Nanomedicine technology for drug delivery in Rheumatoid Arthritis using a synovium-specific targeting peptide

尽管有许多有效的治疗剂可用于治疗类风湿性关节炎(RA)，但相当大一部分患者(约30%-40%)对当前的药物治疗没有反应。此外，由于全身暴露导致潜在的免疫抑制以及与非靶标不良反应相关的问题，目前治疗的风险/收益比需要改进。解决这一问题的一种方法是利用已知存在于正常组织和疾病组织中的组织特异性寻址蛋白分子作为靶点，在疾病组织部位传递/浓缩药物(Ferrari等人，2016)。在癌症等其他疾病中，这已被证明是药物输送系统的强大工具，允许更具体的疾病组织靶向，减少药物剂量要求，并改善安全性。在类风湿性关节炎的案例中，Pitzalis的团队在移植了人滑膜的严重联合免疫缺陷(SCID)小鼠中使用体内噬菌体展示选择，已经为这种疾病组织识别出一个独特的靶点，这种疾病组织由环状EPTA-多肽KSTHDRL识别，称为多肽3.1(Lee等人，2002年)，受形成二硫键的两个末端半胱氨酸限制。同一小组以前发表的工作也证明了多肽3.1能够有效地与抗炎细胞因子IL-4融合并直接传递到移植到SCID小鼠的RA滑膜，并在功能上抑制移植组织中的炎症(Wythe等人，2013年)。Macor教授的团队(的里雅斯特大学)也测试了这种多肽独特的滑膜靶向潜力，他们将其连接到C5或肿瘤坏死因子的中和抗体上，并使用抗原诱导的关节炎大鼠模型(AIA)在体内进行了测试，进一步支持了多肽3.1可以可行地用于专门针对病变关节的药物输送的概念(Macor等人，2012年；科伦坡等人，2016)。同一团队还开发了一种纳米医学方法，用多肽3.1武装可生物降解的纳米颗粒，以便将药物如甲氨蝶呤(MTX)输送到病变关节。纳米粒子-3.1被证明在体外和体内都能特异性地结合其靶点，并通过需要较低的治疗药物剂量来提高MTX的疗效，以达到与游离MTX相似的效果。此外，他们通过免疫荧光跟踪纳米颗粒-3.1在体内的分布证明，这种方法可以用于慢性长期给药，并作为早期检测潜在患者关节炎症的诊断工具(科伦坡等人，2016)。然而，这些武装的纳米颗粒只在AIA大鼠模型等动物模型中进行了测试。因此，为了转化用于治疗人类类风湿关节炎的新型特异性治疗剂的开发，我们建议使用我们实验室常规运行超过17年的人-RA/SCID小鼠模型(Wahid等人，2000年)来测试纳米颗粒-3.1优先向人类滑膜组织运送货物(例如MTX)的能力，而不是皮肤组织(对照)。为了实现这一目标，我们与Macor小组(的里雅斯特大学)和Cruz小组(莱顿大学医学中心)建立了合作关系，这两个小组生产了纳米粒子-3.1，并成功地将其用于传统动物模型，例如AIA。该项目的主要目标是优化这种携带多肽3.1的纳米颗粒在负载RA药物(例如MTX)时的药代动力学，并建立其在人-RA/SCID移植模型中的体内生物分布以及将治疗药物输送和释放到其炎症组织中的特定靶点的效率，以确定其临床使用的潜力。技能优先调整：先进治疗学、数量生物学、整体生物

Despite the availability of numerous effective therapeutic agents for the treatment of rheumatoid arthritis (RA), a sizable group of patients (approximately 30-40%) do not respond to current medication. In addition, the risk/benefit ratio profile of current therapies, requires improvement due to systemic exposure leading to potential immune suppression and issues related to off-target adverse effects. One way to combat this is to exploit tissue specific addressin molecules, known to be present in both normal and disease tissues, as targets to deliver/concentrate drugs at the disease tissue site (Ferrari et al., 2016). This has been shown in other conditions such as cancer, to be a powerful tool for drug delivery systems allowing more specific disease tissue targeting, a reduced drug dosage requirement and improved safety profile. In the case of RA, the Pitzalis' group, using in vivo phage display selection in severe combined immunodeficient (SCID) mice transplanted with human synovium, has identified a distinct target for such disease tissue recognised by a cyclic epta-peptide KSTHDRL, known as peptide 3.1 (Lee et al., 2002) constrained by two terminal cysteines that form a disulphide bond. Previous published work from the same group, has also demonstrated the capability of peptide 3.1 to effectively fuse to and deliver the anti-inflammatory cytokine IL-4, directly to RA synovium transplanted into SCID mice and functionally inhibit inflammation in the grafted tissue (Wythe et al., 2013). The distinct synovial targeting potential of this peptide was also tested by Professor Macor's group (University of Trieste) who conjugated it to a neutralising antibody for C5 or TNF, and tested it in vivo using the antigen-induced arthritis rat model (AIA), further supporting the notion that peptide 3.1 can be feasibly used for drug delivery specifically to the diseased joints (Macor et al., 2012; Colombo et al., 2016)The same group also developed a nanomedicine approach arming biodegradable nanoparticles with peptide 3.1 in order to deliver drugs such as Methotrexate (MTX) to diseased joints. Nanoparticle-3.1 was shown to specifically bind its target both in vitro and in vivo and increase MTX efficacy by requiring a lower therapeutic drug dose in order to achieve a similar effect to free-MTX. In addition, they demonstrated by following the distribution of Nanoparticle-3.1 in vivo by immunofluorescence, that it is possible to use this approach for chronic long-term drug delivery and as a diagnostic tool for early detection of inflammation in the joints of potential patients (Colombo et al., 2016). However, these armed nanoparticles have only been tested in animal models such as the AIA rat model. Therefore, in order to translate the development of novel specific therapeutic agents for the treatment of human RA, we propose to use the human-RA/SCID mouse model that has been routinely run in our laboratory for over 17 years (Wahid et al., 2000) to test the capacity of Nanoparticle-3.1 to deliver cargo (e.g. MTX) preferentially to human synovial tissue over skin tissue (control). In order to achieve this, we have established a collaboration with the Macor group (University of Trieste) and the Cruz group (Leiden University Medical Centre) who have produced Nanoparticle-3.1 and successfully used it in conventional animal models e.g AIA. This project's main objectives are to optimise the pharmokinectics of this nanoparticle armed with peptide 3.1 while loaded with an RA drug (e.g. MTX) and establish its in vivo biodistribution in the human-RA/SCID transplantation model and its efficiency delivering and releasing the therapeutic drug to its specific target in inflamed tissue in order to identify its potential for clinical use. Skills Priority Alignment: Advanced Therapeutics, Quantitative Biology, Whole Organism