IN VITRO INACTIVATION OF VIRUSES IN BLOOD COMPONENTS

描述(改编自申请人的摘要)本申请 建议开展研究以评估金丝桃素的开发潜力 (Hy)和许多结构上相关的芳香族多环二酮， 作为红细胞中的病毒灭活剂用于输血。这个 研究是基于令人鼓舞的初步数据，这些数据表明缺乏 在有效杀毒剂量下对红细胞(RBC)的不良影响。 它们的目的是定义获得完整的 灭活血液中极高滴度的无细胞病毒 环境。金丝桃素是一种有效的杀病毒剂，它直接 灭活大范围的包膜病毒。金丝桃素是一种 光动力分子，它在体内也是活跃的，推测是由于 红色/牛色电势低。因此，HY目前正在进行评估 艾滋病患者的临床试验，以及试验的第一阶段证实 HY的输血性。艾滋病病毒感染的双重传播问题 通过无细胞病毒和通过潜伏感染的PBMC将得到解决 通过评估HY和白细胞过滤的组合实现 完全无菌，并有可能造成光动力 病变转移到感染的PBMC。该应用程序将处理 HY的杀灭病毒作用机制。病毒中的修饰 针对光动力学的HIV p55gag、p24和rt蛋白 HY的交联性将被表征，以及其他受影响的病毒 确定了蛋白质。病毒RNA在RNA中的潜在参与- 将对蛋白质共价复合体进行评估。调查人员建议 阐明HY的作用是否与病毒基因组发生共价交叉 RNA，带有RT和其他核心蛋白。HY核糖核酸的扩增 用RNA聚合酶链式反应处理过的病毒粒子将被用来鉴定基因组 由HY交叉链接的区域。问题是HY本身还是仅仅 光动力产生的激发氧物种影响衣壳 将通过分离完整的艾滋病毒核心和检查 它们对HY和光的敏感性。这些研究具有重要的意义 对于非包膜病毒(如B19)的潜在灭活 细小病毒)在血液中传播。RT活性与衣壳蛋白迁移 将使用SDS-PAGE和Western blotts上的图案作为分析方法。 具有甲基取代和改变的结构HY类似物 将对多环芳香族骨架进行潜在的评估 提高了疗效，减少了与红细胞的结合。杀病毒的人 HY离子对与阳离子金属、碱胺和氨基的活性 酸及其与生理转运蛋白的相互作用， 将与标准HY-Na进行比较研究。潜在的 对血液成分的不良反应，包括临床意义 红细胞膜完整性参数，(2，3-DPG，溶血， ABO、Rh、少量血型抗原的正常表达及可能 免疫球蛋白或补体沉积在红细胞上)，通过HY治疗将是 检查过了。去除病毒后血液中HY的可能方法 灭活，加入疏水树脂和其他方法将 被评估。 申请组织将与以下站点合作： 1)纽约血液中心，2)俄克拉荷马血液机构，3)魏茨曼 科学研究所。

DESCRIPTION (Adapted from the applicant's abstract) This application proposes studies to evaluate the potential for development of hypericin (HY) and of numerous structurally related aromatic polycyclic diones, as virus inactivators in red cell concentrates for transfusion. The studies are based on encouraging preliminary data which show lack of adverse effects to red blood cells (RBC) at effective virucidal doses. They aim to define the optimal conditions for obtaining complete inactivation of very high titers of cell free virus in a blood environment. Hypericin is an effective virucidal agent which directly inactivates a broad range of enveloped viruses. Hypericin is a photodynamic molecule, which is also active in vivo, presumably due to a low red/ox potential. Thus, HY is currently being evaluated in clinical trials in AIDS patients, and phase I of the trials confirmed the transfusibility of HY. The problem of dual transmission of HIV infection via cell-free virus and through latently infected PBMC will be addressed by evaluating combinations of HY and leukocyte filtration in achieving complete sterility, and the possibility of inflicting photodynamic lesions to infected PBMC. The application will address aspects of the mechanism of the virucidal action of HY. The modifications in viral proteins HIV p55gag, p24 and RT, which are targeted for photodynamic cross-linking by HY will be characterized, and other affected virus proteins identified. The potential involvement of viral RNA in RNA- protein covalent complexes will be evaluated. The investigators propose clarify whether the action of HY covalently cross links viral genomic RNA, with RT and other core proteins. Amplification of RNA from HY treated virions using RNA PCR will be utilized to identify genomic regions cross linked by HY. The question whether HY itself or only photodynamically generated excited oxygen species affect the capsids will be examined by isolation of intact HIV cores and examination of their susceptibility to HY and light. These studies have implications for potential inactivation of non enveloped viruses (such as B19 parvovirus) in blood by HY. RT activity and capsid protein migration patterns on SDS-PAGE and Western blots will be used as assays. Structural HY analogs with methyl group substitutions and alterations in the polycyclic aromatic skeleton will be evaluated for potentially improved efficacies and for reduced binding to RBC. The virucidal activities of HY ion pairs with cationic metals, basic amines and amino acids, and their interactions with physiological transport proteins, will be studied in comparison with the standard HY-Na+. Potential for adverse effects to blood components including clinically significant parameters of red cell surface membrane integrity, (2,3-DPG, hemolysis, normal expression of ABO, Rh, minor blood group antigens and possible deposition of IgG or complement on RBC), by treatment with HY will be examined. Potential methods for removal of HY from blood after virus inactivation, incorporating hydrophobic resins and other methods will be evaluated. The applicant organization will collaborate with the following sites: 1) New York Blood Center, 2) Oklahoma Blood Institution, and 3) Weizmann Institute of Science.