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）抑制剂是一种低分子量化合物，针对HIV复制途径中的多个步骤，但主要功能是在暴露于活性化合物时立即特异性灭活无细胞HIV，并抑制病毒感染细胞的感染性HIV的产生。这些ncp7靶向的病毒灭活化合物通过剥离感染性病毒粒子或成熟病毒颗粒中核衣壳（NC）蛋白上的配位锌离子起作用。在这个过程中，这些化合物不可逆地与核衣壳蛋白交联，使病毒粒子无传染性和有缺陷。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。在本建议的R21阶段，我们建议开发新的由高分子前药组成的杀微生物剂，用于递送samt。这种递送机制限制了SAMT的组织吸收，直到它通过附着在高分子量生物相容性聚合物上与精液中的病毒接种物接触。我们将通过酶-可切割键将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物。这种递送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无细胞病毒和细胞相关病毒，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，(2)我们将在聚合物主链中添加一些片段，通过Donnan效应降低偶联药物的局部pH值，从而增加SAMT抑制剂的稳定性。(3)由于女性将在多年内反复使用杀微生物剂，聚合前药方法将能够精确控制组织浓度和抗艾滋病毒化合物的暴露，从而限制病毒产生耐药性的机会并限制毒性。对于这种前药方法的发展至关重要的是，将进行生物学评估，以确认samt在精液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估化合物的酶促活性，以模拟阴道内必须发生的事件，并在精液和其他适当的生物基质存在下量化药物激活和病毒失活的动力学。最后，将评估精液和阴道液的生物学特性对艾滋病毒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.