Abstract
PR-104A is a dual hypoxia/nitroreductase gene therapy prodrug by virtue of its ability to undergo either one- or two-electron reduction to its cytotoxic species. It has been evaluated extensively in pre-clinical GDEPT studies, yet off-target human aldo-keto reductase AKR1C3-mediated activation has limited its use. Re-evaluation of this chemical scaffold has previously identified SN29176 as an improved hypoxia-activated prodrug analogue of PR-104A that is free from AKR1C3 activation. However, optimization of the bystander effect of SN29176 is required for use in a GDEPT setting to compensate for the non-uniform distribution of therapeutic gene transfer that is often observed with current gene therapy vectors. A lipophilic series of eight analogues were synthesized from commercially available 3,4-difluorobenzaldehyde. Calculated octanol-water partition coefficients (LogD7.4) spanned > 2 orders of magnitude. 2D anti-proliferative and 3D multicellular layer assays were performed using isogenic HCT116 cells expressing E. coli NfsA nitroreductase (NfsA_Ec) or AKR1C3 to determine enzyme activity and measure bystander effect. A variation in potency for NfsA_Ec was observed, while all prodrugs appeared AKR1C3-resistant by 2D assay. However, 3D assays indicated that increasing prodrug lipophilicity correlated with increased AKR1C3 activation and NfsA_Ec activity, suggesting that metabolite loss from the cell of origin into media during 2D monolayer assays could mask cytotoxicity. Three prodrugs were identified as bono fide AKR1C3-negative candidates whilst maintaining activity with NfsA_Ec. These were converted to their phosphate ester pre-prodrugs before being taken forward into in vivo therapeutic efficacy studies. Ultimately, 2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyl dihydrogen phosphate possessed a significant 156% improvement in median survival in mixed NfsA_Ec/WT tumors compared to untreated controls (p = 0.005), whilst still maintaining hypoxia selectivity comparable to PR-104A.
Highlights
Cancer gene therapy involves the transfer of foreign genetic material into target cells for therapeutic benefit
In a gene-directed enzyme prodrug therapy (GDEPT) setting, heterogeneous gene therapy vector distribution and/or constrained vector geometry necessitates the optimization of prodrug bystander activity
Having previously identified compound 1 as a hypoxia-activated prodrug that was free from off-mechanism activation by human aldo-keto reductase 1C3 (AKR1C3) [22], we reasoned this was an ideal scaffold to utilize for the generation of a series of lipophilic analogues
Summary
Cancer gene therapy involves the transfer of foreign genetic material into target cells for therapeutic benefit. In the case of gene-directed enzyme prodrug therapy (GDEPT), a transcriptional unit encoding an exogenous enzyme is delivered to cells, expression of which is able to sensitize transfected tumor cells to an otherwise inert prodrug [1,2]. Depending on the tissue penetration capacity of the active metabolite(s), cytotoxic products can diffuse into and kill metabolically naïve neighboring cells, a phenomenon termed the ‘bystander effect’ [3,4]. A major advantage to this class of compound is the stability of the cytotoxic products following reduction; the active metabolites have an appreciable half-life and are able to diffuse out of the cell of origin, resulting in bystander kill of neighboring cells
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