Preclinical efficacy of the bioreductive alkylating agent RH1 against paediatric tumours

Average 5–year survival rates for childhood cancers have improved dramatically over the past few decades, such that 75% of children with cancer can now expect to be cured (Gatta et al, 2005). However, cancer remains the second most common cause of death in children between the ages of 1 and 11 years. Although the overall survival for this group of patients is excellent, there remain selected tumour types in which survival is extremely poor, and for which acquired drug resistance is a major clinical problem. For example, for children over the age of 1 with stage 4 neuroblastoma, the best reported 3-year survival is only 55%, despite intensive chemotherapy, surgery and radiotherapy (Matthay et al, 1999). For those patients who fail to achieve adequate response to induction chemotherapy, survival can be expected to be very low (Katzenstein et al, 2004). Similarly, poor survival is seen in patients with metastatic Ewing's sarcoma and osteosarcoma, despite the vast majority of patients receiving intensive multi-agent chemotherapy in international collaborative trials. Thus, an urgent need exists for novel agents for this group of paediatric cancer patients. Bioreductive agents are a promising area for anticancer drug discovery. These pro-drugs are converted to active intermediates by enzymatic activity either in hypoxic areas of solid tumours (Sartorelli, 1988) or by increased activity of these enzymes in tumour compared with normal tissues (Brown and Giaccia, 1998), and therefore should exhibit tumour-selective cytotoxicity. One- or two-electron reductases, including cytochrome p450 reductase (Bligh et al, 1990), NADH cytochrome b5 reductase (Hodnick and Sartorelli, 1993), and diphtheria toxin-diaphorase (DTD), also known as NAD(P)H:quinine oxidoreductase (NQ01) (Siegel et al, 1990; Gibson et al, 1992), can activate bioreductive drugs. The two-electron reductase DTD is particularly interesting, as its activity is elevated in a wide range of adult tumour types compared with normal tissue, and is not dependant upon tumour hypoxia (Cresteil and Jaiswal, 1991; Malkinson et al, 1992; Cummings et al, 1998). RH1 (2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone) was identified through its very high affinity for DTD (Beall et al, 1995; Winski et al, 1998). Reduction of RH1 facilitates the formation of GCC sequence-specific DNA interstrand cross-linked adducts (Berardini et al, 1993), and the induction of apoptosis within 6 h of exposure (Dehn et al, 2005). Diphtheria toxin-diaphorase activity and expression correlate with RH1-induced cytotoxicity in colon cancer and non-small-cell lung cancer cell lines in vitro (Winski et al, 1998; Sharp et al, 2000). In the NCI60 tumour cell line panel cell lines expressing NQ01 show marked sensitivity to RH1, but, in addition, a number of tumour types, particularly leukaemia and lymphoma cell lines, are sensitive to RH1 despite relatively low DTD activity (Tudor et al, 2005). RH1 is a substrate for other reductase enzymes, including cytochrome p450, although recent work suggests that this is unlikely to play a major role in its activation in cells with normal p450 activity (Begleiter et al, 2007), and that the one-electron reductase NRH:quinine oxidoreductase 2 (NQ02) may be responsible for the toxicity of RH1 in leukaemia and lymphoma cells (Yan et al, 2008). RH1 is active against human tumours in xenograft models in mice (Cummings et al, 2003; Dehn et al, 2004) where DNA cross-linking could be detected rapidly after RH1 dosing (Ward et al, 2005). Acquired tumour cell resistance to RH1 seems to be because of changes in drug uptake/efflux in line with other diazardinylbenzoquinone-resistant cell lines (T Ward, personal communication) (Ward et al, 1995). RH1 has recently completed Phase 1 study in adults in the United Kingdom and further clinical evaluation is ongoing (Danson et al, 2007a, 2007b). The dose-limiting toxicity in this UK study was myelosuppression. To date, there has been no report on the efficacy of RH1 against paediatric tumour cell lines. Here we have investigated the anti-tumour activity of RH1 against neuroblastoma, Ewing's sarcoma, and osteosarcoma cell lines in vitro and against Ewing's sarcoma and osteosarcoma xenograft models.