2-5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1-4-benzoquinone and Lung-Neoplasms

The diaziridiny/benzoquinone RH1 is shortly to enter a phase I clinical trial. The drug was originally designed as a substrate for the enzyme DT-diaphorase (DTD) such that metabolic activation of the drug would lead to toxicity. To evaluate this, we have measured the toxicity of RH1 in a pair of non-small cell lung cancer (NSCLC) cell lines of widely differing levels of DTD and in MDA231 breast cancer cells which have been engineered to overexpress DTD. In addition, we have explored the importance of the putative one-electron reductase, P450 reductase, by assessing the toxicity of RH1 in MDA231 cells engineered to overexpress the enzyme. All drug exposures were carried out under hypoxic and aerobic conditions. Those cells with the highest levels of DTD, i.e. D7 versus MDA231 wt and H460 versus H596, are substantially more sensitive to RH1 than the cell lines expressing low DTD activity. Those cells with the lowest levels of DTD activity, i.e. MDA231 wt, R4 and H596, show much greater sensitivity to RH1 under hypoxic conditions compared to aerobic conditions. Finally, overexpression of P450 reductase, i.e. comparing MDA231 wt with R4, has little, if any, impact on the toxicity of RH1 under hypoxic or aerobic conditions. In summary, RH1 can be effective in killing cells containing high levels of DTD and may be useful in treating tumors expressing this enzyme.

Topics: Aerobiosis; Antineoplastic Agents; Aziridines; Benzoquinones; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Colony-Forming Units Assay; Humans; Inhibitory Concentration 50; Lung Neoplasms; NAD(P)H Dehydrogenase (Quinone); NADPH-Ferrihemoprotein Reductase; Spectrophotometry; Tirapazamine; Transfection; Triazines

RH1 (2,5-diaziridinyl-3-(hydroxylmethyl)-6-methyl-1,4-benzoquinone) has shown preferential activity against human tumour cell lines which express high levels of DTD (EC 1.6.99.2; NAD(P)H:quinone oxidoreductase, NQO1, DT-diaphorase) and is a candidate for clinical trials. EO9 (3-hydroxy-5-aziridinyl-1-methyl-2-[1H indole-4,7-dione]prop-beta-en-alpha-ol) is a known substrate for DTD but clinical trials were disappointing, as a result of rapid plasma clearance and reversible dose-limiting kidney toxicity. It is an obvious concern that RH1 does not exhibit the same limitations. We therefore describe the antitumour activity and pharmacology of RH1 in mice and compare its pharmacological characteristics to those of EO9. Significant antitumour activity (P = 0.01) was seen for RH1 (0.5 mg/kg, i.p.) against the high DTD-expressing H460 human lung carcinoma. Pharmacokinetic analysis of RH1 in mice showed a t1/2 of 23 min with an area under the curve of 43.0 ng hr mL(-1) resulting in a calculated clearance of 5.1 mL min(-1), 10-fold slower than EO9. RH1 was also more stable than EO9 in murine blood, where the breakdown was thought to be DTD-related. NADH-dependent microsomal metabolism of RH1 and EO9 in both liver and kidney was slow (<100 pmol/min/g tissue), reflecting the low microsomal DTD expression (<35 nmol/mg/min). Liver cytosol metabolism was rapid for both compounds (>4500 pmol/min/g tissue), although DTD levels were low (21.4+/-0.6 nmol/mg/min). DTD activity in the kidney cytosol was high (125+/-8.2 nmol/mg/min) and EO9 was rapidly metabolised (4396+/-1678 pmol/min/g), but the metabolic rate for RH1 was 7-fold slower (608+/-86 pmol/min/g), even though RH1 was shown to be an excellent substrate for DTD (Vmax = 800 micromol/min/mg and a Km of 11.8 microM). The two DTD substrates RH1 and EO9 are clearly metabolised differently, suggesting that RH1 may have different pharmacological properties to those of EO9 in the clinic.

Topics: Animals; Antineoplastic Agents; Aziridines; Benzoquinones; Drug Screening Assays, Antitumor; Drug Stability; Humans; Indolequinones; Indoles; Kinetics; Lung Neoplasms; Mice; NAD(P)H Dehydrogenase (Quinone); Neoplasm Transplantation