PR-104 in patients with solid tumours was three.4- to 9.6-fold reduce than may be accomplished in pre-clinical murine models, as defined by total plasma exposure (AUCfree ) to unbound mGluR custom synthesis prodrug PR-104A (Figure 1) [22,25]. This toxicokinetic disconnect is unusual for alkylating agents which ordinarily scale within a predictable 1:1 fashion involving murine and human subjects [53]. The hypersensitivity of human bone marrow progenitor cells to aerobic PR-104A exposure in vitro (Figure 2) indicates a powerful causal link together with the clinical myelotoxicity observations. PR-104A is designed to stay inert below normoxia; aerobic cytotoxicity thus discloses the presence of off-target metabolic activity. A crucial endogenous catalytic function of AKR1C3 is as a prostaglandin D2 11-ketoreductase (prostaglandin F synthase) which regulates maturation of CD34+ myeloid progenitor cells [17,26]. AKR1C3 also acts as a unique aerobic nitro reductase which will bioactivate PR-104A under oxygenated situations [16]. This overlapping functionality in early lineage bone marrow cells is as a result thought to become a major supply in the grade 3/4 myelotoxicity reported at low doses of PR-104 in clinical trials. According to this proof, we synthesised and characterised an analogue of PR-104 (SN35141, Scheme 1, Figure 3A) that is resistant to activation by AKR1C3, thereby reinstating the original style idea of selective activation below hypoxia. In vitro metabolism and 2D (low-density) cytotoxicity assays confirmed that MGMT list SN29176 is just not a substrate for two-electron reduction by human AKR1C3 (Figure 3B ). The scale of this distinction was most apparent when employing high-cell-density in vitro 3D MCL models (Figure 3E) and in vivo tumour models (Figure 6C,D), reflecting the tendency from the lipophilic metabolites to redistribute locally (bystander impact). Given that it was confirmed that SN29176 just isn’t a substrate for AKR1C3, murine tolerance to the pre-prodrug SN35141 should really, in principle, greater predict the exposures achievable in human subjects [53]. Critically, the hypoxia-selective properties of SN29176 stay intact with HCR values ranging from 9 to 145 (Figure 4B), indicating the 2-nitro, 4-methylsulfone design functions as intended. To confirm the partnership amongst one-electron reduction with the prodrug and resulting DNA harm, we compared the formation of H2AX foci in prodrug-exposed WT and POR-expressing HCT116 cells under anoxia, with or without having the flavoenzyme inhibitor DPI. Both PR-104A and SN29176 exposure amplified the DNA damage response inside a POR-dependent manner, a phenomenon prevented by prior DPI exposure (Figure 4C). Additional, comparable accumulation in G2/M, indicative of stalled DNA replication forks (Figure 4D), as previously observed for PR-104A [33], suggest a conserved mechanism of action for PR-104A and SN29176 under hypoxia. This conserved hypoxia-selective activity can also be observed in vivo, with SN35141 treatment providing greater sterilisation of radiation-resistant hypoxic tumour cells relative towards the international tumour cell population (Figure 5C), an effect which was amplified by POR expression. Right here, for example, a modest 0.five log cell kill with single-agent SN35141 was magnified to two.2 log cell kill post radiation (Figure 5C), with efficacy exceeding that of PR-104 or tirapazamine in the HCT116 sPOR#6 tumour model setting. Within a second tumour model, SiHa, the therapeutic activity of SN35141 post radiation was too excellent to detect any surviving colonies from five of ten tumou
