New generation of long acting nucleos(t)ides and immune stimulant for treatment of chronic hepatitis B

全球的目标是减少慢性B型肝炎（CH B）感染的负担， HBV相关的终末期肝病和癌症的发展。现有治疗方法的改进 预计将有助于实现这一目标。具体来说，使用一次两个月注射核苷（t） 类似物与免疫调节性抗病毒化合物的组合而不是终身每日药丸具有 强大的潜力，以帮助实现功能性治愈慢性乙型肝炎。为此，我们建议将水溶性 抗病毒药物，一线药物替诺福韦（TFV）和恩替卡韦（ETV），以及免疫调节药物替唑尼特 (TIZ)变成疏水亲脂的结晶前药。我们将把它们配制成纳米混悬剂， 肌肉注射纳米晶体的物理化学性质的有效优化有望 改善其药代动力学（PK）和药效学（PD）特征。这种优化将提高 前药纳米晶体被肝巨噬细胞和肝细胞摄取以确保缓慢释放， 在B型肝炎病毒复制部位维持治疗药物浓度。 预期长效TFV、ETV和TIZ治疗可降低给药频率，限制毒性， 并有助于持续的病毒抑制和治疗。HBV的功能性治愈有望实现 通过多因素机制，包括抑制病毒聚合酶，防止cccDNA形成， 通过TIZ刺激宿主先天免疫清除HBV微染色体。因此， 本提案的目的是开发临床上可转化的、长效的、可注射的抗病毒药物 纳米制剂以增加粘附性并增强药物递送至持续HBV感染部位， 从而促进持续的病毒抑制和有限的治愈。为此，提出了三个具体目标： 目的1：研制长效抗乙肝病毒前体药物纳米制剂并评价其疗效。在这里，我们将 应用前核苷酸（ProTide）和改良的HepDirect前药技术，将现有药物转化为 适于配制成纳米混悬剂以获得延长治疗活性的疏水前药 肝细胞中的药物浓度。这有望改善药物在感染肝细胞中的生物分布 而不影响药物效力和安全性。前药制剂将在体外筛选， 人巨噬细胞作为潜在的药物储存库，感染的肝细胞作为最终靶点。抗hbv 将使用HBV感染的人源化小鼠在体外和体内检测前药纳米晶体的活性。目的 2：开发长效TIZ纳米制剂并评价TIZ抑制HBV的机制 在感染的肝细胞中复制。目的3：评价所选长效TIZ的协同效应 和NUC制剂，并评估该组合从肝细胞中消除HBV cccDNA的能力 并显著降低HBsAg浓度。

There is a global aim to reduce the burden of chronic hepatitis B (CHB) infection and prevent the development of HBV-associated end-stage liver disease and cancer. The improvement of existing therapeutics is expected to help achieve this goal. Specifically, the usage of once two-month injectable nucleos(t)ide analogs in combination with immunomodulating antiviral compounds instead of life-long daily pills has the strong potential to help to achieve a functional cure for CHB. To this end, we propose to transform water-soluble antiviral drugs, first-line drugs tenofovir (TFV) and entecavir (ETV), and immunomodulating drug tizoxanide (TIZ) into hydrophobic lipophilic crystalline prodrugs. We will formulate them as nanosuspensions suitable for intramuscular injection. The efficient optimization of physicochemical properties of nanocrystals is expected to improve their pharmacokinetics (PK) and pharmacodynamics (PD) profiles. This optimization will enhance uptake of the prodrug nanocrystals by liver macrophages and hepatocytes to ensure a slow release and sustained therapeutic drug concentrations at the site of hepatitis B viral replication. The treatment with long-acting TFV, ETV, and TIZ is expected to decrease dosing frequency, limit toxicity, and facilitate sustained viral suppression and treatment. A functional cure for HBV is expected to be achieved via multifactorial mechanisms, including inhibition of viral polymerase, prevention of cccDNA formation, and the clearance of HBV micro-chromosomes via stimulation of host innate immunity by TIZ. Thus, the overall objective of this proposal is to develop clinically translatable, long-acting, injectable, antiviral drug nanoformulations to increase adherence and enhance drug delivery to sites of persistent HBV infection, thereby facilitating sustained viral suppression and finite cure. To this end, three specific aims are proposed: Aim 1: Develop long-acting anti-HBV prodrug nanoformulations and evaluate the drug efficacy. Here, we will apply pronucleotide (ProTide) and a modified HepDirect prodrug technology to transform existing drugs into hydrophobic prodrugs suitable for formulation as nanosuspensions to achieve prolonged therapeutic active drug concentrations in hepatocyte. This is expected to improve drug biodistribution to infected hepatocytes without compromising drug potency and safety profile. The prodrug formulations will be screened in vitro in human macrophages as a potential drug depot and in infected hepatocytes as final targets. The anti-HBV activity of prodrug nanocrystals will be examined in vitro and in vivo using HBV-infected humanized mice. Aim 2: To develop long-acting TIZ nanoformulation and evaluate the mechanisms by which TIZ suppresses HBV replication in infected hepatocytes. Aim 3: To evaluate the synergistic efficacy of the selected long-acting TIZ and NUC formulations and evaluate the ability of this combination to eliminate HBV cccDNA from hepatocytes and significantly reduce the concentration of HBsAg.