Formation of N-(5-nitro-2-thiazolyl)-N'-carboxymethylurea from 5-hydroxyniridazole. Role of aldehyde dehydrogenase in the oxidative metabolism of niridazole.

N-(5-nitro-2-thiazolyl)-N'-carboxymethylurea (NTCU) has been identified as a urinary metabolite of the antischistosomal drug niridazole [1-(5-nitro-2-thiazolyl)-2-imidazolidinone]. When DBA/2J mice were treated with [14C]niridazole, a metabolite comprising 12-14% of the total radioactivity in 24-hr urine samples was resolved by HPLC. The compound was subsequently isolated from pooled urine of niridazole-treated patients. It was identified as NTCU by mass spectrometry, and the deduced structure was confirmed by chemical synthesis. NTCU is unique among known niridazole metabolites, because it lacks an intact imidazolidinone ring. Its structure allows for a ketoenol tautomerism in which the enolate is stabilized by conjugation with the nitrothiazole ring, as evidenced by a pH-dependent 80-nm red shift in the absorption spectrum. We hypothesized that NTCU arises via oxidation of an acyclic aldehyde tautomer of 5-hydroxyniridazole, one of two proximate oxidative niridazole metabolites. Indirect evidence for the aldehyde tautomer included the fact that 5-hydroxyniridazole displayed the same pH-dependent spectral shift as NTCU with a single isobestic point at 388 nm. The proposed precursor-product relationship was confirmed when we found that NTCU formation from 5-hydroxyniridazole was catalyzed by NAD(+)-dependent aldehyde dehydrogenase (EC 1.2.1.3). The activity copurified with benzaldehyde dehydrogenase activity from mouse liver cytosol. Furthermore, benzaldehyde was a competitive inhibitor of 5-hydroxyniridazole dehydrogenase activity. These results demonstrate that 5hydroxyniridazole is not an end product of niridazole metabolism. Because biotransformation of niridazole to its 4- and 5-hydroxy derivatives has been implicated in the drug's carcinogenicity and central nervous system toxicity, NTCU formation appears to represent a detoxication pathway in mammals.

Studies of embryotoxic mechanisms of niridazole: evidence that oxygen depletion plays a role in dysmorphogenicity.

Previous study has shown that niridazole (NDZ) is dysmorphogenic to rat embryos between days 10 and 11 under culture conditions including 5% oxygen. Other studies have found that reductive embryonic biotransformation is required but that covalent binding is not a major basis of this embryotoxicity. In research presented here, NDZ exposure of homogenates prepared from day 10 rat embryos resulted in stimulation of oxygen uptake from incubation media. Further studies showed that a large percentage of this increased oxygen uptake was associated with the generation of superoxide anion radical and hydrogen peroxide. These findings led us to hypothesize that redox cycling forms the basis of the in vitro dysmorphogenicity of NDZ. The basic premise of this hypothesis is that as a result of redox cycling, oxygen is depleted from the sensitive tissues of embryos. In order to investigate it, we devised a technique for carefully controlling and monitoring oxygen tensions in embryo cultures. We found that when oxygen concentrations of 4% were established, a highly significant incidence of asymmetric defects resulted. These defects appeared analogous to those induced by NDZ exposure, consisting of asymmetric necrosis of mesenchymal tissue near the cephalic end of the neural tube and thinning of the neuroepithelium on the right. We concluded that the hypoxia induced by redox cycling of NDZ and related nitroheterocycles represents a major embryotoxic principle of action.

[Synthesis of acylates of niridazole and its analogs as schistosomicides].

In order to decrease the toxicity and enhance the curative effect of niridazole against Schistosomiasis japonica, a series of acylates of niridazole has been prepared through acylation of niridazole, 2-substituted acetamido-5-nitrothiazoles and 1-(5-nitro-2-thiazolyl)-4-acylpiperazine were also prepared. The products has been tested against Schistosomiasis japonica in mice. Preliminary test results showed that the majority of aliphatic acylates of niridazole exhibited marked schistosomicidal effect against adult worms as well as larva (compounds 2, 4-8, 12, 13, 15, 18, 19), a few of aromatic and heterocyclic acylates (compounds 14, 22) and the analogs of niridazole (compounds 26, 39, 49) showed weak activity.

On the capacity of nitroheterocyclic compounds to elicit an unusual axial asymmetry in cultured rat embryos.

A series of nitroheterocyclic compounds with antimicrobial and radiosensitizing properties was tested for embryotoxicity in cultured Sprague-Dawley rat embryos, and their effects were compared with various other five-membered heterocycles. Nifuroxime, furazolidone, nitrofurazone, niridazole, 2-nitroimidazole, and ronidazole each elicited a striking malformation characterized by asymmetrical, right-sided hypoplasia when coincubated with embryos for 26 hr. Minimum concentrations required to elicit this unusual defect ranged from 0.01 mM with nifuroxime and furazolidone to 0.5 mM with ronidazole and were roughly correlated with single electron redox potentials; i.e., agents with relatively high redox potentials were generally more effective than those with low potentials. Nitrofurantoin failed to elicit this unusual malformation but exhibited an extremely steep dose-response curve for embryolethality. Metronidazole and 4-nitroimidazole, nitroheterocyclic agents with relatively low redox potentials, did not produce the asymmetric abnormality nor were they highly embryotoxic, even at concentrations up to 2 mM. 2-Amino-5-nitrothiazole and 4'-methylniridazole also failed to evoke the asymmetric malformation but produced significant embryotoxicity as manifested by decreased growth parameters and elicitation of other kinds of malformations. Heterocyclic compounds not bearing a nitro group (furosemide, 2-aminothiazole, and 2-aminoimidazole) failed to elicit axial asymmetry at concentrations up to 1.0 mM but produced other signs of embryotoxicity at the highest concentrations tested. The results suggest that the presence of a reducible nitro group is critical for generation of the unusual malformation and that the single-electron redox potential of the nitro group plays a dominant but not exclusive role in the capacity of these chemicals to evoke axial asymmetry in cultured rat embryos.

Concentration-time course of niridazole and six metabolites in the serum of four Filipinos with Schistosoma japonicum infection.

Niridazole and six of its metabolites have been quantitated by high-pressure liquid chromatography in sera of four male Filipino patients with mild Schistosoma japonicum infections given single oral doses of niridazole (15 mg/kg) on two occasions 10 days apart. Of the five oxidative metabolites measured, 4-hydroxyniridazole and 4-ketoniridazole achieved the highest concentrations, reaching peak values of 0.9 +/- 0.3 microgram/ml of serum (mean +/- S.D., n = 4) and 0.7 +/- 0.1 microgram/ml of serum within 1 to 4 hr. 4-Ketoniridazole achieved peak serum levels 1 hr after the other oxidative metabolites in three of four patients and was the predominant metabolite in the serum of all patients 6 to 10 hr after dosing. By 24 hr, both 4-ketoniridazole and 4-hydroxyniridazole had largely disappeared from the serum. Niridazole and three other oxidative metabolites, 4,5-dihydroxyniridazole, 5-hydroxyniridazole and 4,5-dehydroniridazole, appeared within 1 hr in serum but failed to exceed 0.4 microgram/ml; none of these compounds were detected in the 24-hr serum samples. The pharmacokinetic pattern of niridazole and the oxidative metabolites showed marked interindividual variation but was quite reproducible in the same individual studied 10 days later. 1-Thiocarbamoyl-2-imidazolidinone was analyzed in serum samples by a different high-pressure liquid chromatographic procedure. This reductive metabolite attained maximal levels of 50 to 150 ng/ml of serum 6 to 12 hr after drug administration and remained at 40% or more of its peak concentration even after 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Keto niridazole: a major niridazole metabolite with central nervous system toxicity different than niridazole.

4-Keto niridazole, isolated by high-pressure liquid chromatography, was identified by high resolution electron impact mass spectral analysis as a major drug metabolite of niridazole in the serum or plasma of rats and mice treated orally or i.p. with niridazole. This metabolite has a pKa of 5.8 and is approximately 40% bound at physiologic pH to serum proteins of mice receiving therapeutic doses of niridazole. After i.p. injection of niridazole (160 mg/kg), peak serum levels of 4-keto niridazole (10.4 micrograms/ml) were reached within 6 hr in DBA/2J mice. The acute LD50 for 4-keto niridazole i.p. was 55 mg/kg in DBA/2J mice and 51 mg/kg in C57BL/6J mice; the comparable value for niridazole was 220 mg/kg in DBA/2J mice. Signs of acute 4-keto niridazole toxicity were different from those of niridazole toxicity and consisted of profound sedation and labored, irregular breathing terminating in respiratory arrest. Daily i.p. injection of 30 mg/kg of 4-keto niridazole for 5 days into DBA/2J mice resulted in no evidence of cumulative toxicity. The serum and brain concentrations of 4-keto niridazole after a 70-mg/kg i.p. LD90 dose of this compound were 93 micrograms/ml and 7.5 micrograms/g just before death. If an LD90 dose of niridazole (285 mg/kg) was injected into DBA/2J mice, the serum and brain concentrations of 4-keto niridazole just before death were 15 and 5%, respectively, of those found after an LD90 dose of 4-keto niridazole. Thus, 4-keto niridazole does not appear to account for the central nervous system toxicity of niridazole.

The formation of 1-thiocarbamoyl-2-imidazolidinone from niridazole in mouse intestine.

This study was designed to identify the site of formation of 1-thiocarbamoyl-2-imidazolidinone (TCI), a potent immunoactive metabolite of the antihelminthic drug, niridazole. When niridazole was administered intragastrically to C57Bl/6J mice, a 4-hr delay was observed before TCI was detected in the serum. By contrast, 4-hydroxyniridazole, a marker of hepatic niridazole metabolism, appeared in the serum within 30 min. Changing the route of niridazole administration from intragastric to intracaecal abolished the lag period in the rise of serum TCI concentrations relative to the 4-hydroxyniridazole marker. Pretreatment of mice with neomycin sulfate reduced the amount of TCI excreted in the urine by about 90% over a 24-hr period, but did not affect the amount of 4-hydroxyniridazole excreted. Injection of niridazole into isolated segments of mouse intestine resulted in TCI production, with the greatest conversion noted in the caecum. Subsequent incubation of niridazole with suspensions of mouse caecum contents in vitro also resulted in the formation of TCI, but not 4-hydroxyniridazole. Attempts to demonstrate TCI formation in vitro with various fractions of mouse liver were unsuccessful. These results indicate a dissociation of TCI formation from the major hepatic pathway of niridazole metabolism and support the view that TCI is formed from niridazole in the gastrointestinal tract as a result of the action of intestinal microflora.

Molecular orbital studies of antischistosomal agents.

Molecular orbital calculations were used to investigate the antischistosomal agent, niridazole, and an inactive derivative, 1-(5-nitro-2-thiazolyl)-2-ethylurea. The CNDO/2 calculations revealed that the inactive derivative had a preferred conformation stabilized by an intramolecular hydrogen bond. The molecular profile, the relative three-dimensional arrangement of constituent atoms, of the inactive derivative was different than that of the niridazole compound. The likelihood of similar intramolecular interactions rendering niridazole derivatives inactive is discussed. The results of the calculations suggest select structural modifications that might increase the efficacy of niridazole derivatives.