Institute of Medicinal Chemistry, Hoshi University, Japan.
J Control Release. 2013 Mar 10;166(2):139-46. doi: 10.1016/j.jconrel.2012.12.027. Epub 2013 Jan 5.
The development of antiviral agents that have novel mechanisms of action is urgently required in the topical therapy of herpes simplex virus type 2 (HSV-2) infections. We reported previously that topical application of branched 3610-Da polyethylenimine (PEI) exhibited preventative antiviral activity. In this study, to develop therapeutic anti-HSV-2 agents, the most potent PEI combined with ~200 nm-sized liposomes with or without oleic acid (liposomes/PEI) was selected in vitro and further evaluated using in vivo studies. The mechanism of action in vivo was elucidated using PEIs with decreased numbers of primary amine residues, resulting from ethylene carbonate treatment, and polyallylamine, a linear polyamine consisting of primary amines. Cytotoxicity and antiviral activity in vitro, and the appearance of acute herpetic disease and virus yields in mice intravaginally administered with liposomes/PEI were evaluated in cell culture assays and a mouse genital herpes model, respectively. In addition, the cellular association of liposome/PEI was examined by flow cytometry and confocal microscopy. PEI showed higher antiviral activity postinfection than preinfection in vivo. Liposome/PEI and PEI with decreased numbers of primary amine residues at a dose of 0.2 mg PEI/mouse exhibited more potent therapeutic antiviral activity than acyclovir and PEI alone without acute lesion appearance or toxicity pre- or postinfection, but polyallylamine was moderately effective only preinfection. Liposome concentrations were important for the effectiveness of liposome/PEI. This finding suggests that PEI combined with liposomes and with slightly decreased numbers of primary amines may be an effective vaginally administrated antiviral drug, and secondary and tertiary amine residues of PEI may contribute to the inhibitory efficiency against viral infection.
开发具有新型作用机制的抗病毒药物是治疗单纯疱疹病毒 2 型（HSV-2）感染的当务之急。我们之前曾报道过，局部应用支化的 3610-Da 聚亚乙基亚胺（PEI）具有预防抗病毒活性。在这项研究中，为了开发治疗性抗 HSV-2 药物，选择了最有效的 PEI 与~200nm 大小的脂质体结合，无论是否含有油酸（脂质体/PEI），并在体内研究中进一步评估。通过使用经碳酸亚乙酯处理导致伯胺基数减少的 PEI 和由伯胺组成的线性多胺聚烯丙胺，阐明了体内作用机制。在细胞培养测定和小鼠生殖器疱疹模型中，评估了细胞毒性和体外抗病毒活性，以及经阴道给予脂质体/PEI 的小鼠急性疱疹性疾病和病毒产量的出现。此外，通过流式细胞术和共聚焦显微镜检查了脂质体/PEI 的细胞关联。PEI 在体内感染后比感染前显示出更高的抗病毒活性。与单独使用阿昔洛韦和 PEI 相比，脂质体/PEI 和伯胺基数减少的 PEI（剂量为 0.2mg PEI/只）在感染前或感染后均表现出更强的治疗性抗病毒活性，而没有急性病变出现或毒性，但聚烯丙胺仅在感染前有效。脂质体浓度对脂质体/PEI 的有效性很重要。这一发现表明，PEI 与脂质体结合并略微减少伯胺基数可能是一种有效的阴道给药抗病毒药物，PEI 的仲胺和叔胺残基可能有助于抑制病毒感染的效率。
