Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand.
Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand.
Biochem Pharmacol. 2018 Oct;156:265-280. doi: 10.1016/j.bcp.2018.08.027. Epub 2018 Aug 19.
Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug with proven efficacy against hypoxic cells in preclinical tumour models. TH-302 is designed to release the DNA crosslinking agent bromo-isophosphoramide mustard (Br-IPM) when reduced in hypoxic tissue. Br-IPM is considered to diffuse locally from hypoxic regions, eliciting additional tumour cell killing, but the latter 'bystander effect' has not been demonstrated directly. Previous studies with multicellular co-cultures that included cells expressing the E. coli nitroreductase NfsA as a model TH-302 reductase have provided clear evidence of a bystander effect (which we confirm in the present study). However, NfsA is an oxygen-insensitive two-electron reductase that is not expected to generate the nitro radical intermediate that has been demonstrated to fragment to release Br-IPM. Here, we use mass spectrometry methods to characterise TH-302 metabolites generated by one-electron reduction (steady-state radiolysis by ionising radiation and cellular metabolism under hypoxia, including HCT116 cells that overexpress P450 oxidoreductase, POR) or by NfsA expressed in HCT116 cells under oxic conditions, and investigate the stability and cytotoxicity of these products. Br-IPM is shown to have very low cytotoxic potency when added to extracellular culture medium and to be rapidly converted to other hydrophilic products including dichloro-isophosphoramide mustard (IPM). Only traces of Br-IPM or IPM were detected in the extracellular medium when generated by cellular metabolism of TH-302. We identify, in NfsA-expressing cells, the hydroxylamine metabolite of TH-302, and downstream products resulting from rearrangement or hydration of the imidazole ring, and demonstrate that formation of these candidate bystander effect mediators is suppressed by hypoxia. This characterisation of the cellular pharmacology of TH-302 implies that bystander effects from hypoxic activation of TH-302 are unlikely to contribute to its anticancer activity.
依氟鸟氨酸（TH-302）是一种临床阶段的缺氧激活前药，已在临床前肿瘤模型中证明对缺氧细胞有效。TH-302 旨在在缺氧组织中还原时释放 DNA 交联剂溴异磷酰胺 mustard（Br-IPM）。Br-IPM 被认为从缺氧区域局部扩散，引发额外的肿瘤细胞杀伤，但后者的“旁观者效应”尚未直接证明。以前使用包括表达大肠杆菌硝基还原酶 NfsA 的细胞的多细胞共培养物进行的研究为旁观者效应提供了明确的证据（我们在本研究中证实了这一点）。然而，NfsA 是一种氧不敏感的两电子还原酶，预计不会产生已证明会断裂释放 Br-IPM 的硝基自由基中间体。在这里，我们使用质谱方法来表征由单电子还原（通过电离辐射和缺氧下的细胞代谢产生的稳态辐射分解，包括过表达细胞色素 P450 氧化还原酶 POR 的 HCT116 细胞）或在有氧条件下表达 NfsA 的 HCT116 细胞生成的 TH-302 代谢物，并研究这些产物的稳定性和细胞毒性。当添加到细胞外培养基中时，Br-IPM 显示出非常低的细胞毒性效力，并且很快转化为其他亲水性产物，包括二氯异磷酰胺 mustard（IPM）。当由 TH-302 的细胞代谢生成时，在细胞外培养基中仅检测到痕量的 Br-IPM 或 IPM。我们在表达 NfsA 的细胞中鉴定了 TH-302 的羟胺代谢物，以及咪唑环重排或水合的下游产物，并证明缺氧抑制了这些候选旁观者效应介质的形成。TH-302 的细胞药理学特征表明，TH-302 缺氧激活的旁观者效应不太可能有助于其抗癌活性。
