Pharmacological properties of a new aziridinylbenzoquinone, RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), in mice

doi:10.1016/S0006-2952(99)00391-3

Biochemical Pharmacology

Volume 59, Issue 7, 1 April 2000, Pages 831-837

https://doi.org/10.1016/S0006-2952(99)00391-3 Get rights and content

Abstract

RH1(2,5-diaziridinyl-3-(hydroxymethyl)-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-β-en-α-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 t_1/2 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 (V_max = 800 μmol/min/mg and a K_m of 11.8 μM). 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.

Section snippets

Antitumour activity

All animal experiments were carried out under a project licence approved by the Home Office, London, U.K., and UK CCCR guidelines [20] were followed throughout. The H460 is a high diaphorase-expressing lung carcinoma [21] and was grown subcutaneously as a xenograft in NCR-Nu mice of approximately 25 g (supplied by the NCI). H460 tumours were transplanted as fragments by trocar. Groups of 5–10 tumour-bearing mice were treated with RH1 at the previously established single i.v. maximum tolerated

Antitumour activity

Figure 2 shows the in vivo antitumour activity of RH1 against the high DTD-expressing H460 tumour. There was a significant (P = 0.01) growth delay of 2.5 days for the tumour doubling time following i.p. administration of RH1 at a dose of 0.5 mg/kg.

Chromatography

Chromatographic separation was good using the system described with no interfering substances apparent in the control (drug-free) samples tested, which included blood, plasma, liver, and kidney extracts. Calibration curves were linear over the range

Discussion

RH1 is a novel aziridinylbenzoquinone that was synthesised as a substrate for DTD and is shortly to enter clinical trial. It is unusually potent and we describe here the first report of its significant antitumour activity against the high DTD-expressing H460 human lung carcinoma. This study has also investigated several of the factors that are likely to influence the pharmacokinetics of RH1 and compared these to the other DTD substrate EO9. It has been acknowledged for some time that the

Acknowledgements

This work was supported by War on Cancer, Bradford, U.K. We would also like to acknowledge the helpful contribution of Dr. John Butler, Paterson Institute for Cancer Research, Manchester, U.K., to this study. Dr. Butler also provided the HPLC conditions used. EO9 was a gift from the Screening and Pharmacology Group of the EORTC.

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Unlike mitomycin C, RH1 quinone reduction products are more stable, less likely to generate toxic reactive oxygen species and less likely to be influenced by hypoxia than mitomycin C [16]. In keeping with RH1 being an excellent substrate for DTD, it is significantly more cytotoxic in vitro in cells that overexpress DTD, naturally or through transfection with NQO1, than in cells with low DTD [13, 17–20]. Preclinical in vivo xenograft testing demonstrated significant activity in NSCLC, breast, colorectal and ovarian xenograft models [12, 19].
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Eighteen patients of World Health Organization performance status of zero to one with advanced refractory solid malignancies were enrolled. MTD was 1430 μg/m²/day with reversible bone marrow suppression being dose limiting. Plasma pharmacokinetic analysis showed RH1 is rapidly cleared from blood (t_1/2 = 12.3 min), with AUC increasing proportionately with dose. The comet-X assay demonstrated dose-related increases in DNA cross linking in PBMCs. DNA cross linking was demonstrated in tumours, even with low levels of DTD. Only one patient was homozygous for NQO1 polymorphism precluding any conclusion of its effect.
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Chemotherapy and Metabolic InhibitorsPharmacological properties of a new aziridinylbenzoquinone, RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), in mice

Abstract

Section snippets

Antitumour activity

Antitumour activity

Chromatography

Discussion

Acknowledgements

Biochem Pharmacol

Eur J Cancer

Eur J Cancer

Ann Oncol

Eur J Cancer

Int J Radiat Oncol Biol Phys

Eur J Cancer

Biochem Pharmacol

Anal Biochem

Biochem Pharmacol

J Chromatogr B Biomed Sci App

The unique physiology of solid tumorsOpportunities (and problems) for cancer therapy

Cancer Res

DT-diaphorase; A historical review

Chem Scripta

Cytosolic NAD(P)H:(quinone-acceptor)oxidoreductase in human normal and tumor tissueEffects of cigarette smoking and alcohol

Int J Cancer

Chemotherapy and Metabolic Inhibitors
Pharmacological properties of a new aziridinylbenzoquinone, RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), in mice