Development of a Novel Semen-Activated Prodrug as an Anti-HIV Microbicide

说明(申请人提供)：S-酰基-2-硫代苯甲酰胺硫酯(SAMT)抑制剂是一种低分子化合物，针对艾滋病毒复制途径中的多个步骤，但主要作用是在接触到活性化合物后立即特异性地灭活无细胞艾滋病毒，并抑制病毒感染细胞产生传染性艾滋病毒。这些以NCp7为靶点的病毒灭活化合物通过从感染性病毒粒子或成熟病毒颗粒中的核衣壳(NC)蛋白中剥离配位锌离子来发挥作用。在这个过程中，这些化合物不可逆地使核衣壳蛋白交联，使病毒粒子不具感染性和缺陷。因此，NCp7抑制剂干扰了两种潜在的病毒传播机制，这两种机制是感染阴道环境中的靶细胞所必需的。在这项提议的R21阶段，我们建议开发由聚合物前药组成的新的杀微生物剂，用于SAMTS的递送。这种递送机制限制了SAMT的组织吸收，直到它与精液中的病毒接种物接触，将其附着到高分子量生物相容聚合物上。我们将通过酶可裂解的连接将SAMT抑制剂偶联到聚合物载体上，在精液中存在特定酶的情况下释放活性药物产品。这种给药方法在杀微生物剂作用的背景下提供了几个优势，因为(1)NCp7抑制剂可以灭活精液中的游离和细胞相关病毒，我们将在病毒以感染形式扩散到组织或进入组织之前将其作为靶点，(2)我们将在聚合物主链上添加部分，通过Donnan效应降低结合药物的局部pH值来增加SAMT抑制剂的稳定性，(3)由于妇女将在多年内反复使用杀菌剂，因此聚合物前药方法将能够精确控制组织浓度和抗HIV化合物的暴露，从而降低产生病毒耐药性的机会和限制毒性。对于这种前药方法的发展至关重要，将进行生物学评估，以确认SAMTS在精浆和阴道液存在的情况下的疗效。此外，将在特别设计的体外试验中评估该化合物从其前体药物形式的酶激活，以模拟必须发生在阴道中的事件，并在精液和其他适当的生物基质存在的情况下量化药物激活和病毒灭活的动力学。最后，将评估精液和阴道液的生物学特性对HIV传播到靶细胞的效率，以确定精液成分的抗病毒活性与硫酸酯抑制剂的生物活性之间的潜在协同作用。

DESCRIPTION (provided by applicant): The S-acyl-2-mercaptobenzamide thioester (SAMT) inhibitors are low molecular weight compounds which target multiple steps in the HIV replication pathway, but primarily function to specifically inactivate cell-free HIV immediately upon exposure to the reactive compounds and to suppress the production of infectious HIV from virus-infected cells. These NCp7-targeted, virus inactivating compounds act by stripping coordinated zinc ions from the nucleocapsid (NC) protein in the infectious virion or maturing virus particle. In the process, the compounds irreversibly cross-link the nucleocapsid proteins rendering the virion noninfectious and defective. Thus, the NCp7 inhibitors interfere with two potential virus transmission mechanisms required for the infection of target cells in the vaginal environment. In the R21 phase of this proposal we propose to develop new microbicides composed of polymeric prodrugs for delivery of the SAMTs. This delivery mechanism limits the tissue absorption of the SAMT until it comes in contact with the viral inoculum in semen by attaching it to a high molecular weight biocompatible polymer. We will conjugate the SAMT inhibitors to the polymer carrier through enzyme-cleavable linkages that will release the active drug product in the presence of specific enzymes in semen. This delivery approach offers several advantages in the context of microbicide action since (1) the NCp7 inhibitors can inactivate cell-free and cell-associated virus in semen, we will target the virus before it can diffuse in an infectious form to or into tissue, (2) we will add moieties to the polymer backbone that will increase the stability of the SAMT inhibitors by decreasing the pH local to the conjugated drug by the Donnan effect, and (3) since microbicides will be used by women repeatedly over many years, a polymeric prodrug approach will allow precise control over the tissue concentrations and exposure to anti-HIV compounds, limiting the chance to develop viral resistance and limiting toxicity. Critical to the development of this prodrug approach, biological evaluations will be performed to confirm the efficacy of the SAMTs in the presence of seminal plasma and vaginal fluids. Additionally, the enzymatic activation of the compound from its prodrug form will be evaluated in specially designed in vitro assays to mimic the events which must occur in the vagina and to quantify the kinetics of drug activation and virus inactivation in the presence of semen and other appropriate biological matrices. Finally, the biological properties of both semen and vaginal fluids on the efficiency of transmission of HIV to target cells will be evaluated to define the potential synergies between the antiviral activity of constituents of semen and the biological activity of the thioester inhibitors.