Journal of Antimicrobial Chemotherapy (1977) 3, 205-212
Metronidazole: review of uses and toxicity
F. J. c. Roe
The development of a trichomonacide In 1954 Rhone-Poulenc decided to develop a drug effective in the treatment of Trichomonas vaginalis infection. The availability of in vitro and in vivo (mouse) systems for evaluating potential trichomonacides facilitated research and in due course enabled the discovery that azomycin (2-nitroimidazole), isolated from Streptomyces 6670, had weak trichomonacidal activity. Over 200 chemicals related to azomycin were then systematically synthesized and screened for activity. In terms of activity relative to toxicity, metronidazole (l-hydroxyethyl-2-methyl-5-nitroimidazole, 'FlagyP) emerged as the most promising agent and the first evaluation of its efficacy against T. vaginalis infection began in France in 1958. The value of the drug, which was the first systemically active trichomonacide, was immediately apparent (Durel, Roiron, Siboulet & Borel, 1959) and within a few years its use became widespread all over the world. In France, the usual practice has been to combine oral treatment with vaginal pessaries. In other countries, pessaries are much less prescribed because there is no real evidence that they usefully supplement the effects of oral tablets and treatment with pessaries alone often fails because the drug does not reach trichomonads harbouring in the genitourinary tract. Cure usually results from total doses of the drug rather less than those hitherto thought to be necessary. Thus cure rates of 95 % or better can be expected from regimens of 200 mg three times per day for 7 days or a single dose of 2 g by mouth. Parallel treatment of male consorts with the drug at the same dosage as given to the patient is necessary to prevent reinfection. Remarkably there is almost no confirmed evidence of resistance of trichomonads to metronidazole, and in the very few patients in whom failure of treatment cannot be attributed to reinfection or non-taking of tablets, poor absorption of the drug from the gut or its excessive destruction by vaginal flora are the commonest explanations. Amoebiasis and giardiasis The original biological evaluation of metronidazole at Rhone-Poulenc (Cosar, Ganter & Julou, 1961) indicated that metronidazole might be useful against E. histolytica, but it was not until 5 years later that its value in this regard was realized (Powell, MacLeod, Wilmot & Elsdon Dew, 1966). Relatively low dosage regimes were enough to kill vegetative amoebae in the gut wall and liver but higher dosages were found to be necessary 205
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4, Kings Road, Wimbledon, London SW19 8QN, England
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Anaerobic bacterial infections During the last few years the clinical value of metronidazole in the treatment and prevention of other diseases has been becoming increasingly apparent. The most important advance relates to the highly specific effect of metronidazole against obligatory anaerobic micro-organisms. It is only during the last decade that methods of obtaining uncontaminated samples of pus and techniques for growing and identifying strict anaerobes have been perfected and become widely available (Holdeman & Moore, 1972). As a consequence, many of those brain abscesses, pelvic abscesses and weeping skin wounds once thought to be yielding 'sterile pus' are now known to be due to infection with a variety of anaerobes such as Bacteroides fragilis, Clostridia, Peptostreptococci, Fusobacteria and many others (Gorbach & Bartlett, 1974; Finegold, 1974). It was a serendipitous observation in 1962 that led eventually to the realization of the value of metronidazole in the treatment of infections involving obligatory anaerobes: Shinn (1962) and Shinn, Squires & McFadzean (1965) reported the cure of long-standing Vincent's infection in a woman who received a course of treatment with metronidazole for T. vaginalis infection. Laboratory studies (Davies, McFadzean & Squires, 1964) undertaken in the wake of Shinn's observation showed that the drug is intensely active against certain species of anaerobic bacteria and its possible value in the prevention and treatment of tetanus and gas
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for the elimination of trophozoites from the lumen of the gut and thereby prevent parasitological relapse. Adams (1977) reported findings in a series of 5057 cases of amoebiasis seen in the King Edward VIII Hospital, Durban over a 20 year period. Death rates for uncomplicated amoebic dysentery or liver abscess were less than 1 % but complicating peritonitis or pericarditis were associated with greatly increased risk of fatal outcome. The results of a series of clinical trials have shown that a course of 800 mg metronidazole 3 times per day for 5 days was equal to the best of all other treatment regimens for amoebic dysentery, and, combined with aspiration, highly efficacious in the treatment of amoebic liver abscess. Tetracycline, which is very effective for uncomplicated amoebic dysentery, is not a tissue amoebicide and therefore of no value in cases of liver abscess or where there are complications. Although emetine and chloroquine alone or combined, are highly effective against liver abscess, they cause more unpleasant side effects. The therapeutic activity of metronidazole in human giardiasis was first fully demonstrated by Schneider (1961). The development of techniques for jejunal aspiration and jejunal mucosal impression smears from jejunal biopsy specimens (Kamath & Murgasu, 1974) has clarified the role of Giardia lamblia as a cause of diarrhoeal disease in both tropical (Anati, Desai, Jeejeethoy, Kane & Borkar, 1966) and temperate climates (Moore et ah, 1969; Brodsky, Spencer & Schultz, 1974), particularly in immunodeficient subjects (Ament & Rubin, 1972). Wright (1977) reported the eradication of the parasite in 28 out of 31 subjects following a 3 day course of metronidazole in which 2 g was given as a single dose once each day. Metronidazole, which has only slight activity against Trypanosoma cruzi, has given equivocal results in the treatment of chronic Chagas' disease and a similar clinical situation obtains in the treatment of cutaneous leishmaniasis although anti-leishmania activity has not been demonstrated experimentally. Against Balantidium coli, however, metronidazole is highly active and clinical and parasitological cures have been obtained.
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Willis (1977) obtained equally good results with rectal metronidazole in colonic surgery and Eykyn (1977) reported the use of intravenous metronidazole, given alone or with antibiotics, in the treatment of 24 patients with severe anaerobic infections; no side effects were observed and improvement, sometimes dramatic and life-saving, was seen in over 70%. Recently, Pye & Burton (1976) reported a favourable response to oral metronidazole in cases of papulopustular rosacea. Radiosensitization and anticancer activity Hypoxia increases resistance to ionizing radiation and the selective survival of hypoxic cells is thought to reduce response to radiotherapy. Accordingly, agents which sensitize such cells to ionizing radiation have been sought. Under both in vitro conditions and in vivo in mice, a number of nitroimidazoles, including metronidazole, have been found to be effective in this regard (Chapman, Reuvers & Borsa, 1973; Asquith, Foster, Willson & Study Group, 1974; Denekamp, Michael & Harris, 1974; Stone & Withers, 1974). Willson, Cramp & Ings, (1974) and Foster, Conroy, Searle & Willson (1976) have considered the mechanisms underlying radio-sensitization by metronidazole and it is interesting to speculate that the drug may have some direct cytocidal effect on tumour cells that have adopted an anaerobic way of life. Thus, Urtasun et al. (1976) has reported promising prolongation of survival in a trial of combined effects of ionizing radiation and massive doses of metronidazole (6 g/m2 thrice weekly for 3 weeks). Toxicity and side effects Acute and sub-acute toxicity studies in mice and rats have indicated LD50 dosages in the 1 to 5 g/kg range.
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gangrene was postulated (Freeman, McFadzean & Whelan, 1968), but it was not until 1972 that Tally, Sutter and Finegold reported its successful use in the treatment of Bacteroides infections. Today metronidazole takes its place alongside chloramphenicol, lincomycin and clindamycin in the treatment of anaerobic bacterial infections. Metronidazole has no activity against aerobic organisms and this might be thought to be a disadvantage in the case of mixed infections. However, it is compatible with the commonly used antibacterial antibiotics (Salem, Jackson & McFadzean, 1975) and the specificity of its activity against anaerobes makes it a useful tool for the investigation, if not the treatment, of conditions such as ulcerative colitis and Crohn's disease (Ursing & Kamme, 1975). The last few years have seen reports on the value of metronidazole in the treatment of various anaerobic infections including septicaemia, necrotizing pneumonia, and pulmonary abscess (Mitre & Rotheram, 1974; Tally, Sutter & Finegold, 1975), post-hysterectomy pelvic cellulitis and anaerobic intra-uterine infections (Willis and Study Group, 1974), brain abscess (Ingham et al., 1975; Ingham, Selkon, So & Weiser, 1975) and breast abscess (Hale, Perinpanayagam & Smith, 1976). Pre- and post-operative oral treatment with metronidazole greatly reduced the incidence of post-operative non-clostridial anaerobic infections after hysterectomy (Willis & Study Group, 1975) and analogous treatment with metronidazole in rectal suppositories prevented anaerobic sepsis after appendicectomy (Willis & Study Group, 1976). Workers in Glasgow (Goldring, Scott, McNaught & Gillespie, 1975) reported good results with pre-operative oral metronidazole and kanamycin in the prevention of sepsis after colonic surgery.
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More recently metronidazole has been given in higher dosages and for several weeks or months and some adverse reactions have occurred with greater frequency and severity; in patients given dosages of over 180 mg/kg/day as an adjunct to radiotherapy, severe anorexia, nausea and vomiting, which tended to persist for 24 to 48 h after the last dose, sometimes necessitated withdrawal of the drug (Deutsch, Foster, McFadzean & Parnell, 1975). Epileptiform seizures have been noted by other investigators in a few cases. Some patients undergoing prolonged metronidazole therapy, e.g. for the treatment of Crohn's disease and, in one instance, for brain abscess, have experienced paraesthesiae and signs of peripheral neuropathy which disappeared when dosage was reduced or stopped (Ursing & Kamme, 1975; Ingham et al, 1975). As a nitro compound in widespread therapeutic use, metronidazole attracted the attention of oncologists with special interests in tumorigenicity and mutagenicity, and Rustia & Shubik (1972) reported an increased incidence of lung tumours in male and female Swiss mice and an increased incidence of malignant lymphoma in female mice only in response to metronidazole given continuously in the diet for 18 months. Both types of neoplasm occur in high incidence spontaneously and the increased incidences seen were only of the same order as those that can be achieved by simple dietary manipulation (Roe & Tucker, 1974). Rust (1977) has recently reported that two other studies in mice confirmed the effect on lung tumour incidence but not that on the incidence of malignant lymphoma and that in rats a slight but not significant excess of mammary tumours was seen in one small study (Cohen et al., 1973) but not confirmed in a much
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In chronic toxicity studies in rats, daily dosages of 75 or 150 mg/kg given orally for 26 to 80 weeks produced no adverse reactions, but dosages of 300 or 600 mg/kg/day caused testicular dystrophy and prostatic atrophy. Chronic toxicity studies in dogs showed them to be peculiarly susceptible to ataxia, muscular rigidity and tremors at dosages of over 75 mg/kg/day. However, monkeys medicated for one year with dosages of 45, 100 or 225 mg/kg/day, showed no evidence of chronic toxicity except for minimal non-specific changes in the livers of a few animals receiving the highest dosage. Conventional animal tests for embryopathy and effects on reproduction have given negative results (Searle Labs. Investigational Brochure, 1973). During the 15 years of its widespread use for the treatment of T. vaginalis infections, metronidazole has earned a reputation of being remarkably safe. Occasional side effects at the dosages ordinarily used for the treatment of this condition include nausea, an unpleasant taste in the mouth, furring of the tongue and gastrointestinal upsets; headache, dizziness, ataxia, depression and skin eruptions have been reported but rarely. A mild to moderate disulfiram-like effect can occur when alcohol is taken during metronidazole therapy; the severity of the reaction is proportional to the amount of alcohol ingested (Semer, Friedland, Vaisberg & Greenberg, 1966). Despite the profound effect of the drug on the population of anaerobic bacteria of the gut, diarrhoea has not featured prominently among its side effects. In animals enlargement of the caecum has been recorded suggesting an inhibitory effect on the metabolism of certain complex carbohydrates and other substances (probably by gut flora). Transient leucocytopenia has been observed in humans (Willson, 1974) but there have been no reports of serious blood dyscrasias. It should, perhaps, not be given to women during the first trimester of pregnancy or during lactation, since it freely passes the placental barrier and is excreted in milk. On the other hand no case of damage to the foetus or suckling child has been reported where the drug has been administered at these times. Experience indicates that the drug may be safely administered to children (Rubidge, Scragg & Powell, 1970).
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Metabolism biochemistry, and mechanisms of action
As with many other drugs which have been found effective in therapy, precise knowledge of how metronidazole acts is limited. It is clear that metronidazole enters aerobic bacteria just as readily as it enters anaerobic ones, but whereas in the former it remains unchanged and its concentration equilibrates with that in the medium, in the latter it is biochemically reduced, the equilibrium is disturbed and more drug diffuses into the cell (Ings, McFadzean & Ormerod, 1974). Reduction is probably effected by low redox potential electron transport proteins (e.g. ferredoxin or flavodoxin) (O'Brien & Morris, 1972) which play a major role in the metabolism of anaerobes and photosynthetic organisms but have a limited role in aerobes (Yoch & Valentine, 1972). What happens to the reduced metronidazole is largely a mystery, as is the basis of the drug's cytocidal effect. However, it seems that some of the reduced drug binds to cell constituents and that the bound products are non-dialysable. Inhibition of nucleic acid synthesis has been reported (Ings, McFadzean & Ormerod, 1974). In both animals and humans similar ranges of metabolites have been identified (Ings, Law & Parnell, 1966; Stambaugh, Feo & Manthei, 1968) and in humans the urine is the main route of clearance (Schwartz & Jeunet, 1976). Despite this, McHenry (1977) found no evidence that a single oral dose of 500 mg metronidazole was handled differently in volunteers with absent or severely impaired renal function than in those with normal renal function. References Adams, E. B. Amoebiasis and its treatment. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S., McFadzean, J. A. & Roe, F. J. C. Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press.
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larger one. Two carcinogenicity studies in hamsters gave entirely negative results in respect of tumorigenicity (Rustia, 1975; Roe, Ingham & Lowe, 1976). The effect on tumour incidence in mice reported by Rustia and Shubik involved the administration throughout the lives of the animals of total doses, on a mg/kg body weight basis, equivalent to between 350 and 3000 times that given to patients in the form of a 10 day course for the treatment of trichomoniasis. In 1974, metronidazole was reported as having increased the spontaneous mutation rates of certain strains of aerobic bacteria grown in vitro (Voogd, Van der Stel & Jacobs, 1974) and later Rosenkranz (1977) reported that in animals and in humans, the drug can be bio-transformed to substances which are mutagenic for a strain of S. typhimurium. On the other hand Bost (1977) reported that conventional dominant lethal tests in rats and in mice had given negative results. Recently, Mitelman, Hartley-Asp & Ursing (1976) reported chromosomal abnormalities in the circulating lymphocytes of patients with Crohn's disease receiving metronidazole. In the light of the animal data a small and indefinite cloud hangs over metronidazole as it does over many other drugs, in relation to possible carcinogenicity and mutagenicity. However, in the case of metronidazole it is important to take into account the likelihood that cure of trichomoniasis reduces risk of development of cancer of the uterine cervix. It is well established that the risk of this form of cancer is greatly influenced by sexual hygiene standards {British MedicalJournal 1965) so that it is very likely that the elimination of T. vaginalis infection and of secondary non-specific vaginal infections which accompany it reduces cervical cancer risk.
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Ament, M. E. & Rubin, C. Relation of giardiasis to abnormal intestinal structure and function in gastrointestinal immunodeficiency syndromes Gastroenterology 62: 216-27 (1972). Anati, F. P., Desai, H. G., Jeejeethoy, K. N., Kane, M. P. & Borkar, V. V. Indian Journal of Medical Science 20: 471 (1966). Asquith, J. C , Foster, J. L., Willson, R. L. & Study Group. Metronidazole ('Flagyl'). A radiosensitizer of hypoxic cells. British Journal of Radiology 47:474-81 (1974). British Medical Journal. Cervical cancer: early diagnosis or prevention (Leading article) i: 1327-8 (1965). Bost, R. G. Dominant Lethal Test. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S., McFadzean, J. A. & Roe, F. J. C. Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press. Brodsky, R. E., Spencer, H. C. & Schultz, M. G. Giardiasis in American travellers to the Soviet Union. Journal of Infectious Diseases 130: 319-23 (1974). Chapman, J. D., Reuvers, A. P. & Borsa, J. Effectiveness of nitrofuran derivatives in sensitizing hypoxic mammalian cells to X-rays. British Journal of Radiology 46: 623-30 (1973). Cohen, S. M., Erturk, E., Crovetti, A. J. & Bryan, C. T. Carcinogenicity of 5-nitroimidazoles,4nitrobenzenes and related compounds. Journal of the National Cancer Institute 51: 403-17 (1973). Cosar, C , Ganter, P. & Julou, L. Etude experimentale du metronidazole (8823 R.P.). Activites trichomonacide et amoebicide. Toxicit6 et proprietes pharmacologiques generates. Presse Midicale 69: 1069-72 (1961). Davies, A. H., McFadzean, J. A. & Squires, S. Treatment of Vincent's stomatitis with metronidazole. British Medical Journal i: 1149-50 (1964). Denekamp, J., Michael, B. D. & Harris, S. R. Hypoxic cell radiosensitizers: Comparative tests of some electron affinic compounds using epidermal cell survival in vivo. Radiation Research 60: 119(1974). Deutsch, G., Foster, J. L., McFadzean, J. A. & Parnell, M. Human studies with 'high dose' metronidazole: a non-toxic radiosensitizer of hypoxic cells. British Journal of Cancer 31:75-80(1975). Durel, P., Roiron, V., Siboulet, H. & Borel, L. J. Trial of an anti-trichomonal derivative of imidazole (8823 RP). Comptes Rendus de la Societi Francaise de Gynicologie 29: 36, Jan. (1959). Eykyn, S. The value of intravenous metronidazole in the treatment of patients with anaerobic infections. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A. & Roe, F. J. G, Eds.). Journal of Infectious Diseases 1977 (Suppl.): in press. Finegold, S. M. Intra-abdominal, genito-urinary, skin and soft tissue infections due to non-spore forming anaerobic bacteria. Infection with non-sporing anaerobic bacteria, (Phillips & Sussman, Eds.). Churchill/Livingstone, London (1974), pp. 160-87. Foster, J. L., Conroy, P. J., Searle, A. J. & Willson, R. L. Metronidazole ('Flagyl'): characterization as a cytotoxic drug specific for hypoxic tumour cells. British Journal of Cancer 33: 485-90 (1976). Freeman, W. A., McFadzean, J. A. & Whelan, J. P. F. Activity of metronidazole against experimental tetanus and gas gangrene. Journal of Applied Bacteriology 31: 443-7 (1968). Goldring, J., Scott, A., McNaught, W. & Gillespie, G. Prophylactic oral antimicrobial agents in elective colonic surgery: a controlled trial. Lancet ii: 997-1000 (1975). Gorbach, S. L. & Bartlett, J. C. Anaerobic infections (third of three parts). New England Journal of Medicine 290: 1289-94 (1974). Hale, J. E., Perinpanayagam, R. M. & Smith, G. Bacteroides: an unusual case of breast abscess. Lancet ii: 70-1 (1976). Holdeman, L. V. & Moore, W. E. C. Anaerobic Laboratory Manual. The Virginia Polytechnic Institute Anaerobe Laboratory, Blacksburg, VA. U.S.A. (1972). Ingham, H. R., Rich, G. E., Selkon, J. B., Hale, J. H., Roxby, C. M., Betty, M. J., Johnson R. W. C. & Uldall, P. R. Treatment with metronidazole of three patients with serious infections due to Bacteroides fragilis. Journal of Antimicrobial Chemotherapy 1:235-42 (1975). Ingham, H. R., Selkon, J. B., So, S. C. & Weiser, R. Brain abscess. British Medical Journal iv: 39-40 (1975).
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Ings, R. M. J., Law, G. L. & Parnell, E. W. The metabolism of metronidazole. Biochemistry and Pharmacology 15: 515-9 (1966). Ings, R. M. J., McFadzean, J. A. & Ormerod, W. E. The mode of action of metronidazole in Trichomonas vaginalis and other micro-organisms. Biochemistry Pharmacology 23: 1421-9 (1974). Kamath, K. R. & Murgasu, R. A comparative study of four methods for detecting Giardia lamblia in children with diarrhoeal disease and malabsorption. Gastroenterology 66:16-21 (1974). McHenry, M. C. Use in impaired renal function. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A. & Roe, F. J. C. Eds.) Journal of Infectious Disease* 1977 (Suppl.) in press. Mitelman, F., Hartley-Asp, B. & Ursing, B. Chromosome aberrations and metronidazole. Lancet ii: 802 (1976). Mitre, R. J. & Rotheram, E. B. Anaerobic septicaemia from thrombo-phlebitis of the internal jugular vein: successful treatment with metronidazole. Journal of the American Medical Association 230: 1168-9 (1974). Moore, G. T., Cross, W. M., McGuire, D., Mollohan, C. S., Gleason, W. N., Healy, G. R. & Newron, L. H. Epidemic giardiasis at a ski resort. New England Journal of Medicine 281: 402-7 (1969). O'Brien, R. W. & Morris, J. G. Effect of metronidazole on hydrogen production by Clostridium acetobutylicum. Archiv fur Mikrobiologie 84: 225-33 (1972). Powell, S. J., McLeod, I., Wilmot, A. J. & Elsdon Dew, R. Metronidazole in amoebic dysentery and amoebic liver abscess. Lancet ii: 1329-31 (1966). Pye, R. J. & Burton, J. L. Treatment of rosacea by metronidazole. Lancet i: 1211-2 (1976). Roe, F. J. C. & Tucker, M. J. Excerpta Medica International Congress Series 311: 171 (1974). Roe, F. J. C , Ingham, B. & Lowe, C. Y. Metronidazole: tumorigenicity study in hamsters. Report submitted for publication. Rosenkranz, H. S. Bacterial mutagenicity studies. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A. & Roe, F. J. C , Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press. Rubidge, C. J., Scragg, J. N. & Powell, S. J. Treatment of children with acute amoebic dysentery. Comparative trial of metronidazole against a combination of dehydroemetine, tetracycline and diloxanide furoate. Archives of Disease in Childhood 45: 196-7 (1970). Rust, J. H. Tumorigenicity studies—mouse and rat. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A. & Roe, F. J. C , Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press. Rustia, M. & Shubik, P. Induction of lung tumours and malignant lymphomas in mice by metronidazole. Journal of the National Cancer Institute 48: 721-9 (1972). Rustia, M. Personal communication (1975). Salem, A. R., Jackson, D. D. & McFadzean, J. A. An investigation of interactions between metronidazole ('Flagyl') and other antibacterial agents. Journal of Antimicrobial Chemotherapy 1: 387-91 (1975). Schneider, J. Treatment of giardiasis (lambliasis) with metronidazole. Bulletin de la Sociiti de Pathologie Exotique 54: 84-95 (1961). Searle Laboratories. Metronidazole for anaerobic infections. Investigational Brochure (1973). Semer, J. M., Friedland, P., Vaisberg, M. & Greenberg, A. The use of metronidazole in the treatment of alcoholism. American Journal of Psychiatry 123: 722-4 (1966). Schwartz, D. E. & Jeunet, F. Comparative pharmacokinetic studies of ornidazole and metronidazole in men. Chemotherapy 22: 19-29 (1976). Shinn, D. L. S. Metronidazole in acute ulcerative gingivitis. Lancet i: 1191 (1962). Shinn, D. L. S., Squires, S. & McFadzean, J. A. The treatment of Vincent's disease with metronidazole. Dental Practitioner 15: 275-80 (1965). Stambaugh, J. E., Feo, L. G. & Manthei, R. W. The isolation and identification of the urinary oxidative metabolites of metronidazole in man. Journal of Pharmacology and Experimental Therapeutics 161: 373-81 (1968). Stone, H. B. & Withers, H. R. Tumour and normal tissue response to metronidazole and irradiation in mice. Radiology 113: 441-44 (1974).
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Tally, F. P., Sutter, V. L. &Finegold, S. M. Metronidazole versus anaerobes. California Medicine 117: 22-6 (1972). Tally, F. P., Sutter, V. L. & Finegold, S. M. Treatment of anaerobic infections with metronidazole. Antimicrobial Agents and Chemotherapy 7: 672-5 (1975). Ursing, B. & Kamme, C. Metronidazole for Crohn's disease. Lancet i: 775-7 (1975). Urtasun, R., Band, P., Chapman, J. D., Feldstein, M. L., Mielke, B. & Fryer, C. Radiation and high-dose metronidazole in supratentorial glioblastomas. New England Journal of Medicine 294: 1364-7(1976). Voogd, C. E., Van der Stel, J. J. & Jacobs, J. A. The mutagenic action of nitroimidazoles metronidazole, nimorazole, dimetridazole and ronidazole. Mutation Research 26: 483-90 (1974). Willis, A. T. & Study Group. Metronidazole in the prevention and treatment of Bacteroides infections in gynaecological patients. Lancet ii: 1540-3 (1974). Willis, A. T. & Study Group. An evaluation of metronidazole in the prophylaxis and treatment of anaerobic infections in surgical patients. Journal of Antimicrobial Chemotherapy 1: 393-401 (1975). Willis, A. T. Prophylactic use in patients undergoing hysterectomy and appendectomy. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A & Roe, F. J. C. Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press. Willis, A. T. & Study Group. Metronidazole in prevention and treatment of Bacteroides infections after appendicectomy. British Medical Journal i: 318-21 (1976). Willson, R. L., Cramp, W. A. & Ings, R. M. J. Metronidazole ('Flagyl') mechanisms of radiosensitization. International Journal of Radiation Biology 26: 557-69 (1974). Willson, R. L. Acute drug administration and cancer control. Lancet i: 810—1 (1974). Wright, S. A. Giardiasis and its treatment. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A. & Roe, F. J. C , Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press. Yoch, D. C. & Valentine, R. C. Ferredoxin and flavodoxins of bacteria. Annual Review of Microbiology 26: 139-62 (1972).
Metronidazole: review of uses and toxicity
F. J. c. Roe
The development of a trichomonacide In 1954 Rhone-Poulenc decided to develop a drug effective in the treatment of Trichomonas vaginalis infection. The availability of in vitro and in vivo (mouse) systems for evaluating potential trichomonacides facilitated research and in due course enabled the discovery that azomycin (2-nitroimidazole), isolated from Streptomyces 6670, had weak trichomonacidal activity. Over 200 chemicals related to azomycin were then systematically synthesized and screened for activity. In terms of activity relative to toxicity, metronidazole (l-hydroxyethyl-2-methyl-5-nitroimidazole, 'FlagyP) emerged as the most promising agent and the first evaluation of its efficacy against T. vaginalis infection began in France in 1958. The value of the drug, which was the first systemically active trichomonacide, was immediately apparent (Durel, Roiron, Siboulet & Borel, 1959) and within a few years its use became widespread all over the world. In France, the usual practice has been to combine oral treatment with vaginal pessaries. In other countries, pessaries are much less prescribed because there is no real evidence that they usefully supplement the effects of oral tablets and treatment with pessaries alone often fails because the drug does not reach trichomonads harbouring in the genitourinary tract. Cure usually results from total doses of the drug rather less than those hitherto thought to be necessary. Thus cure rates of 95 % or better can be expected from regimens of 200 mg three times per day for 7 days or a single dose of 2 g by mouth. Parallel treatment of male consorts with the drug at the same dosage as given to the patient is necessary to prevent reinfection. Remarkably there is almost no confirmed evidence of resistance of trichomonads to metronidazole, and in the very few patients in whom failure of treatment cannot be attributed to reinfection or non-taking of tablets, poor absorption of the drug from the gut or its excessive destruction by vaginal flora are the commonest explanations. Amoebiasis and giardiasis The original biological evaluation of metronidazole at Rhone-Poulenc (Cosar, Ganter & Julou, 1961) indicated that metronidazole might be useful against E. histolytica, but it was not until 5 years later that its value in this regard was realized (Powell, MacLeod, Wilmot & Elsdon Dew, 1966). Relatively low dosage regimes were enough to kill vegetative amoebae in the gut wall and liver but higher dosages were found to be necessary 205
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4, Kings Road, Wimbledon, London SW19 8QN, England
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Anaerobic bacterial infections During the last few years the clinical value of metronidazole in the treatment and prevention of other diseases has been becoming increasingly apparent. The most important advance relates to the highly specific effect of metronidazole against obligatory anaerobic micro-organisms. It is only during the last decade that methods of obtaining uncontaminated samples of pus and techniques for growing and identifying strict anaerobes have been perfected and become widely available (Holdeman & Moore, 1972). As a consequence, many of those brain abscesses, pelvic abscesses and weeping skin wounds once thought to be yielding 'sterile pus' are now known to be due to infection with a variety of anaerobes such as Bacteroides fragilis, Clostridia, Peptostreptococci, Fusobacteria and many others (Gorbach & Bartlett, 1974; Finegold, 1974). It was a serendipitous observation in 1962 that led eventually to the realization of the value of metronidazole in the treatment of infections involving obligatory anaerobes: Shinn (1962) and Shinn, Squires & McFadzean (1965) reported the cure of long-standing Vincent's infection in a woman who received a course of treatment with metronidazole for T. vaginalis infection. Laboratory studies (Davies, McFadzean & Squires, 1964) undertaken in the wake of Shinn's observation showed that the drug is intensely active against certain species of anaerobic bacteria and its possible value in the prevention and treatment of tetanus and gas
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for the elimination of trophozoites from the lumen of the gut and thereby prevent parasitological relapse. Adams (1977) reported findings in a series of 5057 cases of amoebiasis seen in the King Edward VIII Hospital, Durban over a 20 year period. Death rates for uncomplicated amoebic dysentery or liver abscess were less than 1 % but complicating peritonitis or pericarditis were associated with greatly increased risk of fatal outcome. The results of a series of clinical trials have shown that a course of 800 mg metronidazole 3 times per day for 5 days was equal to the best of all other treatment regimens for amoebic dysentery, and, combined with aspiration, highly efficacious in the treatment of amoebic liver abscess. Tetracycline, which is very effective for uncomplicated amoebic dysentery, is not a tissue amoebicide and therefore of no value in cases of liver abscess or where there are complications. Although emetine and chloroquine alone or combined, are highly effective against liver abscess, they cause more unpleasant side effects. The therapeutic activity of metronidazole in human giardiasis was first fully demonstrated by Schneider (1961). The development of techniques for jejunal aspiration and jejunal mucosal impression smears from jejunal biopsy specimens (Kamath & Murgasu, 1974) has clarified the role of Giardia lamblia as a cause of diarrhoeal disease in both tropical (Anati, Desai, Jeejeethoy, Kane & Borkar, 1966) and temperate climates (Moore et ah, 1969; Brodsky, Spencer & Schultz, 1974), particularly in immunodeficient subjects (Ament & Rubin, 1972). Wright (1977) reported the eradication of the parasite in 28 out of 31 subjects following a 3 day course of metronidazole in which 2 g was given as a single dose once each day. Metronidazole, which has only slight activity against Trypanosoma cruzi, has given equivocal results in the treatment of chronic Chagas' disease and a similar clinical situation obtains in the treatment of cutaneous leishmaniasis although anti-leishmania activity has not been demonstrated experimentally. Against Balantidium coli, however, metronidazole is highly active and clinical and parasitological cures have been obtained.
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Willis (1977) obtained equally good results with rectal metronidazole in colonic surgery and Eykyn (1977) reported the use of intravenous metronidazole, given alone or with antibiotics, in the treatment of 24 patients with severe anaerobic infections; no side effects were observed and improvement, sometimes dramatic and life-saving, was seen in over 70%. Recently, Pye & Burton (1976) reported a favourable response to oral metronidazole in cases of papulopustular rosacea. Radiosensitization and anticancer activity Hypoxia increases resistance to ionizing radiation and the selective survival of hypoxic cells is thought to reduce response to radiotherapy. Accordingly, agents which sensitize such cells to ionizing radiation have been sought. Under both in vitro conditions and in vivo in mice, a number of nitroimidazoles, including metronidazole, have been found to be effective in this regard (Chapman, Reuvers & Borsa, 1973; Asquith, Foster, Willson & Study Group, 1974; Denekamp, Michael & Harris, 1974; Stone & Withers, 1974). Willson, Cramp & Ings, (1974) and Foster, Conroy, Searle & Willson (1976) have considered the mechanisms underlying radio-sensitization by metronidazole and it is interesting to speculate that the drug may have some direct cytocidal effect on tumour cells that have adopted an anaerobic way of life. Thus, Urtasun et al. (1976) has reported promising prolongation of survival in a trial of combined effects of ionizing radiation and massive doses of metronidazole (6 g/m2 thrice weekly for 3 weeks). Toxicity and side effects Acute and sub-acute toxicity studies in mice and rats have indicated LD50 dosages in the 1 to 5 g/kg range.
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gangrene was postulated (Freeman, McFadzean & Whelan, 1968), but it was not until 1972 that Tally, Sutter and Finegold reported its successful use in the treatment of Bacteroides infections. Today metronidazole takes its place alongside chloramphenicol, lincomycin and clindamycin in the treatment of anaerobic bacterial infections. Metronidazole has no activity against aerobic organisms and this might be thought to be a disadvantage in the case of mixed infections. However, it is compatible with the commonly used antibacterial antibiotics (Salem, Jackson & McFadzean, 1975) and the specificity of its activity against anaerobes makes it a useful tool for the investigation, if not the treatment, of conditions such as ulcerative colitis and Crohn's disease (Ursing & Kamme, 1975). The last few years have seen reports on the value of metronidazole in the treatment of various anaerobic infections including septicaemia, necrotizing pneumonia, and pulmonary abscess (Mitre & Rotheram, 1974; Tally, Sutter & Finegold, 1975), post-hysterectomy pelvic cellulitis and anaerobic intra-uterine infections (Willis and Study Group, 1974), brain abscess (Ingham et al., 1975; Ingham, Selkon, So & Weiser, 1975) and breast abscess (Hale, Perinpanayagam & Smith, 1976). Pre- and post-operative oral treatment with metronidazole greatly reduced the incidence of post-operative non-clostridial anaerobic infections after hysterectomy (Willis & Study Group, 1975) and analogous treatment with metronidazole in rectal suppositories prevented anaerobic sepsis after appendicectomy (Willis & Study Group, 1976). Workers in Glasgow (Goldring, Scott, McNaught & Gillespie, 1975) reported good results with pre-operative oral metronidazole and kanamycin in the prevention of sepsis after colonic surgery.
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More recently metronidazole has been given in higher dosages and for several weeks or months and some adverse reactions have occurred with greater frequency and severity; in patients given dosages of over 180 mg/kg/day as an adjunct to radiotherapy, severe anorexia, nausea and vomiting, which tended to persist for 24 to 48 h after the last dose, sometimes necessitated withdrawal of the drug (Deutsch, Foster, McFadzean & Parnell, 1975). Epileptiform seizures have been noted by other investigators in a few cases. Some patients undergoing prolonged metronidazole therapy, e.g. for the treatment of Crohn's disease and, in one instance, for brain abscess, have experienced paraesthesiae and signs of peripheral neuropathy which disappeared when dosage was reduced or stopped (Ursing & Kamme, 1975; Ingham et al, 1975). As a nitro compound in widespread therapeutic use, metronidazole attracted the attention of oncologists with special interests in tumorigenicity and mutagenicity, and Rustia & Shubik (1972) reported an increased incidence of lung tumours in male and female Swiss mice and an increased incidence of malignant lymphoma in female mice only in response to metronidazole given continuously in the diet for 18 months. Both types of neoplasm occur in high incidence spontaneously and the increased incidences seen were only of the same order as those that can be achieved by simple dietary manipulation (Roe & Tucker, 1974). Rust (1977) has recently reported that two other studies in mice confirmed the effect on lung tumour incidence but not that on the incidence of malignant lymphoma and that in rats a slight but not significant excess of mammary tumours was seen in one small study (Cohen et al., 1973) but not confirmed in a much
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In chronic toxicity studies in rats, daily dosages of 75 or 150 mg/kg given orally for 26 to 80 weeks produced no adverse reactions, but dosages of 300 or 600 mg/kg/day caused testicular dystrophy and prostatic atrophy. Chronic toxicity studies in dogs showed them to be peculiarly susceptible to ataxia, muscular rigidity and tremors at dosages of over 75 mg/kg/day. However, monkeys medicated for one year with dosages of 45, 100 or 225 mg/kg/day, showed no evidence of chronic toxicity except for minimal non-specific changes in the livers of a few animals receiving the highest dosage. Conventional animal tests for embryopathy and effects on reproduction have given negative results (Searle Labs. Investigational Brochure, 1973). During the 15 years of its widespread use for the treatment of T. vaginalis infections, metronidazole has earned a reputation of being remarkably safe. Occasional side effects at the dosages ordinarily used for the treatment of this condition include nausea, an unpleasant taste in the mouth, furring of the tongue and gastrointestinal upsets; headache, dizziness, ataxia, depression and skin eruptions have been reported but rarely. A mild to moderate disulfiram-like effect can occur when alcohol is taken during metronidazole therapy; the severity of the reaction is proportional to the amount of alcohol ingested (Semer, Friedland, Vaisberg & Greenberg, 1966). Despite the profound effect of the drug on the population of anaerobic bacteria of the gut, diarrhoea has not featured prominently among its side effects. In animals enlargement of the caecum has been recorded suggesting an inhibitory effect on the metabolism of certain complex carbohydrates and other substances (probably by gut flora). Transient leucocytopenia has been observed in humans (Willson, 1974) but there have been no reports of serious blood dyscrasias. It should, perhaps, not be given to women during the first trimester of pregnancy or during lactation, since it freely passes the placental barrier and is excreted in milk. On the other hand no case of damage to the foetus or suckling child has been reported where the drug has been administered at these times. Experience indicates that the drug may be safely administered to children (Rubidge, Scragg & Powell, 1970).
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Metabolism biochemistry, and mechanisms of action
As with many other drugs which have been found effective in therapy, precise knowledge of how metronidazole acts is limited. It is clear that metronidazole enters aerobic bacteria just as readily as it enters anaerobic ones, but whereas in the former it remains unchanged and its concentration equilibrates with that in the medium, in the latter it is biochemically reduced, the equilibrium is disturbed and more drug diffuses into the cell (Ings, McFadzean & Ormerod, 1974). Reduction is probably effected by low redox potential electron transport proteins (e.g. ferredoxin or flavodoxin) (O'Brien & Morris, 1972) which play a major role in the metabolism of anaerobes and photosynthetic organisms but have a limited role in aerobes (Yoch & Valentine, 1972). What happens to the reduced metronidazole is largely a mystery, as is the basis of the drug's cytocidal effect. However, it seems that some of the reduced drug binds to cell constituents and that the bound products are non-dialysable. Inhibition of nucleic acid synthesis has been reported (Ings, McFadzean & Ormerod, 1974). In both animals and humans similar ranges of metabolites have been identified (Ings, Law & Parnell, 1966; Stambaugh, Feo & Manthei, 1968) and in humans the urine is the main route of clearance (Schwartz & Jeunet, 1976). Despite this, McHenry (1977) found no evidence that a single oral dose of 500 mg metronidazole was handled differently in volunteers with absent or severely impaired renal function than in those with normal renal function. References Adams, E. B. Amoebiasis and its treatment. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S., McFadzean, J. A. & Roe, F. J. C. Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press.
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larger one. Two carcinogenicity studies in hamsters gave entirely negative results in respect of tumorigenicity (Rustia, 1975; Roe, Ingham & Lowe, 1976). The effect on tumour incidence in mice reported by Rustia and Shubik involved the administration throughout the lives of the animals of total doses, on a mg/kg body weight basis, equivalent to between 350 and 3000 times that given to patients in the form of a 10 day course for the treatment of trichomoniasis. In 1974, metronidazole was reported as having increased the spontaneous mutation rates of certain strains of aerobic bacteria grown in vitro (Voogd, Van der Stel & Jacobs, 1974) and later Rosenkranz (1977) reported that in animals and in humans, the drug can be bio-transformed to substances which are mutagenic for a strain of S. typhimurium. On the other hand Bost (1977) reported that conventional dominant lethal tests in rats and in mice had given negative results. Recently, Mitelman, Hartley-Asp & Ursing (1976) reported chromosomal abnormalities in the circulating lymphocytes of patients with Crohn's disease receiving metronidazole. In the light of the animal data a small and indefinite cloud hangs over metronidazole as it does over many other drugs, in relation to possible carcinogenicity and mutagenicity. However, in the case of metronidazole it is important to take into account the likelihood that cure of trichomoniasis reduces risk of development of cancer of the uterine cervix. It is well established that the risk of this form of cancer is greatly influenced by sexual hygiene standards {British MedicalJournal 1965) so that it is very likely that the elimination of T. vaginalis infection and of secondary non-specific vaginal infections which accompany it reduces cervical cancer risk.
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Ament, M. E. & Rubin, C. Relation of giardiasis to abnormal intestinal structure and function in gastrointestinal immunodeficiency syndromes Gastroenterology 62: 216-27 (1972). Anati, F. P., Desai, H. G., Jeejeethoy, K. N., Kane, M. P. & Borkar, V. V. Indian Journal of Medical Science 20: 471 (1966). Asquith, J. C , Foster, J. L., Willson, R. L. & Study Group. Metronidazole ('Flagyl'). A radiosensitizer of hypoxic cells. British Journal of Radiology 47:474-81 (1974). British Medical Journal. Cervical cancer: early diagnosis or prevention (Leading article) i: 1327-8 (1965). Bost, R. G. Dominant Lethal Test. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S., McFadzean, J. A. & Roe, F. J. C. Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press. Brodsky, R. E., Spencer, H. C. & Schultz, M. G. Giardiasis in American travellers to the Soviet Union. Journal of Infectious Diseases 130: 319-23 (1974). Chapman, J. D., Reuvers, A. P. & Borsa, J. Effectiveness of nitrofuran derivatives in sensitizing hypoxic mammalian cells to X-rays. British Journal of Radiology 46: 623-30 (1973). Cohen, S. M., Erturk, E., Crovetti, A. J. & Bryan, C. T. Carcinogenicity of 5-nitroimidazoles,4nitrobenzenes and related compounds. Journal of the National Cancer Institute 51: 403-17 (1973). Cosar, C , Ganter, P. & Julou, L. Etude experimentale du metronidazole (8823 R.P.). Activites trichomonacide et amoebicide. Toxicit6 et proprietes pharmacologiques generates. Presse Midicale 69: 1069-72 (1961). Davies, A. H., McFadzean, J. A. & Squires, S. Treatment of Vincent's stomatitis with metronidazole. British Medical Journal i: 1149-50 (1964). Denekamp, J., Michael, B. D. & Harris, S. R. Hypoxic cell radiosensitizers: Comparative tests of some electron affinic compounds using epidermal cell survival in vivo. Radiation Research 60: 119(1974). Deutsch, G., Foster, J. L., McFadzean, J. A. & Parnell, M. Human studies with 'high dose' metronidazole: a non-toxic radiosensitizer of hypoxic cells. British Journal of Cancer 31:75-80(1975). Durel, P., Roiron, V., Siboulet, H. & Borel, L. J. Trial of an anti-trichomonal derivative of imidazole (8823 RP). Comptes Rendus de la Societi Francaise de Gynicologie 29: 36, Jan. (1959). Eykyn, S. The value of intravenous metronidazole in the treatment of patients with anaerobic infections. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A. & Roe, F. J. G, Eds.). Journal of Infectious Diseases 1977 (Suppl.): in press. Finegold, S. M. Intra-abdominal, genito-urinary, skin and soft tissue infections due to non-spore forming anaerobic bacteria. Infection with non-sporing anaerobic bacteria, (Phillips & Sussman, Eds.). Churchill/Livingstone, London (1974), pp. 160-87. Foster, J. L., Conroy, P. J., Searle, A. J. & Willson, R. L. Metronidazole ('Flagyl'): characterization as a cytotoxic drug specific for hypoxic tumour cells. British Journal of Cancer 33: 485-90 (1976). Freeman, W. A., McFadzean, J. A. & Whelan, J. P. F. Activity of metronidazole against experimental tetanus and gas gangrene. Journal of Applied Bacteriology 31: 443-7 (1968). Goldring, J., Scott, A., McNaught, W. & Gillespie, G. Prophylactic oral antimicrobial agents in elective colonic surgery: a controlled trial. Lancet ii: 997-1000 (1975). Gorbach, S. L. & Bartlett, J. C. Anaerobic infections (third of three parts). New England Journal of Medicine 290: 1289-94 (1974). Hale, J. E., Perinpanayagam, R. M. & Smith, G. Bacteroides: an unusual case of breast abscess. Lancet ii: 70-1 (1976). Holdeman, L. V. & Moore, W. E. C. Anaerobic Laboratory Manual. The Virginia Polytechnic Institute Anaerobe Laboratory, Blacksburg, VA. U.S.A. (1972). Ingham, H. R., Rich, G. E., Selkon, J. B., Hale, J. H., Roxby, C. M., Betty, M. J., Johnson R. W. C. & Uldall, P. R. Treatment with metronidazole of three patients with serious infections due to Bacteroides fragilis. Journal of Antimicrobial Chemotherapy 1:235-42 (1975). Ingham, H. R., Selkon, J. B., So, S. C. & Weiser, R. Brain abscess. British Medical Journal iv: 39-40 (1975).
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Ings, R. M. J., Law, G. L. & Parnell, E. W. The metabolism of metronidazole. Biochemistry and Pharmacology 15: 515-9 (1966). Ings, R. M. J., McFadzean, J. A. & Ormerod, W. E. The mode of action of metronidazole in Trichomonas vaginalis and other micro-organisms. Biochemistry Pharmacology 23: 1421-9 (1974). Kamath, K. R. & Murgasu, R. A comparative study of four methods for detecting Giardia lamblia in children with diarrhoeal disease and malabsorption. Gastroenterology 66:16-21 (1974). McHenry, M. C. Use in impaired renal function. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A. & Roe, F. J. C. Eds.) Journal of Infectious Disease* 1977 (Suppl.) in press. Mitelman, F., Hartley-Asp, B. & Ursing, B. Chromosome aberrations and metronidazole. Lancet ii: 802 (1976). Mitre, R. J. & Rotheram, E. B. Anaerobic septicaemia from thrombo-phlebitis of the internal jugular vein: successful treatment with metronidazole. Journal of the American Medical Association 230: 1168-9 (1974). Moore, G. T., Cross, W. M., McGuire, D., Mollohan, C. S., Gleason, W. N., Healy, G. R. & Newron, L. H. Epidemic giardiasis at a ski resort. New England Journal of Medicine 281: 402-7 (1969). O'Brien, R. W. & Morris, J. G. Effect of metronidazole on hydrogen production by Clostridium acetobutylicum. Archiv fur Mikrobiologie 84: 225-33 (1972). Powell, S. J., McLeod, I., Wilmot, A. J. & Elsdon Dew, R. Metronidazole in amoebic dysentery and amoebic liver abscess. Lancet ii: 1329-31 (1966). Pye, R. J. & Burton, J. L. Treatment of rosacea by metronidazole. Lancet i: 1211-2 (1976). Roe, F. J. C. & Tucker, M. J. Excerpta Medica International Congress Series 311: 171 (1974). Roe, F. J. C , Ingham, B. & Lowe, C. Y. Metronidazole: tumorigenicity study in hamsters. Report submitted for publication. Rosenkranz, H. S. Bacterial mutagenicity studies. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A. & Roe, F. J. C , Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press. Rubidge, C. J., Scragg, J. N. & Powell, S. J. Treatment of children with acute amoebic dysentery. Comparative trial of metronidazole against a combination of dehydroemetine, tetracycline and diloxanide furoate. Archives of Disease in Childhood 45: 196-7 (1970). Rust, J. H. Tumorigenicity studies—mouse and rat. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A. & Roe, F. J. C , Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press. Rustia, M. & Shubik, P. Induction of lung tumours and malignant lymphomas in mice by metronidazole. Journal of the National Cancer Institute 48: 721-9 (1972). Rustia, M. Personal communication (1975). Salem, A. R., Jackson, D. D. & McFadzean, J. A. An investigation of interactions between metronidazole ('Flagyl') and other antibacterial agents. Journal of Antimicrobial Chemotherapy 1: 387-91 (1975). Schneider, J. Treatment of giardiasis (lambliasis) with metronidazole. Bulletin de la Sociiti de Pathologie Exotique 54: 84-95 (1961). Searle Laboratories. Metronidazole for anaerobic infections. Investigational Brochure (1973). Semer, J. M., Friedland, P., Vaisberg, M. & Greenberg, A. The use of metronidazole in the treatment of alcoholism. American Journal of Psychiatry 123: 722-4 (1966). Schwartz, D. E. & Jeunet, F. Comparative pharmacokinetic studies of ornidazole and metronidazole in men. Chemotherapy 22: 19-29 (1976). Shinn, D. L. S. Metronidazole in acute ulcerative gingivitis. Lancet i: 1191 (1962). Shinn, D. L. S., Squires, S. & McFadzean, J. A. The treatment of Vincent's disease with metronidazole. Dental Practitioner 15: 275-80 (1965). Stambaugh, J. E., Feo, L. G. & Manthei, R. W. The isolation and identification of the urinary oxidative metabolites of metronidazole in man. Journal of Pharmacology and Experimental Therapeutics 161: 373-81 (1968). Stone, H. B. & Withers, H. R. Tumour and normal tissue response to metronidazole and irradiation in mice. Radiology 113: 441-44 (1974).
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Tally, F. P., Sutter, V. L. &Finegold, S. M. Metronidazole versus anaerobes. California Medicine 117: 22-6 (1972). Tally, F. P., Sutter, V. L. & Finegold, S. M. Treatment of anaerobic infections with metronidazole. Antimicrobial Agents and Chemotherapy 7: 672-5 (1975). Ursing, B. & Kamme, C. Metronidazole for Crohn's disease. Lancet i: 775-7 (1975). Urtasun, R., Band, P., Chapman, J. D., Feldstein, M. L., Mielke, B. & Fryer, C. Radiation and high-dose metronidazole in supratentorial glioblastomas. New England Journal of Medicine 294: 1364-7(1976). Voogd, C. E., Van der Stel, J. J. & Jacobs, J. A. The mutagenic action of nitroimidazoles metronidazole, nimorazole, dimetridazole and ronidazole. Mutation Research 26: 483-90 (1974). Willis, A. T. & Study Group. Metronidazole in the prevention and treatment of Bacteroides infections in gynaecological patients. Lancet ii: 1540-3 (1974). Willis, A. T. & Study Group. An evaluation of metronidazole in the prophylaxis and treatment of anaerobic infections in surgical patients. Journal of Antimicrobial Chemotherapy 1: 393-401 (1975). Willis, A. T. Prophylactic use in patients undergoing hysterectomy and appendectomy. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A & Roe, F. J. C. Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press. Willis, A. T. & Study Group. Metronidazole in prevention and treatment of Bacteroides infections after appendicectomy. British Medical Journal i: 318-21 (1976). Willson, R. L., Cramp, W. A. & Ings, R. M. J. Metronidazole ('Flagyl') mechanisms of radiosensitization. International Journal of Radiation Biology 26: 557-69 (1974). Willson, R. L. Acute drug administration and cancer control. Lancet i: 810—1 (1974). Wright, S. A. Giardiasis and its treatment. In Proceedings of Conference on Metronidazole, Montreal, 26-28th May, 1976 (Finegold, S. M., McFadzean, J. A. & Roe, F. J. C , Eds.). Journal of Infectious Diseases 1977 (Suppl.) in press. Yoch, D. C. & Valentine, R. C. Ferredoxin and flavodoxins of bacteria. Annual Review of Microbiology 26: 139-62 (1972).
