Flash photolysis of misonidazole and metronidazole

Laser flash photolysis at 355 nm of misonidazole or metronidazole in aqueous solutions produced the relatively long-lived nitro radical anion as the only observable transient species. When 266 nm excitation was used, a small yield of solvated electron was observed. It is suggested that the nitroimidazole first undergoes photoionization and the photoelectrons are scavenged by ground state nitroimidazole molecules to produce the nitro radical anion. Alternatively, added EDTA or carbonate ion acted as an electron donor to the excited state nitroimidazole molecule, thereby increasing the yield of nitro radical anion. The transient yield from metronidazole was about half that from misonidazole, while the phosphorescence intensity of metronidazole in an ethanol glass was about 20 times that of misonidazole. The misonidazole n, pi* triplet state is more easily reduced than that of metronidazole and, in the presence of an electron donor, the radical anion is postulated to result from electron transfer to the triplet state of the nitroimidazole.     

Association of the radiosensitizers metronidazole and misonidazole (RO-07-0582) with bovine serum albumin. Effect of urea and ionic strength

The stability of association of nitroimidazole radiosensitizers (metronidazole and misonidazole) with bovine serum albumin (BSA) was examined in aqueous solutions by 1H n.m.r. spectroscopy in the presence of urea (0-8M) as denaturant, or high salt concentration (NaCl0-5M). A broadening of n.m.r. lines of the two radiosensitizers observed in the presence of BSA disappeared with increasing urea concentration. An especially large narrowing effect was observed for the lines attributed to the methylene group near to the hydroxyl in the side chain of misonidazole. The results suggest a release of both radiosensitizers from their binding sites on unfolding by urea of the polypeptide chain of BSA. The increase of ionic strength I caused a monotonic enhancement of broadening by BSA of the metronidazole lines. For misonidazole, the enhancement of broadening was observed at values of I greater than 1, but at low (less than 1 M) concentrations of NaCl the broadening disappeared. Thus, the results obtained in the systems with salt reflect quantitative changes in hydrophobic and hydrogen-bonded contributions to binding of aliphatic moieties of radiosensitizers to BSA.     

Simultaneous determination of seven nitroimidazole residues in meat by using HPLC-UV detection with solid-phase extraction

A method was developed for the determination of the seven nitroimidazoles including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole (ONZ), secnidazole (SNZ) and the common metabolite of RNZ and hydroxydimetridazole (DMOHZ) in poultry and pork muscles by high-performance liquid chromatography (HPLC) with ultraviolet detection (UV). After extraction with ethyl acetate and evaporation, the nitroimidazoles were redissolved in ethyl acetate and purified using strong cation exchange (SCX) solid-phase extraction (SPE) column. The HPLC separation was carried through on a C(18) bonded silica column with a deionized water-methanol-acetonitrile mobile phase using a gradient elution procedure. The limit of detection of all the seven nitroimidazoles was 0.2 microg/kg. The recoveries of the seven nitroimidazoles for chicken, pork and bacon samples spiked with 1-20 microg/kg were in the range of 71.4-99.5%. The linearity is satisfactory with a correlation coefficient of >0.998 at concentrations ranging from 0.7 to 60 microg/kg. The relative standard deviations of 10 measurements for spiked chicken, pork and bacon samples at the concentration of 1 and 20 microg/kg were in the range of 6.2-13.9% and 4.0-8.7%, respectively. The intra-day precision (n=5) for nitroimidazoles residues in chicken spiked at 20 microg/kg is 6.9%, and the inter-day precision for 5 days (n=25) is 11%. The method is capable of identifying nitroimidazole residues at > or =0.7 microg/kg levels and was applied in the determination of nitroimidazole residues in meat sample.     

On the mutagenicity of nitroimidazoles

Regarding mutagenicity, metronidazole is one of the best-investigated compounds of the nitroimidazoles. This drug is mutagenic on bacteria, especially if base-pair tester strains are used and bacterial nitroreductases are present. The serum levels attained in man after intake of this drug are sufficient to cause mutations in bacteria. Furthermore, interaction with and binding to DNA occurs under anaerobic conditions and sometimes DNA breaks are observed. However, metronidazole does not show mutagenic activity in mammalian cells in vitro; the micronucleus test is negative and chromosome aberrations are only found under anaerobic conditions. With microbial systems the mutagenicity of 47 nitroimidazoles has been investigated. Only 4 compounds were always negative in the applied test systems. Because with base-pair tester strains mutagenicity was assessed, this class of compounds should be regarded as a base-pair mutagen. In fungi, some compounds (e.g. ZK 26173 and azathioprine) are potent mutagens, whilst with most investigated nitroimidazoles only a weak or no mutagenic activity could be detected. Somewhat similar observations have been made in tests with Drosophila melanogaster, a test for gene mutations in mammalian cells, the micronucleus test, cytogenic tests and the dominant lethal test. The reduction products of metronidazole, misonidazole and 1-methyl-2-nitro-5-vinylimidazole, cause DNA damage if the nitro group is reduced in the presence of DNA. Reduction products are formed by microbes in the gut or by mammalian cells under anaerobic conditions. No teratological effect due to metronidazole or most other nitroimidazoles has been observed. Metronidazole is carcinogenic in mice and rats, and dimetridazole in rats. Up to the present, no carcinogenic effects have been observed in man. Azathioprine is probably carcinogenic for man. It is unlikely that the therapeutic applications of the presently used nitroimidazoles, except for azathioprine, will cause an increase in the tumor incidence in man or will cause other genotoxic effects, although such effects cannot be excluded with certainty.     

The influence of thiols on the pre-irradiation incubation effect of nitroimidazoles in E. coli cells

The increase in the degree of radiosensitization of Escherichia coli cells following prolonged pre-irradiation incubation with nitroimidazoles is not correlated with the loss of intracellular non-protein thiols (NPSH) alone. The rates of reduction of the nitro compounds and the NPSH removal do not show strong dependencies on the lipophilicities of the nitroimidazoles whereas the highly lipophilic compound RGW-609 effects an increase in radiosensitization in a much shorter incubation time than the other nitroimidazoles. Exogenous dithiothreitol (DTT) increased the rate of reduction of misonidazole in the cells but did not alter the fraction converted to the amine. Added DTT (0.15 mmol dm-3) completely protected against the pre-irradiation incubation effect of misonidazole (2.5 mmol dm-3) when added at the start of the incubation but only partially protected when added before irradiation. It is suggested that NPSH can intercept metabolite(s) (or their precursors) of nitroimidazoles which can potentiate cell killing by radiation.     

Postirradiation modification of tumor blood flow: a method to increase the effectiveness of chemical radiosensitizers

The effect of postirradiation hypoxia induced by administration of the vasodilator hydralazine on the efficacy of misonidazole and RSU-1069 used in combination with radiation has been evaluated. Studies with the Lewis lung carcinoma indicate that hydralazine at a dose of 5 mg/kg reduces tumor blood flow and consequently increases the amount of hypoxia in the tumor tissue. Administration of hydralazine immediately after radiation treatment increased the amount of cell kill. However, the increase in cell kill was more pronounced when hydralazine was used in treatment regimes in which misonidazole (0.2 mg/g) or RSU-1069 (0.02 mg/g) was administered pre- or postirradiation. The finding that similar effects are observed if the nitroimidazoles were administered either before or after radiation in the regimes involving hydralazine suggests that the enhanced cell killing observed is due to hypoxic cell cytotoxicity. In contrast to the effects of hydralazine on the response of tumors to radiation plus misonidazole or RSU-1069, it has no effect on the response of mouse intestine to such treatment regimes. Thus therapeutic gain may accrue from the use of hydralazine in radiation treatments which incorporate the nitroimidazole radiosensitizers misonidazole and RSU-1069.     

Protection against light-activated photofrin II killing of tumor cells by nitroimidazoles.

Nitroimidazoles are good quenchers of triplet state porphyrins in chemical systems, thereby inhibiting singlet oxygen formation and type II photodynamic reactions. Photobiological studies were performed with EMT-6 tumor cells in vitro utilizing Photofrin II (PII) in combination with etanidazole (ETAN), misonidazole (MISO), and trifluoromisonidazole (TF-MISO). After short-term (1 h) exposure of cells to PII, 5 mM ETAN and MISO had no effect on photoinactivation while 5 mM TF-MISO had a small but significant protective effect. When the intracellular oxygen level was equilibrated with 0.3% oxygen in the gas phase, all three nitroimidazoles produced significant photoprotection at concentrations as low as 0.3 microM. After long-term (24 h) exposure of cells to PII, all three nitroimidazoles demonstrated large photoprotective effects under both aerobic and 0.3% oxygen conditions. At equal concentrations of nitroimidazole, photoprotection was greatest for the most lipophilic compound (TF-MISO) and least effective for the most hydrophilic compound (ETAN). These studies suggest that nitroimidazoles can quench triplet state porphyrins (within cells) to reduce intracellular concentrations of singlet oxygen, the putative toxin in PII photoinactivation. In addition, after long-term exposures to PII when porphyrins have partitioned into cellular membranes and lipid environments, the lipophilicity of this class of photoprotector correlates with effectiveness in these mammalian cells.     

Generation of radical anions from metronidazole, misonidazole and azathioprine by photoreduction in the presence of EDTA

Ultraviolet irradiation of the nitroimidazole derivatives metronidazole, misonidazole, azathioprine and 1-methyl-4-nitroimidazole in aqueous solution with various reductants produced the respective nitro radical anions, as detected by electron spin resonance spectroscopy. The most effective reductant, yielding high concentrations of the radical anions, was EDTA at pH 10. NADH, NADPH, formaldehyde glutathione and methanol were also tested but were less efficient as reductants.     

Nitroimidazoles, Part XXIII--activity of satranidazole series against anaerobic infections.

A large number of nitroimidazoles have been examined for in vitro activity against three anaerobes - Bacteroides fragilis (Bf), a strain of Bf resistant to metronidazole (16a) and Clostridium perfringens and many found to be active. Among these may be mentioned 1-methyl-5-nitroimidazoles carrying N - bound hetetocycles at position 2, such as satranidazole 1a, 1b, 1c, 1k, 1n and 1v which are at least twice as active as metronidazole (16a), ornidazole (16b) and tinidazole (16c). Even more active are 5-nitroimidazolyl benzimidazole 5d, -thiazolidinone 6b and thiadiazolidine dioxide 8a. Many other types of compounds derived from 1-methyl-2-amino-5-nitroimidazole are feebly active. Among 5-nitroimidazoles with a carbon substituent at position 2, 16a, 16b and 16c are equiactive while dimetridazole 14f is more active than 16a against Bf. Some 2-vinyl derivatives are very potent, with 18f and 18i being outstanding. Activity better than that of metronidazole is seen for nitroimidazooxazepines, e.g. 29d. 5-Nitroimidazoles are more active against anaerobes than 4-nitro isomers. Antianaerobic and antiamoebic activities generally run parallel in these classes of compounds. The study has led to the elaboration of the antianaerobic profile of satranidazole 1a.     

Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance

Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica , Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs.     

Toxic and radiosensitizing effect of reduced nitroimidazoles on E. coli B/r cells

The spectrophotometric method was used to study the rate of chemical reduction of misonidazole and metronidazole by NH4Cl and Zn in the atmosphere of argon and oxygen. Reduction of drugs increased their toxicity for hypoxic and oxygenated E. coli B/r. The reduced metronidazole is a more effective radiosensitizer of hypoxic E. coli B/r than the original compound.     

Generation of free radicals from metronidazole and other nitroimidazoles by Tritrichomonas foetus

Metronidazole, ronidazole, secnidazole, benznidazole, and misonidazole are reduced by intact Tritrichomonas foetus cells to nitro anion radicals that can be detected by electron spin resonance spectroscopy. This activity appears to be related to the cellular content of reducing substrates, since nitro anion radical formation is stimulated in the presence of glucose and pyruvate. The nitro anion radicals could not be detected under aerobic conditions. Anaerobic homogenates of T. foetus also reduce metronidazole to the nitro anion radical when pyruvate, NADH, or NADPH is added as the ultimate source of reducing equivalents. Free radical formation may be the basic cause of nitroimidazole toxicity in trichomonads.     

Electron-Affinic Sensitization VII. A Correlation between Structures, One-Electron Reduction Potentials, and Efficiencies of Nitroimidazoles as Hypoxic Cell Radiosensitizers

Radiosensitization efficiencies for seven different 2-nitroimidazoles including Ro-07-0582 and its urinary metabolite, Ro-05-9963, and two 5-nitroimidazoles including metronidazole, have been determined in hypoxic Chinese Hamster cells, line V79-379A, X-irradiated in vitro. All the compounds were active hypoxic cell sensitizers with the enhancement ratios increasing with drug concentration. The 2-nitroimidazoles were all more efficient than the 5-nitroimidazoles. Overall, the efficiencies, defined as the concentration required to give a particular enhancement ratio, varied by a factor of about 200. Electron-affinities of the sensitizers were determined by pulse radiolysis as the one-electron reduction potentials and these correlate well with the sensitization efficiencies of the compounds. The correlation extends beyond the nitroimidazole series as is shown by data for p-nitroacetophenone, nifuroxime (a nitrofuran) and oxygen itself. The nitroimidazoles varied by a factor of 70 in their octanol/water partition coefficients, but the effect of this parameter on sensitizing efficiency is small compared with the influence of electron affinity.     

Electrochemical Characteristics of Nitroheterocyclic Compounds of Biological Interest VI. The Misonidazole Radical Anion

The addition of four aprotic solvents to misonidazole in an aqueous buffer system has been examined electrochemically. Qualitatively they all result in separation of the initial irreversible 4 electron reduction step into two stages, the RNO₂/RNO₂· and RNO₂· /RNHOH couples respectively. Despite some difficulties in achieving measurements for the discrete RNO₂/RNO₂· without interference from the following reduction step, it was clear that the various aprotic solvents influenced the lifetime of the RNO₂· species to different degrees. Resolution of the two processes was best achieved using a water-acetone system and this has been employed to study the lifetimes of the misonidazole radical anion as a function of acetone content and drug concentration. Analysis of the cyclic voltammetric response showed a second order decay pathway, in line with the metronidazole system studied under similar conditions. This has been compared with results from pulse radiolysis work, which suggested a first order reaction of unknown pathway for 2-nitroimidazole radical anions.     

Thiol reactive nitroimidazoles: radiosensitization studies in vitro and in vivo

Using Chinese hamster V79 cells in vitro a study has been made of the radiosensitizing properties of 4- or 5-nitroimidazoles substituted in the 2, 5 or 4 position with various halo, sulphur ether, sulphonamide, sulphonate, ether or nitro groups. Values of E17 (the one-electron reduction potential measured versus the normal hydrogen electrode at pH7) vary in the range -178 to -565 mV. All the compounds, with one exception, are more efficient radiosensitizers than would be predicted from their redox potentials, and the factor, C1.6/C1.6, by which a compound is more efficient has been calculated. The second-order rate constants, k2, for reaction of these nitroimidazoles with glutathione and/or dithiothreitol were determined. Within each class of nitroimidazole there is a trend for k2 to increase with increasing redox potential. However, there is no clear trend between k2 and C1.6/C1.6. The concentration required to cause a 50 per cent depletion of intracellular glutathione was determined for selected compounds, as was the ability of glutathione-S-transferase to catalyse reaction with thiols. These observations suggested that the relative thiol reactivity measured under chemically controlled conditions does not necessarily indicate thiol reactivity intracellularly. Studies using the MT tumour in mice showed that the high levels of radiosensitization seen in vitro could not be duplicated in vivo. This was attributed to thiol reactivity, resulting in low metabolic stability and rapid depletion of sensitizer in vivo.     

Generation of radical anions of nitrofurantoin, misonidazole, and metronidazole by ascorbate

Nitrofurantoin, misonidazole, and metronidazole were reduced to their corresponding nitro anion radicals by ascorbate in anaerobic solutions at high pH. The nitrofurantoin anion radical could be detected at neutral pH. In neutral solutions, the nitro anion radicals of misonidazole and metronidazole were too unstable to be observed by electron spin resonance spectroscopy. At neutral pH, solutions containing ascorbate, nitrofurantoin, or misonidazole consumed oxygen. The addition of superoxide dismutase, catalase, or both superoxide dismutase and catalase decreased the rate of oxygen consumption. These results show that nitro anion radicals are formed by reduction with ascorbate, and superoxide anion radical and hydrogen peroxide are produced by reactions of these radicals with oxygen.     

Inhibition of plant fatty acid synthesis by nitroimidazoles.

1. The effect of the addition of a number of nitroimidazoles was tested on fatty acid synthesis by germinating pea seeds, isolated lettuce chloroplasts and a soluble fraction from pea seeds. 2. All the compounds tested had a marked inhibition on stearate desaturation by lettuce chloroplasts and on the synthesis of very-long-chain fatty acids by pea seeds. 3. In contrast, the effect of the drugs on total fatty acid synthesis from [14C]acetate in chloroplasts was related to the compound's electron reduction potentials. 4. Of the compounds used, only metronidazole had a marked inhibition on palmitate elongation in the systems tested. 5. The mechanism of inhibition of plant fatty acid synthesis by nitroimidazoles is discussed and the possible relevance of these findings to their neurotoxicity is suggested.     

The interaction between radiation and complexes of cis-Pt(II) and Rh(II): studies at the molecular and cellular level

A range of Rh(II) carboxylates and cis-Pt(II) complexes have been examined for their ability to increase the radiation sensitivity of aerobic and hypoxic V79 cells in vitro. The transition metal complexes sensitize in both air and nitrogen, with the greater effect generally occurring in nitrogen. The cis-Pt(II) complexes only show small levels of sensitization with dose modification factors (DMFs) of no more than 1.2. In contrast, the Rh(II) complexes can give DMFs of 2.0. Radiation chemical experiments show the transition metal complexes to have substantially lower redox potentials than metronidazole and, in addition, neither type of complex undergoes electron transfer reaction or adduct formation on interaction with radicals derived from DNA bases. Thus, the inorganic complexes do not operate by mechanisms similar to those occurring with electron affinic or stable free radical sensitizers. The increase in radiation sensitivity for cells treated with the Rh(II) carboxylates, but not the cis-Pt(II) complexes, is attributed to the ability of the Rh compounds to deplete intracellular thiols. Further, the efficiency of sensitization by the Rh(II) complexes and their ability to interact with cellular thiols depends upon the nature of the carboxylate ligand and follows the order butyrate greater than propionate greater than acetate greater than methoxyacetate. The differences between the carboxylates may be due to differences in drug uptake. A combination of the Rh(II) complexes with misonidazole given to hypoxic cells irradiated in vitro gives an additive response. However, it was not possible to demonstrate a similar effect in tumours in mice given the combination of Rh(II) methoxyacetate and the misonidazole analogue RSU 1070.     

Clinical and microbiological efficacy of metronidazole and ornidazole in the treatment of urogenital trichomoniasis in men]

The aim of the study was to compare the efficacy of metronidazole and ornidazole in the treatment of urogenital trichomoniasis in men. The drugs were used randomly in usual doses as monotherapy: 210 patients, metronidazole and 217 patients, ornidazole. The clinical efficacy of metronidazole and ornidazole was stated in 57.6 and 94.5% of the patients and the microbiological efficacy was stated in 77.1 and 98.2% of the patients respectively. The side effects were recorded in 59.0 and 3.7% of the patients respectively. Thus, ornidazole proved to be a more efficient and safe agent vs. metronidazole in the treatment of urogenital trichomoniasis.     

Action of hypoxic sensitizers on the survival of haplont yeasts irradiated with ionizing radiation

A study was made of the oxygen effect and the radiosensitizing action of metronidazole and misonidazole on hypoxic cells of irradiated yeast haplonts. It was shown that metronidazole did not increase the radiosensitivity of all the strains under study while the sensitizing effectiveness of oxygen and misonidazole approximated the values characteristic of different repair-deficient rad-mutants. Possible causes of the radiosensitizing effects observed are discussed.