Validation of the joint-homing and drug delivery attributes of novel peptides in a mouse arthritis model

类风湿关节炎(RA)是一种慢性衰弱疾病，影响着超过1%的美国人。女性会受到影响 发生这种情况的几率大约是男性的3倍。RA也构成了美国退伍军人的一个主要健康问题。这个 退伍军人人口正在老龄化，在军队服役的女性人数正在逐渐增加。在……里面 此外，战区的军事人员在不同的环境中暴露在极端的压力和有毒物质中 表格。这些因素可能直接或间接影响宿主免疫和RA的诱发/加重。这个 由类风湿关节炎引起的疼痛、不适、失去工作日和残疾会对退伍军人的生活质量产生不利影响 在许多方面，以及施加沉重的医疗保健和康复成本。因此，研究提出了新的 类风湿关节炎的治疗方式与美国退伍军人密切相关。使用大鼠佐剂性关节炎 (AA)人RA模型，我们开发了一种多肽导向的治疗关节炎的脂质体给药系统。 心理治疗。为此，我们使用了一种新的联合归巢多肽ART-2来引导脂质体的包封率 地塞米松对大鼠关节炎关节的影响。多肽导向的脂质体DEX对 对大鼠关节炎的抑制作用优于非靶向脂质体药物或游离药物。重要的是，提高的疗效 ART-2脂质体组与同时增加的全身毒性无关。因此，多肽- 与其他两种药物相比，引导脂质体给药改善了DEX的治疗概况(效益/风险比) 医疗模式。然而，与近交系大鼠(用于大鼠再障模型)不同，近交系大鼠的基因是相同的(相同) 而产生免疫反应的主要是热休克蛋白65(Hsp65)，RA患者具有多样性的基因 构成并产生对Hsp65和II型胶原(CII)的免疫应答。因此，我们需要 在另一个独立的第二个模型--小鼠胶原蛋白--中验证我们目前在大鼠AA模型中的发现 诱发性关节炎(CIA)模型，该模型在遗传学和某些疾病参数上不同于AA模型。 这项验证将是降低将该技术应用于RA患者的风险的第一步 在进行进一步的开发和评估以满足研究新药(IND)的标准之前 为食品和药物管理局。这一点意义重大，因为退伍军人事务部(VA)已经就该用途申请了非临时专利 与美国专利商标局合作开发用于治疗关节炎的多肽ART-2。AA和CIA模型 关节炎与人类类风湿性关节炎有一些(但不是全部)特征，但也有几个关键特征不同： 1)与Lewis大鼠(RT-1)不同，近交系野生型小鼠不会患再生障碍性贫血；2)T 细胞/抗体在CIA中为CII，而在AA中为Hsp65；3)抗体在CIA中是致病的，但要么没有作用，要么 对再生障碍性贫血有保护作用；4)大鼠和小鼠的药物代谢不同。我们将使用两个CIA模型：Wild 表达人类白细胞抗原-DR4的DBA/1(H-2Q)型小鼠(H-2Q)和人源化小鼠(H-2b) 类风湿关节炎的基因。[我们将测试甲氨蝶呤(MTX)，RA的黄金标准。我们已经成功地准备好了 脂质体包裹MTX，在亲代大鼠关节炎模型(AA)中的初步测试表明，MTX 脂质体具有生物活性，能有效抑制关节炎。最近，我们已经完成了 在我们实验室建立CIA模型，以验证AA模型的上述发现。]具体的 我们研究基于ART-2和甲氨蝶呤(MTX)的CIA模型的目的是：目标1：(A)到 系统给药的多肽ART-2展示脂质体在体内的选择性归巢 将一种近红外染料(花菁7)包裹到CIA小鼠的关节炎关节；以及(B)检查 脂质体MTX在CIA小鼠体内的药代动力学和药效学研究。目标2：(A) 验证通过多肽ART-2展示脂质体、对照脂质体、 和用于抑制CIA小鼠关节炎的免费药物；以及(B)确定这些药物的相对多器官毒性 三种给药方式，以评估它们的比较治疗效果/毒性概况。我们相信 通过适当的验证，这项技术可以进行调整和进一步改进，以用于患有RA的退伍军人。

Rheumatoid arthritis (RA) is a chronic debilitating disease affecting over 1% of Americans. Women are affected about 3 times more often than men. RA also constitutes a major health problem among the U.S. Veterans. The Veteran population is aging, and the number of women serving in the military is gradually increasing. In addition, military personnel in combat zones are exposed to extreme stress and toxic substances in different forms. All these factors may directly or indirectly influence host immunity and induction/aggravation of RA. The pain, discomfort, lost working days, and disabilities caused by RA can adversely affect Veterans’ quality of life in many ways, as well as impose heavy cost of healthcare and rehabilitation. Therefore, studies advancing new modalities of treatment of RA are highly relevant to the U.S. Veterans. Using the rat adjuvant-induced arthritis (AA) model of human RA, we developed a peptide-guided targeted liposomal drug delivery system for arthritis therapy. For this purpose, we used a novel joint-homing peptide ART-2 to guide liposomes entrapping dexamethasone (DEX) to arthritic joints of rats. Peptide-guided liposomal DEX was more effective in suppressing arthritis in rats than untargeted liposomal drug or free drug. Importantly, the increased efficacy of the ART-2 liposomal group was not associated with concurrent increase in systemic toxicity. Thus, peptide- guided liposomal drug delivery improved the therapeutic profile (benefit/risk ratio) of DEX over the other two modalities. However, unlike inbred rats (used in the rat AA model), which are genetically homogeneous (same) and generate immune response mainly to heat-shock protein 65 (Hsp65), RA patients have diverse genetic make-up and generate immune response to both Hsp65 and type II collagen (CII). Therefore, we need to validate our current findings from the rat AA model in an independent second model, the mouse collagen- induced arthritis (CIA) model, which differs in genetics and certain disease parameters from the AA model. This validation will be the first step to de-risk the translational application of this technology to RA patients before undertaking further development and evaluation to meet the criteria of an investigational new drug (IND) for the FDA. This is significant because the Veterans Affairs (VA) has filed a non-provisional patent on the use of peptide ART-2 for arthritis therapy with the U.S. Patent and Trademark Office. The AA and CIA models of arthritis share some (but not all) features with human RA, but they also differ in a few critical characteristics: 1) unlike Lewis rats (RT-1l), inbred wild type mice do not develop AA; 2) the predominant target of the T cells/antibodies is CII in CIA, but Hsp65 in AA; 3) antibodies are pathogenic in CIA, but either show no effect or are protective in AA; and 4) drug metabolism is different in rats and mice. We will use two CIA models: wild type DBA/1 (H-2q) mice and “humanized” mice (H-2b) expressing HLA-DR4, one of the disease-susceptibility genes for RA. [We will test methotrexate (MTX), the gold-standard for RA. We have successfully prepared liposomes entrapping MTX, and pilot testing in the parent rat arthritis model (AA) showed that MTX entrapped in liposomes is biologically active and effective in suppressing arthritis. Recently, we have completed setting-up of the CIA model in our laboratory to validate the above findings from AA model.] The specific aims of our study in CIA model based on peptide ART-2 and methotrexate (MTX) are: Aim 1: (a) To validate the preferential in-vivo homing of systemically administered peptide ART-2-displaying liposomes entrapping a near-infrared dye (Cyanine 7) to arthritic joints of mice with CIA; and (b) To examine the pharmacokinetic and pharmacodynamic aspects of liposomal MTX delivery in mice having CIA. Aim 2: (a) To validate the comparative efficacy of MTX delivery via peptide ART-2-displaying liposomes, control liposomes, and free drug in suppressing arthritis in CIA mice; and (b) To determine the relative multiorgan toxicity of these three drug delivery modalities to assess their comparative therapeutic efficacy/toxicity profiles. We believe that with appropriate validation, this technology can be adapted and further refined for use in Veterans with RA.