Polarographic measurement of misonidazole in mouse mammary tumors

The sensitizing effect of misonidazole, a hypoxic cell radiosensitizer, is presumed to be due to its strong electron affinity. The concentration of misonidazole in mouse mammary tumors was measured polarographically in vivo. When 1 mg of misonidazole per gram body weight was administered intraperitoneally to C3H/He mice, 0.5-0.75 mM of misonidazole was observed in the tumors 25-60 min later. The distribution of misonidazole in the tumors varied, but the mean concentrations were not significantly different except in the necrotic area and the central-superficial region, where the concentration of misonidazole tended to be lower than in the other regions. No differences in the concentration of misonidazole were observed in tumors with diameters ranging from 4 to 11 mm. The polarographic measurement of misonidazole in vivo is technically simple and can be applied to the study of hypoxic cells in tumor tissue.     

Activation of misonidazole by rat liver microsomes and purified NADPH-cytochrome c reductase

Rat liver microsomes and purified NADPH-cytochrome c reductase metabolized [14C]misonidazole anaerobically to a reactive intermediate that covalently binds to tissue macromolecules. Air strongly inhibited the binding whereas carbon monoxide had no effect, indicating that misonidazole is activated via reduction and not by cytochrome P-450-dependent oxidation. Both systems showed an absolute requirement for NADPH and were stimulated by flavine (FAD) and paraquat. The apparent Km for misonidazole binding to microsomal protein was 0.74 mM the apparent Vmax was 0.64 nmole 14C bound . mg-1 . min-1. At a single substrate concentration, nitrofurantoin, nitrofurazone and desmethylmisonidazole inhibited the covalent binding of misonidazole to microsomal protein by 47, 26, and 38% respectively. The effect of nitrofurantoin on the kinetics of misonidazole binding gave a complex interaction indicative of uncompetitive inhibition. Glutathione reduced the binding of misonidazole to microsomal protein below the level observed for boiled microsomes while ascorbic acid had no effect. Compared to nitrofurantoin and paraquat, misonidazole was a poor stimulator of superoxide production as measured by adrenochrome formation.     

Influence of hypoxia on the metabolism and excretion of misonidazole by the isolated perfused rat liver--a model system

The isolated perfused rat liver was evaluated as a model system for the characterization of misonidazole metabolism under hypoxic conditions. Misonidazole metabolism by livers perfused under aerobic conditions was also examined. The clearance of misonidazole was more than three times greater under anaerobic compared to aerobic conditions (4.94 +/- 1.56 vs 1.27 +/- 0.22 ml/min; means +/- S.D., N = 3). Misonidazole metabolites were detected only in the bile. Analysis of these metabolites by reverse-phase high performance liquid chromatography (HPLC) demonstrated that misonidazole metabolism was also qualitatively changed when anaerobic conditions were employed. Misonidazole beta-glucuronide was the major metabolite detected under aerobic conditions, but it was a minor metabolite in anaerobically perfused livers. The three major metabolites produced under anaerobic conditions were not characterized, but desmethyl misonidazole (RO-07-9963) and the 2-amino-imidazole derivative of misonidazole (1-[2-aminoimidazol-1-yl]-3-methoxy-2-propanol) were excluded as possible structures.     

The effect of hypoxic radiosensitizer after mild hyperthermia in C3H mammary carcinoma

The radiosensitizing effect of misonidazole after hyperthermia was investigated in C3H mammary carcinoma. The tumors transplanted into the flanks of the mice were heated in a 42.3 degrees C water bath for 30 min. When misonidazole was administrated before heating, the subsequent radiation effect was prominently enhanced, whereas post-heating administration of misonidazole did not enhance the radiation effect significantly. The effect of varying the time between heating and radiation with or without misonidazole was as follows. Without misonidazole, the radiation effect was decreased at 6 hours after heating but increased at 12 hours, then it returned to the initial level at 24 hours and remained until 96 hours after heating. With misonidazole administered 30 min before irradiation, the radiosensitizing effect was observed at 24, 48 and 96 hours after heating. However, the total effects of this procedure were almost the same as the results in the combination without heating. Changes in the hypoxic fraction after hyperthermia are also discussed.     

Reductive metabolism and DNA binding of misonidazole

Misonidazole (1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol) is an experimental anticancer drug. Reductive metabolism is thought to be important for the cytotoxicity of misonidazole. In this study, the DNA binding of misonidazole was examined after chemical and enzymatic reduction. Under anaerobic conditions, both rat liver microsomes and cytosol catalyzed the reductive metabolism and DNA binding of misonidazole. The misonidazole utilized in these studies was radiolabeled on the side chain. The adduct(s) formed was too unstable for structural analysis. Little or no metabolism of misonidazole was detected in aerobic incubations. Likewise, very little DNA binding occurred in the presence of oxygen. Xanthine oxidase, a model nitroreductase, also was capable of catalyzing the DNA binding of misonidazole. However, unlike the xanthine oxidase catalyzed DNA binding of carcinogenic nitropolycyclic aromatic hydrocarbons, the DNA binding of misonidazole was not increased at slightly acidic pH. The putative reactive intermediate, the N-hydroxylamine, was synthesized by zinc reduction of misonidazole. The DNA binding of the N-hydroxylamine derivative increased with increasing pH. The observed pH dependence of the reactions with DNA is similar to other heterocyclic N-hydroxylamines, but is in contrast to the reactivity of a number of aromatic N-hydroxylamines.     

Biochemistry of misonidazole reduction by NADPH-cytochrome c (P-450) reductase

The biochemical mechanism for the reduction of misonidazole [1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol] by purified rabbit liver NADPH-cytochrome c (P-450) reductase, the primary nitroreductase of liver, has been studied. Neither the anaerobic nor the futile aerobic reduction velocities exhibited signs of Michaelis-Menten saturation at concentrations less than 5 and 10 mM, respectively. The anaerobic reduction of misonidazole resulted in the formation of glyoxal from fragmentation of the imidazole ring in 25% yield. The rate of glyoxal formation was linear with time and paralleled the reduction of misonidazole, suggesting that it was derived from the partitioning of a reactive intermediate between at least two alternative pathways. Negligible amounts of the 2-amino derivative of misonidazole were formed, however, indicating the existence of alternative reduction/fragmentation pathways.     

Reduction of misonidazole and its derivatives by xanthine oxidase

The nitroimidazole drug misonidazole, now undergoing clinical trials as a radiosensitizer of hypoxic cells, is selectively toxic to hypoxic mammalian cells; this toxicity may be due to metabolic reduction of the drug. Zinc reduction of misonidazole yields its azo and azoxy derivatives [P. D. Josephy, B. Palcic and L. D. Skarsgard, in Radiation Sensitizers (Ed. L. W. Brady), p. 61. Masson, New York (1980)]. We have shown in the present work that misonidazole and its azo and azoxy derivatives were reduced by xanthine oxidase, under hypoxic conditions. The nature of the products of misonidazole reduction was examined; hydroxylamino-misonidazole appeared to be the main product.     

Drug interactions with misonidazole: Effects of dexamethasone and its derivatives on the pharmacokinetics and toxicity of misonidazole in mice

Misonidazole [1-(2-nitroimidazol-1-yl)-3-methoxypropan-2-ol; Ro 07-0582] selectively sensitizes hypoxic cells to radiation and is undergoing clinical trial in the radiation treatment of solid tumours. It has been suggested that the glucocorticoid hormone dexamethasone may reduce the incidence of neurotoxicity, the dose-limiting side effect of misonidazole in man. Here it is shown that the absorption and elimination of misonidazole (1 $\text{g}\text{kg}$ i.p.) in C3H mice are unaffected by pretreatment (i.p. for 5 days) with dexamethasone (10$\text{mg}\text{kg}$/day), dexamethasone acetate (10 $\text{mg}\text{kg}\text{day}$) and dexamethasone phosphate (0.5, 10, 25 and lOO $\text{mg}\text{kg}\text{day}$). The apparent half-life of misonidazole in blood and area under the curve (AUC) of misonidazole concentration × time were unaltered. Likewise O-demethylation was unaffected. In contrast, phenobarbitone pretreatment (80 $\text{mg}\text{kg}\text{day}$) increased misonidazole clearance through induction of demethylation. Dexamethasone phosphate pretreatment increased pentobarbitone sleeping-time and slightly decreased liver weight, whereas phenobarbitone did the opposite. Dexamethasone phosphate (25 $\text{mg}\text{kg}$) given as an i.v. bolus injection immediately before misonidazole also had no effect on the systemic pharmacokinetics of misonidazole. Broadly, pretreatment with dexamethasone derivatives had little effect on brain misonidazole and desmethylmisonidazole. But after 100 $\text{mg}\text{kg}\text{day}$ dexamethasone phosphate the 6 hr misonidazole concentration was reduced 36 per cent. Simultaneous dexamethasone phosphate (25 $\text{mg}\text{kg}$) reduced the concentration at 1 hr by 15 per cent and the brain AUC_(0–6hr) by 14 per cent. Dexamethasone phosphate pretreatment reduced the acute LD₅₀ for misonidazole by up to 19 per cent whereas phenobarbitone increased it by 16 per cent. Simultaneous dexamethasone phosphate had no effect. The drug had little effect on misonidazole-induced hypothermia. The significance of these findings for the putative role of dexamethasone in the protection of misonidazole neurotoxicity is discussed.     

Inhibition of glutathione peroxidase and glutathione transferase in mouse liver by misonidazole

The mechanisms of toxicity and sensitization by the radiosensitizer misonidazole [1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol] are not well understood. We report here on the inhibition of total glutathione peroxidase (GSHPx), selenium-dependent glutathione peroxidase (selenium-GSHPx) and glutathione transferase (GSHTx) activities by misonidazole. Mouse liver cytosol GSHPx and selenium-GSHPx were inhibited in vitro with 0.5 mM misonidazole. On administration of the drug intraperitoneally (800 mg/kg) to mice, it was found that GSHPx, selenium-GSHPx, and GSHTx were inhibited in homogenate, cytosol, and microsomal fractions of mouse liver. GSHPx was depressed in all fractions up to 60-70% of control values, with maximum depression occurring in the cytosol and homogenate fractions in less than 2 hr. Recovery of activity was slower in the microsomes. In general, the pattern of depression of selenium-GSHPx was parallel to that of GSHPx except in microsomes, where GSHPx is minimal. Quantitatively, selenium-GSHPx was least affected. GSHTx was inhibited 70-80% of control values in cytosol and homogenate with recovery by 24 hr, whereas a second period of depression occurred at 24 hr in the microsomes. The inhibition of peroxide-metabolizing enzymes may lead to elevation of intracellular peroxide levels, contributing to the radiosensitizing effect and/or toxicity of misonidazole.     

Comparative misonidazole metabolism in anaerobic bacteria and hypoxic Chinese hamster lung fibroblast (V-79-473) cells

The metabolism of the radiation sensitizer misonidazole was similar in anaerobic cecal contents and hypoxic Chinese hamster lung fibroblasts (V-79-473). Both systems formed the amino derivative of misonidazole, [1-(2-aminoimidazol-1-yl)-3-methoxypropan-2-ol] (AIM), and urea, as well as a metabolite, (2-hydroxy-3-methoxypropyl)-guanidine (G), which has not been described previously. It appears that the nitro group of misonidazole was reduced to form AIM and that this compound was then hydrolyzed to yield either urea or G, the latter in yields of 25% (tissue culture) to 55% (cecal contents). When tested with the Ames tester strain, both G and AIM were slightly mutagenic only for strain TA 98 and then only in the presence of the system for microsomal activation.     

Reduction of nitroheterocyclic compounds by mammalian tissues in vivo

To determine whether nitro group reduction occurs in mammalian tissues, metronidazole (0.021, 0.064 and 10 mg/kg), misonidazole (0.015 mg/kg) and nitrofurazone (0.13 mg/kg), respectively, were administered to germfree rats. A reduced metabolite [1-(2-aminoimidazol-1-yl)-3-methoxypropan-2-ol] and two of its hydrolysis products, urea and (2-hydroxy-3-methoxypropyl)-guanidine, were found in the urines of germfree rats that received misonidazole. When nitrofurazone was administered, a reduced metabolite, 4-cyano-2-oxobutyraldehyde semicarbazone, was detected in the urines. However, acetamide and N-(2-hydroxyethyl)oxamic acid, fragmentation products from the reduction of metronidazole, were not found in significant concentrations in the urine when germfree rats received metronidazole. Apparently metronidazole is reduced so much more slowly than misonidazole and nitrofurazone in the tissues of germfree rats that its reductive metabolites are not detectable. This observation may be explained by the one-electron reduction potentials (E1 7) of these drugs, that of metronidazole (E1 7 = -486 mV) being lower than those of either misonidazole (E1 7 = -389 mV) or nitrofurazone (E1 7 = -257 mV). Under these circumstances, metronidazole reduction is not detected, either because its radical anion forms more slowly than that of the other nitroheterocyclic compounds or because its radical anion interacts more rapidly with oxygen to restore the parent compound.     

A biochemical assessment of the neurotoxicity of the radiosensitizing drug misonidazole in the rat

We have obtained biochemical evidence that misonidazole when administered in large doses to rats produces a sparse dying-back peripheral neuropathy and degenerative changes in the trigeminal ganglia and cerebellum. In our experience these neurotoxic effects of misonidazole cannot be detected reliably by the use of simple behavioural and functional tests, e.g., inclined plane and narrowing bridge tests (Rose and Dewar, unpublished results). Therefore, these methods would be of limited use in the neurotoxicity screening of misonidazole analogues. On the other hand, the biochemical approach provides a convenient quantitative method which could be used as the basis for comparing the neurotoxicity of other candidate radiosensitizing drugs.     

The relationship between misonidazole cytotoxicity and base composition of DNA

The damage induced by electrolytically reduced misonidazole on DNAs of varying base composition has been measured. Damage assessment using a variety of techniques including viscometry, helix renaturation, hydroxyapatite chromatography and agarose gel electrophoresis indicates that damage is related to A + T content, suggesting that misonidazole cytotoxicity involves a specific target in DNA.     

The neurotoxicity of radiosensitizing drugs: a biochemical assessment of desmethylmisonidazole (DMM) in the rat

This study evaluates a major metabolite of misonidazole, desmethylmisonidazole, for its potential to induce peripheral nerve damage using the lysosomal enzyme correlates of neuropathological change, namely beta-glucuronidase and beta-galactosidase. The results showed that desmethylmisonidazole like misonidazole had a similar potential for inducing peripheral nerve damage as measured biochemically, but the dosing regimen had to be maintained for 10 consecutive days as opposed to the 7 days required for misonidazole.     

Synthesis of an (iodovinyl)misonidazole derivative for hypoxia imaging

Nitroimidazoles undergo a bioreduction in viable hypoxic tissue, resulting in trapping within these tissues, as demonstrated by misonidazole. A radioiodinated analogue of misonidazole (IVM, (E)-5-(2-Nitroimidazolyl)-4-hydroxy-1-iodopent-1-ene, 3) has been synthesized by halodestannylation, for evaluation as an imaging agent for hypoxia. A key step in the synthetic sequence involves the use of the Lewis acid BF3.Et2O to promote the nucleophilic ring opening of glycidyl tosylate with (E)-1-lithio-2-(tributylstannyl)ethylene. Direct comparison of IVM versus F-MISO (2) another misonidazole type hypoxic cell marker, in several in vitro cell culture studies, indicates that IVM behaves in analogous fashion to F-MISO and has promise as a hypoxia imaging agent for SPECT.     

Induction of DNA strand breaks by reduced nitroimidazoles. Implications for DNA base damage

Radiation-reduced 2-nitroimidazoles (misonidazole, RSU-1137, Ro.03-8799 and Ro.03-8800) incubated in air with plasmid DNA (pH 7.0, 310K) induce DNA strand breakage, as revealed following subsequent heat or alkali treatment. Only RSU-1137 resulted in the binding of a [2-14C] fragment and significant yields of heat-labile strand breaks (greater than 20% loss of type-I DNA after 48 hr incubation). RSU-1137 was shown to be greater than 6 times more effective than misonidazole at producing alkali-labile breaks. In fact, the efficiency of alkali-induced strand break production is in the order: misonidazole less than Ro.03-8799 approximately Ro.03-8800 less than RSU-1137. Reaction of these reduced 2-nitroimidazoles with 2'-deoxyguanosine (dG) also results in the formation of a common glyoxal-dG product, with its yield and rate of production being dependent upon the 2-nitroimidazole used. It has been demonstrated that these variations are influenced by the N-1 side-chain of the 2-nitroimidazole. Product yields are approximately 5-6 times greater with misonidazole than with RSU-1137. From the evidence presented, it is apparent that formation of glyoxal (or a glyoxal-like product) is not responsible for the DNA strand breakage seen. It is inferred that these breaks are induced by a nitro-reduction product(s) which remains unidentified.     

The pharmacokinetics of a new radiosensitiser,Ro 03-8799 in humans

Summary A new hypoxic cell radiosensitiser, Ro 03-8799 has been administered intravenously to human volunteers and its kinetic parameters derived from plasma and urine data. Good penetration of drug into tumour tissue is found, consistent with its large volume of distribution. The plasma clearance of this compound is rapid due to high metabolic and renal clearances. These parameters combine to produce an elimination half-life of 5.6 h, approximately half that of misonidazole, a well studied radiosensitiser. It is hoped that this decrease in total body exposure will also reduce the cumulative toxicity seen when misonidazole is administered repeatedly.     

Metabolism and radiosensitization of 4,5-dimethylmisonidazole, a ring-substituted analog of misonidazole.

4,5-Dimethylmisonidazole (DMM) is a ring-substituted derivative of the 2-nitroimidazole, misonidazole. 2-Nitroimidazoles are able to sensitize radioresistant hypoxic cells, and to kill them outright through bioreductive metabolism. The toxic process is believed to reflect the consequences of reductively activated drugs forming adducts with cellular (macro)molecules. Both this process and the radiosensitizing activity are thought to correlate with the electron affinity of radiosensitizing agents. In the present study, methyl groups were added to the imidazole ring of misonidazole in order to hinder adduct formation with cellular molecules after reductive-activation of the compound. It was anticipated that this would substantially decrease the hypoxic-cell toxicity of the parent drug. The presence of the two methyl groups reduced the half-wave reduction potential of DMM by about 70 mV, so we expected that its radiosensitizing ability would also decrease. In direct comparison with misonidazole, DMM, at equimolar concentrations, showed dramatically reduced binding to cellular macromolecules under bioreductive conditions, both in vivo, using a liver perfusion system, and in vitro, using tissue culture cells incubated under extreme hypoxia. However, DMM was only moderately less toxic than the parent compound, and showed greatly diminished radiation sensitization capacity. Since the decrease in toxicity was much less than expected, and the decrease in radiosensitization was much more than expected, this compound may be an important drug for continuing studies on the mechanisms of radiation sensitization, binding and cytotoxicity caused by electron affinic drugs.     

Effects of huperzine A on acetylcholinesterase isoforms in vitro: comparison with tacrine,donepezil, rivastigmine and physostigmine

Five inhibitors of acetylcholinesterase, huperzine A, donepezil, tacrine, rivastigmine and physostigmine, were compared with regard to their effects on different molecular forms of acetylcholinesterase in cerebral cortex, hippocampus, and striatum from the rat brain. In general, huperzine A preferentially inhibited tetrameric acetylcholinesterase (G4 form), while tacrine and rivastigmine preferentially inhibited monomeric acetylcholinesterase (G1 form). Donepezil showed pronounced selectivity for G1 acetylcholinesterase in striatum and hippocampus, but not in cortex. Physostigmine showed no form-selectivity in any brain region. In cortex, the most potent inhibitors of G4 acetylcholinesterase were huperzine A (K(i) 7 x 10(-9) M) and donepezil (K(i) 4 x 10(-9) M). The potent inhibitors of cortical G1 acetylcholinesterase were donepezil (K(i) 3.5 x 10(-9) M) and tacrine (K(i) 2.3 x 10(-8) M). In hippocampus, huperzine A and physostigmine were the most potent inhibitors of G4 acetylcholinesterase, while donepezil and tacrine were most potent against G1 acetylcholinesterase. In striatum, huperzine A and donepezil were the most potent against G4 acetylcholinesterase, while again donepezil was the most potent against G1. Although the inhibition constants (K(i)) of these acetylcholinesterase inhibitors differed significantly from region to region, the nature of the inhibition did not vary. These results suggest that the use of acetylcholinesterase inhibitors in treatment of Alzheimer's disease must consider both form-specific and region-specific characteristics of acetylcholinesterase inhibition.     

Studies on the action of nitroimidazole drugs. The products of nitroimidazole reduction

The electron requirements for the electrolytic reduction of misonidazole, metronidazole and 4(5)-nitroimidazole have been measured using high-resolution coulometry. Eleven of the labelled final reduction products of metronidazole (a 5-nitroimidazole) have been separated by high-performance liquid chromatography and identified. These appear to be formed without the prior generation of a stable intermediate. In contrast, the reduction products of misonidazole (a 2-nitroimidazole) show little similarity to those of metronidazole but are likely to be formed via the four-electron hydroxylamine derivative. None of the final reduction products show toxicity towards Clostridium bifermentans or Escherichia coli suggesting that the short-lived cytotoxic agent of nitroimidazoles is a reduction product formed by the addition of not more than three electrons.