Microbicide properties of RT inhibitor combinations

描述（由申请人提供）：局部杀微生物剂是减少HIV异性传播的重要策略。几种单药杀微生物剂正在临床试验中，包括一种基于非核苷类逆转录酶抑制剂（NNRTI） UC781的杀微生物剂，我们发现它是一种潜在的杀微生物剂。然而，联合杀微生物剂可能更可取，但目前只有一种联合杀微生物剂正在评估中。还迫切需要确定新的管道杀微生物剂。我们发现核苷类RT抑制剂（NRTI） 4'-乙基-2-氟脱氧腺苷（4' e - 2fda）在随后缺乏外源性药物的情况下，对HIV- 1感染细胞提供了有效和持久的屏障，这一特性以前只在UC781等NNRTI中发现。4'E-2FdA赋予“记忆效应”屏障的药物水平比核苷酸替诺福韦所需的保护水平低几个数量级，目前正在临床评估杀菌剂的使用。我们假设杀菌剂包含不同类别的高效RT抑制剂的组合，即NNRTI UC781和NRTI如4'E-2FdA，将提供HIV-1传播的最佳屏障。因此，我们提出了R21/R33阶段创新应用的这些具体目标：R21目标1。评价NRTI与UC781单独及联合使用的体外（基于细胞的）杀微生物性能。这些研究包括评估NRTIs和UC781单独或联合使用原代细胞（PBMCs、CD4+ t细胞、巨噬细胞）和不同的HIV药物敏感和耐药菌株、分离株和分支的抗病毒活性和“记忆效应”保护。R21目标2。目的：阐明4e - 2fda（及其类似物）在HIV易感细胞中诱导保护性屏障或“记忆效应”的机制。这些研究包括NRTI- tp的摄取、转化为三磷酸和细胞内稳定性的表征，以及NRTI代谢酶对NRTI底物活性的详细动力学评估。R21交付成果：鉴定一种先导NRTI和两种后备NRTI，用于与UC781联合开发杀菌剂。R33目标1；将R21期选择的NRTI/NNRTI组合配制成适合阴道局部使用的给药系统。nrti和nnrti具有不同的化学性质，因此必须确定适当的输送系统，以使活性剂的结合和释放成为可能。我们将制备和评价凝胶和快速溶解膜制剂的联合杀菌剂。2.目标；目的：在离体宫颈移植组织模型中评价复方NRTI/NNRTI组合抗hiv杀微生物活性。这些研究将使用新开发的生理学相关极化宫颈组织模型来评估配方杀菌剂单独使用和联合使用对艾滋病毒传播和传染性的影响。R33交付物：选定NRTI/NNRTI组合的适当给药配方，以进入后续临床前安全性和有效性研究。

DESCRIPTION (provided by applicant): Topical microbicides are an important strategy to minimize heterosexual transmission of HIV. Several single agent microbicides are in clinical trials, including one based on the nonnucleoside reverse transcriptase inhibitor (NNRTI) UC781 that we discovered as a potential microbicidal agent. However, combination microbicides may be preferable, yet only a single combination microbicide is currently under evaluation. There is also an urgent need to identify new pipeline microbicidal agents. We have found that the nucleoside RT inhibitor (NRTI) 4'-ethynyl-2-fluoro-deoxyadenosine (4'E-2FdA) provides a potent and prolonged barrier to HIV- 1 infection of cells in the subsequent absence of exogenous drug, a property previously only noted for NNRTI such as UC781. The "memory effect" barrier is imparted by 4'E-2FdA at drug levels orders of magnitude less than those needed for protection by the nucleotide tenofovir, currently in clinical assessment for microbicide use. We hypothesize that microbicides comprising combinations of different classes of highly potent RT inhibitors, namely the NNRTI UC781 and an NRTI such as 4'E-2FdA, will provide an optimal barrier to HIV-1 transmission. We therefore propose these Specific Aims for this R21/R33 phased innovation application: R21 Aim 1. To evaluate the in vitro (cell-based) microbicidal properties of NRTI and UC781 alone and in combination. These studies include assessment of antiviral activity and "memory effect" protection imparted by NRTIs and UC781 alone and in combination using primary cells (PBMCs, CD4+ T-cells, macrophages) and different HIV drug-sensitive and drug-resistant strains, isolates and clades. R21 Aim 2. To elucidate the mechanism of 4'E-2FdA (and analogs) induced protective barrier or "memory effect" in HIV susceptible cells. These studies include characterization of uptake, conversion to triphosphate and intracellular stability of the NRTI-TPs, as well as detailed kinetic evaluations of the NRTI substrate activity with enzymes involved in metabolism of the NRTIs. R21 Deliverables: Identification of a lead NRTI and two back-ups for use with UC781 for development as a combination microbicide. R33 Aim 1. To formulate the NRTI/NNRTI combinations selected in the R21 phase into an appropriate delivery system for vaginal topical use. NRTIs and NNRTIs have different chemical properties, thus appropriate delivery systems must be identified to enable incorporation and release of the active agents. We will prepare and evaluate both gel and rapidly dissolving film formulations for the combination microbicide. R33 Aim 2. To evaluate the anti-HIV microbicidal activity of formulated NRTI/NNRTI combinations in an ex vivo cervical explant tissue model. These studies will use a newly developed physiologically relevant polarized cervical tissue model to assess the impact of formulated microbicides alone and in combination on HIV transmission and infectivity. R33 Deliverables: Identification of an appropriate delivery formulation for the selected NRTI/NNRTI combination for entry into subsequent preclinical safety and efficacy studies.