tirapazamine and 3-amino-1-2-4-benzotriazine

Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a heterocyclic di-N-oxide that undergoes enzymatic deoxygenation selectively in the oxygen-poor (hypoxic) cells found in solid tumors to generate a mono-N-oxide metabolite. This work explored the idea that the electronic changes resulting from the metabolic deoxygenation of tirapazamine analogues might be exploited to activate a DNA-alkylating species selectively in hypoxic tissue. Toward this end, tirapazamine analogues bearing nitrogen mustard units were prepared. In the case of the tirapazamine analogue 18a bearing a nitrogen mustard unit at the 6-position, it was found that removal of the 4-oxide from the parent di-N-oxide to generate the mono-N-oxide analogue 17a did indeed cause a substantial increase in reactivity of the mustard unit, as measured by hydrolysis rates and DNA-alkylation yields. Hammett sigma values were measured to quantitatively assess the magnitude of the electronic changes induced by metabolic deoxygenation of the 3-amino-1,2,4-benzotriazine 1,4-dioxide heterocycle. The results provide evidence that the 1,2,4-benzotiazine 1,4-dioxide unit can serve as an oxygen-sensing prodrug platform for the selective unmasking of bioactive agents in hypoxic cells.

Topics: Alkylating Agents; Antineoplastic Agents; Cyclic N-Oxides; DNA Damage; Gas Chromatography-Mass Spectrometry; Hypoxia; Mechlorethamine; Molecular Structure; Oxidation-Reduction; Prodrugs; Tirapazamine; Triazines

An on-line microdialysis microbore HPLC method is described for the determination of the bioreductive anti-tumor agent, tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide, SR4233, WIN59075, Tirazone, TPZ) and its two major reduced metabolites, 3-amino-1,2,4-benzotriazine-1-N-oxide (SR4317) and 3-amino-1,2,4-benzotriazine (SR4330). Detection limits of 0.003 microM, 0.005 microM and 0.007 microM were obtained for tirapazamine, SR4317 and SR4330, respectively. Linear ranges of 0.011-20 microM, 0.017-20 microM and 0.025-20 microM for tirapazamine, SR4317 and SR4330 permitted quantitative analysis of all three compounds in microdialysis samples. Typical intra-day reproducibilities (n = 7) of 4.1% (tirapazamine), 6.6% (SR4317), 9.9% (SR4317), and 1.8% (tirapazamine), 2.4% (SR4317) and 2.6% (SR4330) were obtained at the 0.12 microM and 1.2 microM levels, respectively. Inter-day reproducibilities (n = 5) of 3.4% (tirapazamine), 1.8% (SR4317), 4.5% (SR4330) and 2.5% (tirapazamine), 2.5% (SR4317) and 1.7% (SR4330) were obtained at the 0.12 microM and 1.2 microM levels, respectively. The use of an on-line microdialysis HPLC system, permitted the determination of tirapazamine, SR4317 and SR4330 in blood and muscle tissue of rats with a high temporal resolution of sampling. The pharmacokinetics of tirapazamine and its metabolites were studied in the muscle and blood of rats previously administered an intraperitoneal dose of tirapazamine.

Topics: Animals; Antineoplastic Agents; Chromatography, High Pressure Liquid; Electronic Data Processing; Male; Microdialysis; Rats; Rats, Sprague-Dawley; Tirapazamine; Triazines

P450 reductase (NADPH:cytochrome P450 reductase, EC 1.6.2.4) is known to be important in the reductive activation of the benzotriazene-di-N-oxide tirapazamine (SR 4233). Using a panel of six human breast adenocarcinoma cell lines we have examined the relationship between P450 reductase activity and sensitivity to tirapazamine. The toxicity of tirapazamine was found to correlate strongly with P450 reductase activity following an acute (3 h) exposure under hypoxic conditions, the drug being most toxic in the cell lines with the highest P450 reductase activity. A similar correlation was also observed following a chronic (96 h) exposure to the drug in air but not following acute (3 h) exposure in air. We have also determined the ability of lysates prepared from the cell lines to metabolise tirapazamine to its two-electron reduced product, SR 4317, under hypoxic conditions using NADPH as an electron donor. The rate of SR 4317 formation was found to correlate both with P450 reductase activity and with sensitivity to tirapazamine, the highest rates of SR 4317 formation being associated with the highest levels of P450 reductase activity and the greatest sensitivity to the drug. These findings indicate a major role for P450 reductase in determining the hypoxic toxicity of tirapazamine in breast tumour cell lines.

Topics: Antineoplastic Agents; Biotransformation; Breast Neoplasms; Cell Hypoxia; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; Humans; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Oxidoreductases; Oxygen; Prodrugs; Tirapazamine; Triazines; Tumor Cells, Cultured

A sensitive solid-phase-extraction and high-performance liquid chromatography (HPLC) method has been developed to investigate the pharmacokinetics and metabolism of the hypoxic-cell cytotoxic agent tirapazamine (1,2,4-benzotriazine-3-amine 1,4-di-N-oxide; WIN 59075, SR 4233), currently in phase I/II studies in the United Kingdom and United States. A sample extraction and concentration process was devised using strong cation-exchange Bond Elut cartridges. Tirapazamine, the mono and zero-N-oxide metabolites (WIN 64012, WIN 60109) were isocratically resolved using a microBondapak phenyl HPLC column and measured using photodiode-array detection. The minimal quantifiable level (MQL) of tirapazamine was 40 ng/ml in mouse plasma and 20 ng/ml in human plasma. Recovery was consistently greater than 80% for all compounds over the concentration range of 20 ng/ml to 20 micrograms/ml. No significant decomposition was observed following up to three freeze/thaw cycles and storage at -70 degrees C for 52 days. The assay was accurate and reproducible, with measured values lying within the limits of defined acceptance criteria. Additional studies to investigate the degree of plasma protein binding showed that tirapazamine did not bind extensively to plasma proteins (binding, 9.7% +/- 0.1% and 18.7% +/- 1.3% in mouse and human plasma, respectively). These small species differences in protein binding are unlikely to have any major impact on the extrapolation of pharmacokinetic data from mice to humans. The assay has now been successfully applied to investigate the pharmacokinetics and metabolism of tirapazamine in mice and patients as part of a pharmacokinetically guided dose-escalation strategy for phase I clinical trials.

Topics: Animals; Antineoplastic Agents; Biotransformation; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Drug Stability; Humans; Mice; Reproducibility of Results; Sensitivity and Specificity; Tirapazamine; Triazines

3-Amino-1,2,4-benzotriazine-2,4-di-N-oxide (SR4233) is a promising new antineoplastic agent based on reductive activation. SR4233 and its major metabolites (SR4317 and SR4330) are all easily reduced at a carbon electrode. Reductive amperometric detection can therefore provide high selectivity and low detection limits with chromatographic analysis and is an ideal approach to detection of SR4233 in microdialysis samples. However, in order to use amperometric detection in the reductive mode, sample deoxygenation is necessary. This is typically done by purging the sample with either argon or nitrogen prior to injection. This approach is not feasible for microdialysis samples because only 5-10 microliters is usually available. In this report, a microbore liquid chromatographic method with dual-electrode amperometric detection is described for the determination of SR4233 and its metabolites without predeoxygenation. A dual-electrode amperometric detector was used in the series configuration with an upstream potential of -450 mV to reduce SR4233 and its metabolites to a common product and a downstream potential of +400 mV to oxidize this product. Oxygen is only electroactive at the upstream electrode because of its irreversible behavior. This method is compatible with the small sample volumes provided by microdialysis sampling. Linear calibration graphs were obtained up to 55 microM for SR4233, and 140 microM for both SR4317 and SR4330. The detection limits were 70 nM for SR4233, and 50 nM for SR4317 and SR4330. The average intra-day variation over 5 days was 1.8% (SR4233), 1.4% (SR4330), and 1.8% (SR4317), whereas the inter-day variation over 5 days was 14.1% (SR4233), 8.6% (SR4317), and 2.6% (SR4330).

Topics: Animals; Antineoplastic Agents; Biotransformation; Calibration; Chromatography, Liquid; Electrochemistry; Electrodes; Indicators and Reagents; Male; Microdialysis; Muscles; Rats; Tirapazamine; Triazines

The electrochemical properties of the benzotriazine di-N-oxide, tirapazamine (SR4233), and the mono- and zero-N-oxides, SR4317 and SR4330 respectively, have been investigated in dimethylformamide and acetonitrile. The voltammetry of tirapazamine is complicated, with up to 6 reduction steps being identified, depending on the solvent. Both SR4317 and SR4330 show two reduction steps. The first reduction of all three compounds is a reversible or quasi-reversible step, which is assigned to a 1-electron addition. Cyclic voltammetric studies show that the anion radical product is stable, although the tirapazamine 1-electron addition product shows a tendency to participate in a chemical following reaction. Subsequent reduction steps are all highly irreversible in nature. The 2nd electron transfer of SR4317 results in the formation of the free base, SR4330, which is identified voltammetrically. Comparison is made with the voltammetric behaviour of quinoline and quinoline-oxide.

Topics: Antineoplastic Agents; Electrochemistry; Free Radicals; Oxidation-Reduction; Tirapazamine; Triazines

Purified DT-diaphorase [NAD(P)H (quinone acceptor) oxidoreductase (EC.1.6.99.2)] from Walker cells was used to investigate the reductive metabolism of 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) under aerobic and anaerobic conditions. In the presence of NADPH, under aerobic conditions, HPLC analysis showed the four-electron reduction product 3-amino-1,2,4-benzotriazine (SR 4330) was the major reaction product. In contrast, anaerobically, the 2-electron reduction product 3-amino-1,2,4-benzotriazine-1-oxide (SR 4317) was the predominant metabolite. Anaerobic reduction of SR 4233 to the known metabolites SR 4317 and SR 4330, catalyzed by DT-diaphorase, was 3-fold higher than reduction under aerobic conditions. Anaerobically, approximately half of the substrate utilized could not be accounted for by the formation of known products. Aerobically, the majority of the SR 4233 lost could be accounted for by its conversion to SR 4317 and SR 4330. In Walker cells incubated with SR 4233 anaerobically, SR 4317 was the major metabolite formed. Dicoumarol (100 microM) had little effect on the rate of formation of this metabolite in this cell line or in a rat liver epithelial derived (JBJ) cell line. Dicoumarol did however partially reduce the induction of unscheduled DNA synthesis caused by SR 4233 in Walker cells but not in JB1 cells, suggesting the action of dicoumarol may be specific to Walker cells. It is concluded that DT-diaphorase plays only a minor role in the overall reduction of SR 4233 in the two cell lines studied.

Topics: Anaerobiosis; Animals; Antineoplastic Agents; Cell Hypoxia; Cell Line; Dicumarol; DNA; Hydrogen-Ion Concentration; NAD(P)H Dehydrogenase (Quinone); NADP; Oxidation-Reduction; Time Factors; Tirapazamine; Triazines; Tumor Cells, Cultured

SR 4233 or WIN 59075 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel and highly selective hypoxic cell cytotoxin requiring reductive bioactivation for its impressive antitumour effects. Expression of appropriate reductases will contribute to therapeutic selectivity. Here we provide more detailed information on the role of cytochrome P450 and cytochrome P450 reductase in SR 4233 reduction by mouse liver microsomes. Reduction of SR 4233 to the mono-N-oxide SR 4317 (3-amino-1,2,4-benzotriazine-1-oxide) is NADPH, enzyme and hypoxia dependent. An inhibitory antibody to cytochrome P450 reductase decreased the microsomal SR 4233 reduction rate by around 20%. Moreover, studies with purified rat cytochrome P450 reductase showed unequivocally that this enzyme was able to catalyse SR 4233 reduction at a rate of 20-30% of that for microsomes with equivalent P450 reductase activity. Exposure to the specific cytochrome P450 inhibitor carbon monoxide (CO) inhibited microsomal reduction by around 70% and CO plus reductase antibody blocked essentially all activity. Additional confirmation of cytochrome P450 involvement was provided by the use of other P450 ligands: beta-diethylaminoethyl diphenylpropylacetate hydrochloride gave a slight stimulation while aminopyrine, n-octylamine and 2,4-dichloro-6-phenylphenoxyethylamine were inhibitory. Induction of SR 4233 reduction was seen with phenobarbitone, pregnenalone-16-alpha-carbonitrile and beta-napthoflavone, suggesting that cytochrome P450 subfamilies IIB, IIC and IIIA may be involved. Since cytochrome P450 and P450 reductase catalyse roughly 70 and 30%, of mouse liver microsomal SR 4233 reduction respectively, we propose that expression of these and other reductases in normal and tumour tissue is likely to be a major factor governing the toxicity and antitumour activity of the drug.

Topics: Animals; Biotransformation; Carbon Monoxide; Cell Hypoxia; Cell Survival; Cytochrome P-450 Enzyme System; Male; Mice; Mice, Inbred C3H; Microsomes, Liver; NADPH-Ferrihemoprotein Reductase; Phenobarbital; Tirapazamine; Triazines

3-Amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233; WIN 59075) is a highly selective hypoxic cell cytotoxin soon to enter phase I clinical trial. The compound is thought to exert its action through a toxic one-electron reduced free radical intermediate. Preliminary data have suggested that SR 4233 may be metabolized by DT-diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2)] to both two- and four-electron reduced products and that this route of biotransformation may represent a bioprotection pathway. In this study, a highly purified enzyme preparation was employed in order to investigate further the metabolism of SR 4233 by DT-diaphorase and to examine the mechanism of reduction in more detail. Spectrophotometric analysis showed that SR 4233 underwent reduction by DT-diaphorase with an apparent Km of 1.23 +/- 0.27 mM and Vmax of 8.55 +/- 1.67 nmol/min/microgram protein. This reaction was inhibited completely by dicoumarol (100 microM) and partially by an antiserum raised against the purified enzyme. Characterization of the products of SR 4233 reduction by reverse-phase HPLC confirmed that both two- (SR 4317) and four- (SR 4330) electron reduction products were generated, the latter being the predominant metabolite, particularly in prolonged incubations. Further experiments showed that the four-electron reduction product, but not the two-electron reduction product, was also a substrate for DT-diaphorase with an apparent Km of 1.14 mM and a Vmax of 57.12 nmol/min/micrograms protein. The results presented confirm that SR 4233 is indeed a substrate for DT-diaphorase and that a mixture of two-, four- and six-electron reduced products may be formed. The possible toxicological and pharmacodynamic significance of this metabolism is discussed.

Topics: Animals; Carcinoma 256, Walker; Cell Hypoxia; Cell Line; Chromatography, High Pressure Liquid; Kinetics; NAD(P)H Dehydrogenase (Quinone); Oxidation-Reduction; Rats; Tirapazamine; Triazines

The electrochemistry of five benzotriazine di-N-oxides has been examined by cyclic voltammetry and differential pulse and dc polarographies as a function of pH. Between the pH range 8.5 and 2 the trend to less negative potentials with lowering of pH can be described by an equation of the type Ep = -apH + b. Comparison has been made with the mono- and zero-N-oxides which were found to show virtually identical trends in electron affinity with pH. The general electrochemical characteristics for the di- and mono-N-oxides under acidic conditions were found to be comparable with the zero-N-oxide. This was particularly the case on repeat scanning in the cyclic voltammetric mode. The redox mechanism involved reduction by a 4-electron addition step and subsequent loss of the N-oxide group(s) yielding the intact benzotriazine heterocycle. The heterocycle was also redox active, involving a reversible 2-electron reduction. For the di-N-oxides these two stages could be identified as separate processes at alkaline pH, but only a single step at acidic values. The mono-N-oxide in which the electrochemical behaviour was dominated by the triazine, showed only a single reduction step, although the single N-oxide group was redox active.

Topics: Antineoplastic Agents; Electrochemistry; Hydrogen-Ion Concentration; Oxidation-Reduction; Radiation-Sensitizing Agents; Tirapazamine; Triazines