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

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.

类风湿关节炎（RA）是一种慢性衰弱的疾病，影响了1％以上的美国人。妇女受到影响 是男人的频率约3倍。 RA也构成了美国退伍军人的重大健康问题。这 资深人口正在衰老，军队服役的妇女人数正在逐渐增加。在 此外，战斗区的军事人员暴露于不同的压力和有毒物质 表格。所有这些因素可能直接或间接影响RA的宿主免疫力和诱导/加重。 疼痛，不适，工作日失去以及由RA引起的残疾可能会对退伍军人的生活质量产生不利影响 在许多方面，以及施加了大量的医疗保健和康复成本。因此，研究推进了新的 RA的治疗方式与美国退伍军人高度相关。使用大鼠调节引起的关节炎 （AA）人类RA的模型，我们开发了一种肽引导的靶向脂质体药物输送系统的关节炎 治疗。为此，我们使用了一种新型的联合归圣肽ART-2来引导脂质体捕获脂质体 地塞米松（DEX）到大鼠的节推关节。肽引导的脂质体DEX更有效 抑制大鼠的关节炎比未靶向的脂质体药物或游离药物抑制关节炎。重要的是，提高的效率 ART-2脂质体组与全身毒性的同时增加无关。那，胡椒 指导性脂质体药物的递送改善了DEX的治疗概况（益处/风险比） 方式。但是，与近交大鼠（用于大鼠AA模型中）不同，该大鼠是遗传均匀的（相同） RA患者主要产生免疫响应65（HSP65），具有多种通用性 化妆并产生对HSP65和II型胶原蛋白（CII）的免疫反应。因此，我们需要 在独立的第二个模型中，从大鼠AA模型中验证我们目前的发现，小鼠胶原蛋白 诱导关节炎（CIA）模型，其遗传学和某些疾病参数与AA模型不同。 该验证将是去风险转化为RA患者的转化应用的第一步 在进行进一步的开发和评估之前，以满足研究新药的标准（IND） 对于FDA。这很重要，因为退伍军人事务（VA）已就使用申请了非统治专利 美国专利商标办公室用于关节炎治疗的Pepper Art-2。 AA和CIA模型 关节炎与人类RA有一些（但不是全部）特征，但在一些关键特征上也有所不同： 1）与刘易斯大鼠（RT-1L）不同，近交野生型小鼠不会发育AA； 2）t的主要目标 细胞/抗体是CIA中的CII，但AA中的Hsp65； 3）CIA中的抗体是致病性的，但要么没有作用或 在AA中受到保护； 4）药物代谢在大鼠和小鼠中是不同的。我们将使用两种CIA模型：野生 DBA/1（H-2Q）小鼠和表达HLA-DR4的“人性化”小鼠（H-2B），这是疾病敏感性之一 RA的基因。 [我们将测试RA的金标准Methodtrexate（MTX）。我们已经成功准备了 脂质体入口MTX和亲大鼠关节炎模型（AA）中的试验测试表明MTX 进入脂质体具有生物学活性，可有效抑制关节炎。最近，我们已经完成 在我们的实验室中设立中央情报局模型，以验证AA模型的上述发现。] 我们研究基于肽ART-2和Methodotrexate（MTX）的CIA模型研究的目的是：目标1：（a） 验证系统施用的肽Art-2脱脂脂质体的首选体内归位 将近红外染料（氰氨酸7）夹在带有CIA的小鼠的关节中； （b）检查 CIA小鼠脂质体MTX递送的药代动力学和药效学方面。目标2：（a） 通过肽ART-2滴定脂质体验证MTX递送的比较效率，对照脂质体， 和抑制CIA小鼠关节炎的免费药物； （b）确定这些相对多机器人的毒性 三种药物输送方式，以评估其比较治疗效率/毒性谱。我们相信 通过适当的验证，可以对该技术进行调整，并进一步改善用于RA的退伍军人。