Preclinical efficacy of the bioreductive alkylating agent RH1 against paediatric tumours

Abstract

Background: Despite substantial improvements in childhood cancer survival, drug resistance remains problematic for several paediatric tumour types. The urgent need to access novel agents to treat drug-resistant disease should be expedited by pre-clinical evaluation of paediatric tumour models during the early stages of drug development in adult cancer patients.

Methods/results: The novel cytotoxic RH1 (2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone) is activated by the obligate two-electron reductase DT-diaphorase (DTD, widely expressed in adult tumour cells) to a potent DNA interstrand cross-linker. In acute viability assays against neuroblastoma, osteosarcoma, and Ewing's sarcoma cell lines RH1 IC(50) values ranged from 1-200 nM and drug potency correlated both with DTD levels and drug-induced apoptosis. However, synergy between RH1 and cisplatin or doxorubicin was only seen in low DTD expressing cell lines. In clonogenic assays RH1 IC(50) values ranged from 1.5-7.5 nM and drug potency did not correlate with DTD level. In A673 Ewing's sarcoma and 791T osteosarcoma tumour xenografts in mice RH1 induced apoptosis 24 h after a single bolus injection (0.4 mg/kg) and daily dosing for 5 days delayed tumour growth relative to control.

Conclusion: The demonstration of RH1 efficacy against paediatric tumour cell lines, which was performed concurrently with the adult Phase 1 Trial, suggests that this agent may have clinical usefulness in childhood cancer.

Figure 1

Cytotoxicity of RH1 against paediatric tumour cell lines in vitro. (A) Immunoblot showing variation in expression of DTD in paediatric tumour cell lines (top), activity of DTD is shown beneath, expression of NQ02 in the same cell lines (bottom). GAPDH is shown as a loading control. (B) Clonogenic response of paediatric tumour cell lines to RH1 in comparison with cisplatin and doxorubicin. (C) SRB response of paediatric tumour cell lines to RH1 in comparison with cisplatin and doxorubicin. (D) Comparison of IC₅₀ values in paediatric tumour cell lines for RH1, cisplatin and doxorubicin for clonogenic and SRB assays.

Figure 2

Differential induction of apoptosis by RH1 in paediatric tumour cell lines. (A) Differences in SRB IC₅₀ values for RH1 between 791T and U2OS osteosarcoma cell lines and LA1-5S and LA1-55n neuroblastoma cell lines are reflected in differences in the induction of apoptosis as measured by nuclear morphology (bar charts) or cleavage of PARP (immunoblots). (B) Similar induction of apoptosis between the Ewing's sarcoma cell lines A673 and RDES reflects the lack of difference in SRB IC₅₀ values between these two lines, whether measured by nuclear morphology (bar charts) or cleaved PARP (immunoblots).

Figure 3

The combination index (CI) values for combinations of RH1 and cytotoxic agents in paediatric tumour cell lines. (A) Combination index values for RH1 with cisplatin. (B) Combination index values for RH1 with doxorubicin. CI values of 1 indicate additivity, below 1 indicates synergy, and above 1 indicates antagonism. The lower the CI value the greater the degree of synergism.

Figure 4

Induction of apoptosis in paediatric tumour xenografts. (A) Analysis of cells staining for cleaved caspase 3 in A673 tumour xenografts in nude mice 24 h after a single intraperitoneal dose of RH1. (B) Analysis of cells staining for cleaved caspase 3 in 791T xenografts in nude mice 24 h after a single intraperitoneal dose of RH1.

Figure 5

Inhibition of growth of paediatric tumour xenografts by RH1. (A) Mean slopes of tumour growth for individual mice bearing A673 xenografts. Vertical lines represent the group mean. (B) Mean slopes of tumour growth for individual mice bearing 791T xenografts. Vertical lines represent the group means. (C) A forest plot of the comparison between group means for the two experiments, P=0.057 for the pooled comparison between RH1 treated and control groups.