Phase II study of oral docetaxel and cyclosporine in canine epithelial cancer.

The goal of the current study was to determine the efficacy of oral docetaxel in combination with cyclosporine in the treatment of canine epithelial cancer. Requirements for eligibility were histological confirmation of epithelial neoplasia, measurable disease, no chemotherapy treatment within 2 weeks, and a life expectancy of ≥ 3 months. Fifty-one dogs were enrolled. All dogs received 1.625 mg kg(-1) of docetaxel with 5 mg kg(-1) of cyclosporine (DT/CSA) by gavage. Ten dogs had progressive disease at 2 weeks, one dog died, and one dog was withdrawn from the study. Thirty-nine dogs were given a second dose of DT/CSA, three each receiving a third or fourth dose. Eight dogs had a dose reduction (1.5 mg kg(-1)) and six dogs had treatment delays primarily for gastrointestinal toxicity. The overall response rate was 16.7% (8/48 had a partial response there were no complete responses). The highest response rate was seen in dogs with oral squamous cell carcinoma (50%; 6/12).

Phase I and pharmacokinetic evaluation of the combination of orally administered docetaxel and cyclosporin A in tumor-bearing cats.

Intravenously administered docetaxel (DT) is problematic in cats because of the requirement for premedication to ameliorate acute vehicle-induced hypersensitivity reactions. Previously we have revealed that therapeutic plasma concentrations of DT can be achieved in normal and tumor-bearing dogs when DT is administered PO in combination with oral cyclosporin A (CSA). The purpose of this study was to identify the maximally tolerated dosage and characterize the pharmacokinetic disposition of oral DT combined with CSA in cats with tumors. Eighteen tumor-bearing cats were enrolled in this phase I dose escalation and pharmacokinetic study. DT was administered by gavage with CSA (5 mg/kg) twice over a 3-week period. The starting dose of DT was 1.0 mg/kg. Based on the clinical toxicity profile, with gastrointestinal adverse effects and hematologic toxicity the maximal tolerated dose of oral DT was 1.75 mg/kg in combination with 5 mg/kg CSA. Additional studies are necessary to determine the efficacy of DT/CSA in cats with epithelial tumors.

Mammalian cell toxicity and bacterial mutagenicity of nitrosoimidazoles.

It is currently believed that the biological activity of such therapeutic 5-nitroimidazoles as metronidazole is mediated by a short-lived, highly toxic species that arises from nitro group reduction. We found that the 5-nitroimidazole, 1-methyl-4-phenyl-5-nitroimidazole (5-NO2), is at least 1000-fold less cytotoxic for CHO cells and mutagenic for Ames tester strain TA100 than its homologous nitroso compound, 1-methyl-4-phenyl-5-nitrosoimidazole (5-NO). Such evidence, along with previous work showing a similar relative bactericidal potency of these compounds, is consistent with the labile nitrosoimidazole being a biologically active species of the nitroimidazole, and indicates that mammalian cells are very susceptible to such an active form. The high potency of both 5-NO and 1-methyl-4-nitroso-5-phenylimidazole (4-NO), in contrast to the lack of potency of 1-methyl-4-nitro-5-phenylimidazole (4-NO2) relative to 5-NO2, is additional evidence to support the suggestion that the activity of a nitroimidazole is determined mainly by the ease with which it is reduced.

Nitrosoimidazoles: highly bactericidal analogues of 5-nitroimidazole drugs.

It is believed that metronidazole and related 5-nitroimidazoles are activated by reduction of the nitro group and that the active species has a nitrogen functionality of intermediate oxidation state. However, the preparation and isolation of the active forms of the 5-nitroimidazoles used therapeutically have proven elusive. To pursue this problem we have prepared both 1-methyl-4-phenyl-5-nitrosoimidazole (3) and 1-methyl-4-nitroso-5-phenylimidazole (5) from 4(5)-nitroso-5(4)-phenylimidazole (1). We have also prepared the homologous nitroimidazoles. Escherichia coli mutants with defects in DNA repair were found to be sensitive to both 1-methyl-4-phenyl-5-nitroimidazole (4) and metronidazole, but fairly resistant to 1-methyl-4-nitro-5-phenylimidazole (6), a finding in accord with the relative biological activity of 4- and 5-nitroimidazoles examined previously. In contrast, all three nitroso compounds are considerably more bactericidal than their analogous nitro compounds under both aerobic and anaerobic conditions, a finding that provides direct evidence that reduction of the nitro group is responsible for activation of the nitroimidazoles. Further evidence is also consistent with the possibility that the nitrosoimidazoles are themselves biologically active species derived from nitroimidazoles, although a more conservative interpretation is simply that they are more facilely converted to such active species.

Chemical and biological properties of acetyl derivatives of the hydroxylamino reduction products of metronidazole and dimetridazole.

Metronidazole and related 5-nitroimidazoles undergo reduction of their nitro group apparently to produce such reactive species as 5-hydroxylaminoimidazoles. To define the role of these species we have sought ways to prepare them by the catalytic reduction of metronidazole, dimetridazole and flunidazole. Although their respective 5-hydroxylaminoimidazoles were too unstable to be isolated directly, their O,N-diacetyl derivatives were isolable. Of these, the diacetyl derivative of the hydroxylamine derived from dimetridazole, O,N-diacetyl-1,2-dimethyl-5-hydroxylaminoimidazole (DiacDMH), was used for further study. DiacDMH was converted to its monoacetyl derivative, N-acetyl-1,2-dimethyl-5-hydroxylaminoimidazole (AcDMH), by enzymatic deacylation. Both DiacDMH and AcDMH were examined for bactericidal activity against such strains as Bacteroides fragilis, Clostridium perfringens, and Escherichia coli strain SR58, which are known to be sensitive to dimetridazole, as well as a variety of other bacteria. No bactericidal activity was detected, even in the presence of deacetylating enzymes. As the 5-hydroxylaminoimidazole itself could not be shown to form in these bacterial incubations, it remains uncertain whether or not the hydroxylamino functionality of a 5-nitroimidazole has bactericidal activity.

Formation of an amino reduction product of metronidazole in bacterial cultures: lack of bactericidal activity.

To investigate whether the amino reduction product of metronidazole has antibacterial activity, 5-amino-1-beta-hydroxyethyl-2-methylimidazole (AMN) was synthesized and tested against Bacteroides fragilis and Escherichia coli strain SR58, both of which are known to be sensitive to metronidazole. Neither of these strains was found to be sensitive either to AMN or to the equivalent amine derived from dimetridazole, 5-amino-1,2-dimethylimidazole. Both of these amines are relatively stable in the presence of bacteria, making it possible to examine the bacterial reduction of radiolabeled metronidazole in the presence of AMN. This experiment indicated that at least 17% of the metronidazole that disappeared under the reducing conditions of the bacterial medium was converted to AMN. We conclude, therefore, that AMN forms during the activation of metronidazole by bacterial reduction but is not a bactericidal form of metronidazole.

Interaction of metronidazole with DNA repair mutants of Escherichia coli.

It has been proposed that one of metronidazole's partially reduced intermediates interacts either with DNA to exert a bactericidal effect or with water to form acetamide. To test this hypothesis we have examined the effect of metronidazole on several mutants of Escherichia coli that are defective in DNA repair. UV-susceptible RecA- and UvrB- point mutants have an increased susceptibility to metronidazole as manifested by both a decreased minimal inhibitory concentration and a greater bactericidal response to metronidazole in resting cultures. By these criteria, however, we find that UvrB- deletion mutants, which lack the ability to reduce nitrate and chlorate, are no more susceptible to metronidazole than is the wild type. We find, however, that these deletion mutants also lack the ability to reduce metronidazole and thus possibly to form its reactive species. When metronidazole's bactericidal effect is expressed in terms of the concurrent accumulation of acetamide derived from metronidazole, then all RecA- and UvrB- mutants are killed more efficiently than their wild types. The data are consistent, therefore, with metronidazole's lethal effect being mediated by a partially reduced intermediate on the metabolic pathway between metronidazole and acetamide. Defects in other aspects of the DNA repair system do not confer this increased susceptibility to the proposed intermediate. A Tag- mutant, for example, which is defective in 3-methyl-adenine-DNA glycosylase, does not have this increased susceptibility to the presumed precursor of acetamide. Thus, these results provide further support for the hypothesis that the bactericidal effect of metronidazole is mediated by a partially reduced intermediate in the metabolic conversion of metronidazole to acetamide and suggest that this intermediate interacts with DNA to produce a lesion similar to that caused by UV light.

Metronidazole metabolism in cultures of Entamoeba histolytica and Trichomonas vaginalis.

Acetamide forms from metronidazole in cultures of either Entamoeba histolytica or Trichomonas vaginalis as it does in cultures of bacteria which are susceptible to this drug. Under aerobic conditions, the formation of acetamide is more rapid in a strain of T. vaginalis which is more susceptible to metronidazole. Thus, it appears that the formation of acetamide may be associated with the microbiocidal action of metronidazole.

A metronidazole metabolite in human urine and its risk.

Metronidazole is a drug used for the treatment of trichomonal vaginitis, amebiasis, giardiasis, and certain anaerobic bacterial infections in humans. Acetamide and N-(2-hydroxyethyl)oxamic acid are metabolites of metronidazole in the rat, and we find small amounts of both metabolites in the urine of human patients taking the drug. Although acetamide is carcinogenic for rats, we do not believe that our finding further defines metronidazole's risk for humans. That risk can only be estimated from surveillance of people previously exposed to the drug.