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，靶向HIV复制途径中的多个步骤，但主要作用是在暴露于反应性化合物后立即特异性抑制无细胞HIV，并抑制病毒感染细胞产生感染性HIV。这些靶向NCp 7的病毒灭活化合物通过从感染性病毒体或成熟病毒颗粒中的核衣壳（NC）蛋白中剥离配位的锌离子来起作用。在此过程中，化合物不可逆地交联核衣壳蛋白，使病毒粒子无感染性和缺陷。因此，NCp 7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，我们建议开发由用于递送SAMT的聚合物前药组成的新杀微生物剂。这种递送机制限制了SAMT的组织吸收，直到它通过将其附着到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。这种递送方法在杀微生物剂作用的背景下提供了几个优点，因为（1）NCp 7抑制剂可以抑制精液中的无细胞和细胞相关病毒，我们将在病毒以感染性形式扩散到组织或进入组织之前靶向病毒，（二）我们将向聚合物主链添加部分，其将通过降低缀合药物局部的pH来增加SAMT抑制剂的稳定性，Donnan效应，和（3）由于杀微生物剂将被妇女重复使用多年，聚合物前药方法将允许精确控制组织浓度和暴露于抗HIV化合物，限制发展病毒抗性的机会和限制毒性。对于这种前药方法的开发至关重要的是，将进行生物学评价以确认SAMT在精浆和阴道液存在下的功效。此外，将在专门设计的体外试验中评价化合物从其前药形式的酶促活化，以模拟阴道中必须发生的事件，并定量精液和其他适当生物基质存在下药物活化和病毒灭活的动力学。最后，将评价精液和阴道液对HIV向靶细胞传播效率的生物学特性，以确定精液成分的抗病毒活性与硫酯抑制剂的生物学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.