2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT

Abstract

An important feature of gene-directed enzyme-prodrug therapy is that prodrug activation can provide diffusible cytotoxic metabolites capable of generating a local bystander effect in tumours. Activation of the aziridinyl dinitrobenzamide CB 1954 by E. coli nitroreductase (NTR) provides a bystander effect assumed to be due to the potently cytotoxic 4-hydroxylamine metabolite. We show that there are four cytotoxic extracellular metabolites of CB 1954 in cultures of NTR-expressing tumour cells (the 2-and 4-hydroxylamines and their corresponding amines). The 4-hydroxylamine is the most cytotoxic in DNA crosslink repair defective cells, but the 2-amino derivative (CB 10-236) is of similar potency to the 4-hydroxylamine in human tumour cell lines. Importantly, CB 10-236 has much superior diffusion properties to the 4-hydroxylamine in multicellular layers grown from the SiHa human cervical carcinoma cell line. These results suggest that the 2-amine, not the 4-hydroxylamine, is the major bystander metabolite when CB 1954 is activated by NTR in tumours. The corresponding dinitrobenzamide nitrogen mustard SN 23862 is reduced by NTR to form a single extracellular metabolite (also the 2-amine), which has superior cytotoxic potency and diffusion properties to the CB 1954 metabolites. These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts.

The nitroreductase prodrug SN 28343 enhances the potency of systemically administered armed oncolytic adenovirus ONYX-411NTR

“…57,58 However, CB 1954 has poor formulation characteristics 56 that have not been overcome by analog development 59,60 and a limited bystander effect in 3D cell culture models and xenografts. 46,61,62 Of critical importance, CB 1954 provides disappointing plasma concentrations in humans (5.8 mmol h l À1 ) 56 relative to mice (320 mmol h l À1 ) 63 because of dose-limiting hepatotoxicity and diarrhea. 64 Thus, there is a clear requirement for improved NTR prodrugs which has spurred efforts in this area.…”

Targeted cell‐ablation in Xenopus embryos using the conditional, toxic viral protein M2(H37A)

“…With this approach, the nitroreductase enzyme is expressed in the target tissue and its activity is used to convert an added nontoxic prodrug into a toxic agent that will kill cells. The principal limitation with this system is the issue of subsequent diffusion of the toxic metabolites into neighboring cells (Helsby et al, 2004), but this has apparently been solved using a new prodrug, metronidazole. The new studies have produced targeted cell-ablations within the pancreas, liver, and the heart (Curado et al, 2007;Pisharath et al, 2007), although their cardiac cell "damage" phenotype does not appear as extensive as we report here.…”

Studies relating to the synthesis, enzymatic reduction and cytotoxicity of a series of nitroaromatic prodrugs

“…[9][10][11][12][13][14][15][16][17] The reduction of the 2-nitro-group in CB 1954 1 also occurred in the presence of E. coli NR resulting in the ultimate formation of amine derivative 4, a monofunctional alkylating agent which exhibited a significant bystander effect. 18 Analogues of CB 1954 1 have also been prepared and studied as potential cytotoxic agents 19 as have the structurally related nitrogen-mustard derivatives SN 23862 5 and its analogues. [20][21][22][23][24][25][26] The 2-nitro-group in SN 23862 5 is reduced by E. coli NR producing the amine derivative 6 thus facilitating the formation of an aziridinium species 7 from the mustard moiety.…”

“…Compounds 8a-8e (in which R 2 = NO2) would not be expected to produce bifunctional alkylating species, but their corresponding amines (if formed), may exhibit a bystander effect similar to amine 4. 18 Compounds 8a-e were therefore prepared from racemic 1,2,3,4-tetrahydroisoquinoline 10 as outlined in Scheme 1 (see supplementary information for experimental details). Thus, compound 10 was reacted with an appropriate arylfluoride in warm DMSO solution in the presence of K2CO3 yielding, after acidification, the arylated carboxylic acid derivatives 11a-d.…”