delivery to treatment of OA pain by evaluating the effectiveness in sustained release formulations of (i) cationic fusion CPC-IL-1RA (already developed as part of the parent R01 tasks) and (ii) cationic CCR2 receptor antagonist (CCR2RA). We and others have shown that CCL2/CCR2 signaling is key for promoting persistent pain in the destabilization of the medial meniscus (DMM) model and that its local pharmacological blockade decreases pain behavior. These findings suggest that CCL2- CCR2 signaling locally in the joint also contributes to pain in experimental OA, highlighting the need for developing sustained local delivery methods for CCR2 inhibitors such as CCR2 receptor antagonist (CCR2RA), which is a small molecule. CCR2RA will be fused with a cationic cartilage targeting nano-construct developed for delivery of small molecules, multi-arm Avidin (mAv). The central hypothesis is that a one-time IA injection of CPC-IL-1RA and mAv-CCR2RA will provide sustained analgesic relief over 4 weeks in a rat medial meniscal transection (MMT) OA model. We have a team with expertise in OA pain and in musculoskeletal joint drug delivery that will test this hypothesis in the following aims, Aim 1: Synthesize an intra-cartilage depot for a month- long sustained release of CCR2RA using cationic mAv using hydrolysable ester linkers. Drug release rates and bioactivity of the conjugate will be measured and compared with unmodified drug in-vitro. Aim 2: Test the efficacy of a single IA injection of cationic CPC-IL-1RA and mAv-CCR2RA in the rat MMT model. Male and female rats will receive an IA injection of saline, CPC-IL-1RA, unconjugated IL-1RA, mAv-CCR2RA, or unconjugated CCR2RA. Weight-bearing asymmetry and knee hyperalgesia will be assessed; DRG changes will be evaluated by IHC and RNAscope; joint histopathology will be assessed, including assessment of knee innervation changes and immune cell infiltration. This competitive urgent revision will broaden the impact of the already designed cationic therapeutics for sustained OA pain suppression over a month with only a single IA administration thereby providing long-term pain relief, in addition to disease modification (which was addressed in the original R01).