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

描述（由申请人提供）：S-酰基-2-巯基苯甲酰胺硫酯（SAMT）抑制剂是低分子量化合物，针对 HIV 复制途径中的多个步骤，但主要功能是在接触反应性化合物后立即特异性灭活无细胞 HIV，并抑制病毒感染细胞产生感染性 HIV。这些靶向 NCp7 的病毒灭活化合物通过从感染性病毒颗粒或成熟病毒颗粒的核衣壳 (NC) 蛋白中剥离配位的锌离子来发挥作用。在此过程中，这些化合物不可逆地交联核衣壳蛋白，使病毒颗粒不具有传染性且有缺陷。因此，NCp7抑制剂干扰阴道环境中靶细胞感染所需的两种潜在病毒传播机制。在该提案的 R21 阶段，我们建议开发由聚合物前药组成的新型杀菌剂，用于传递 SAMT。这种输送机制限制了 SAMT 的组织吸收，直到它通过将其附着在高分子量生物相容性聚合物上而与精液中的病毒接种物接触。我们将通过酶可裂解的连接将 SAMT 抑制剂与聚合物载体结合，在精液中存在特定酶的情况下释放活性药物产品。这种递送方法在杀菌剂作用方面具有多种优势，因为 (1) NCp7 抑制剂可以灭活精液中的无细胞和细胞相关病毒，我们将在病毒以传染性形式扩散到或进入组织之前将其靶向，(2) 我们将在聚合物主链中添加部分，通过唐南效应降低缀合药物的局部 pH 值，从而增加 SAMT 抑制剂的稳定性，以及 (3) 因为杀菌剂将被女性多次重复使用。多年来，聚合前药方法将允许精确控制组织浓度和抗 HIV 化合物的暴露，限制产生病毒耐药性的机会并限制毒性。对于这种前药方法的开发至关重要，将进行生物学评估以确认 SAMT 在精浆和阴道液存在下的功效。此外，化合物从其前药形式的酶促活化将在专门设计的体外测定中进行评估，以模拟阴道中必须发生的事件，并量化在精液和其他适当的生物基质存在下药物活化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 向靶细胞传播效率的影响，以确定精液成分的抗病毒活性与硫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.