Niridazole Research Articles

Niridazole Biodegradable Inserts for Local Long-Term Treatment of Periodontitis: Possible New Life for an Orphan Drug

Periodontal pocket inserts of niridazole (NZ) made with Resomer® (grades RG 503H and RG858, designated as RH and RG, respectively) were studied. Various formulation variables were evaluated to obtain a biodegradable delivery systems showing device degradation and drug depletion parallel to each other in vitro. Drug release from the prepared inserts was evaluated using a static dissolution setup (for 1 month). Incorporation of 3 parts of RG in 1 part of RH inserts caused a 50% decrease in the initial release rate. The RH-NZ inserts showed a spurt in release around the 10th day of the study, which coincided with the decrease in device weight, suggesting onset of device degradation. Pilot-scale clinical trials in 12 patients indicated improvements in clinical indices from the baseline values. The average pocket depth was reduced significantly (α = 0.05) from 6.34 ± 1.86 mm at baseline to 5.94 ± 0.28 mm after 28 days of treatment.

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.

Studies of mechanisms of niridazole-elicited embryotoxicity: evidence against a major role for covalent binding.

Studies reported here were designed to examine the hypothesis that covalent binding of reactive intermediates to macromolecules of the conceptus represents a major mechanism for the embryotoxicity of niridazole (NDZ). The roles of embryonic thiol content and oxygenation on: 1) malformation incidence; 2) reductive metabolism; and 3) covalent binding to embryonic macromolecules of metabolites resulting from reductive biotransformation of NDZ were studied. Results were compared with those from studies with the nondysmorphogenic analog of NDZ, 4'-methylniridazole (MNDZ). Day 10 rat embryos were pretreated for 5 hours in vitro with either L-buthionine-S, R-sulfoximine (BSO) or N-acetylcysteine (NAC) to modulate their glutathione (GSH) content. BSO reduced GSH levels, but NAC was ineffective. Following pretreatment, embryos were cultured for an additional 15 hours in the presence of [14C]NDZ or [14C]MNDZ with an initial oxygen concentration of 5%. At the end of the culture period (day 11, AM), those embryos with active heartbeat and vitelline circulation were examined for asymmetric malformations. Drug metabolites were subjected to multiple extractions from the culture medium and subjected to quantitative high-performance liquid chromatography (HPLC) analysis. Homogenates of the embryos were extracted with trichloroacetic acid (TCA) to estimate the covalent binding of radiolabeled parent compound/metabolites. Autoradiographic analyses were performed on other embryos. BSO pretreatment, which reduces embryonic GSH tissue levels, dramatically increased both the conversion of NDZ to 1-thiocarbamoyl-2-imidazolidinone (TCI) (generated via reductive metabolism of NDZ) and covalently bound label but failed to increase embryotoxicity. NAC, by contrast, did not significantly affect embryonic GSH levels, TCI generation, or covalent binding. Because both rates of metabolism of NDZ to TCI and covalent binding could vary independently of malformation incidence, we concluded that they do not represent critical mechanistic factors for the embryotoxicity of NDZ and related nitroheterocycles.

Niridazole metabolism by rat embryos in vitro.

We report the results of studies on the reductive activation of the schistosomicidal agent, niridazole (NDZ). Intact rat embryos in vitro reduced this compound, generating a stable metabolite in the presence of 5% O2. By contrast, embryo and yolk sac homogenates or liver microsomes appeared to require anaerobiasis. Malformation incidence--specifically, axial asymmetry--showed a strong correlation with nitroreductase activity rates when the latter were modulated by oxygen tension. Data presented here suggest that when embryos are exposed to NDZ under conditions of low oxygen in vitro, redox cycling ensues with molecular oxygen serving to oxidize early reduction products. This process continues, regenerating the parent compound until oxygen is depleted locally. The basis of this localized depletion is unknown, but inability of the immature supply system to replete oxygen or demand by precociously aerobic tissues may be involved. Once local anaerobiasis is attained, further reduction could generate toxic metabolites capable of covalently binding cellular macromolecules. Localized hypoxia represents another potential mechanism of dysmorphogenesis.

The toxicity of niridazole in rat embryos in vitro.

The antischistosomal drug niridazole (NDZ) was found to be teratogenic in a concentration-dependent manner from 10 to 50 micrograms/ml (47-233 microM) in rat embryos cultured from day 10 to day 11. A striking malformation consisting of axial asymmetry in which the right side of the embryo showed severe necrosis was the predominant malformation. The drug showed significantly greater dysmorphogenic activity at low (5%) compared to high (20%) oxygen tensions in cultures. Coincubation of embryos and NDZ with an exogenous source of metabolic enzymes and cofactors (NADPH) failed to modify teratogenicity. Inclusion of CO in the culture atmosphere significantly reduced the malformation incidence as did preincubation of the drug with S-9 and cofactors in medium with low O2 tension. Treatment of gravida with NDZ up to and including the maternal-lethal dose failed to result in observable malformations despite the use of several routes of exposure. These data lead us to hypothesize that rat embryos are capable of performing the reductive activation of NDZ in a fashion analogous to the schistosome but that reductive extraembryonic metabolism may result in teratogenic bioinactivation.

The effect of oxygen concentration on the teratogenicity of salicylate, niridazole, cyclophosphamide, and phosphoramide mustard in rat embryos in vitro

Comparisons were made of rat embryos cultured at 5% or 20% oxygen in the presence of salicylate (SAL), cyclophosphamide (CP), niridazole (NDZ), or phosphoramide mustard (PM). Multiple regression analyses were used to compare the effects of drug concentration, oxygen concentration, and the product of drug times oxygen concentration on malformation incidence, viability, and protein content of embryos cultured for 24 hours. Drug concentration significantly affected malformation incidence or severity and protein content (P less than 0.001) for the four drugs tested. Oxygen concentration significantly affected protein content for the four compounds (P less than 0.001) but affected malformation incidence only with NDZ. Furthermore, the interaction of oxygen concentration and drug concentration significantly affected the malformation incidence in the presence of NDZ (P less than 0.001), and protein content (P less than 0.001) and viability (P less than 0.001) in the presence of CP. The pattern of significant effects of the independent variables (drug concentration, oxygen concentration, and drug times oxygen concentration) is consistent with the hypotheses of oxygen-dependent metabolism (or lack of metabolism) of the drugs in question. NDZ, which is thought to be converted to reactive intermediates by an oxygen-inhibited nitroreductase, was more toxic at reduced oxygen tension. CP, which is activated by an oxygen-dependent P-450 system, was more toxic with increased oxygen tension. Significant effects of the independent variables on embryos exposed to SAL or PM were consistent with the effects on control embryos, notably, increased protein content with increased oxygen.

Covalent binding of niridazole (ambilhar) to tissue proteins of the rat

(1) In vivo and in vitro experiments have shown that [ 14C]niridazole (NDZ) can covalently bind to the proteins of rat liver, kidney and testes, but not to the DNA in these tissues. (2) The covalent binding was dose dependent, and the greatest amount of binding was found in the microsomal fraction. (3) The binding of [ 14C]NDZ to microsomal protein was linear with time and with protein concentration. (4) Reduced nicotinamide adenine dinucleotide phosphate was necessary for the binding, while cobaltous chloride pretreatment inhibited it, demonstrating that a cytochrome P-450 dependent mixed function oxidase mediated the binding. (5) Pretreatment of rats with other compounds, such as phenobarbital, 3-methylcholanthrene and chloracetamide which alter the rate of metabolism of [ 14C]NDZ similarly affected the extent of hepatic binding of the radiolabelled metabolite. (6) The possible relationships between these results and the cytotoxic effects of NDZ have been discussed.

Enzymatic reduction of niridazole by rat liver microsomes

Investigations were carried out with liver microsomes to identify the metabolites and to characterize the enzyme responsible for the reduction of niridazole (NDZ) [1-(5-nitro-2-thiazolyl)-2-imidazolidinone]. Data obtained from gas chromatography—mass spectrometry indicated that a major microsomal metabolite of NDZ was the hydroxylamine derivative (hydroxyaminothiamidazol). The metabolite was shown to be extremely susceptible to the presence of oxygen and readily reconverted to NDZ by exposure to air. The following evidence suggests that the conversion of NDZ to hydroxyaminothiamidazol by liver microsomes is mediated by NADPH-cytochrome c reductase: (1) the microsomal nitroreductase was not blocked by carbon monoxide and not associated with the cytochrome P-450; (2) the rate of nitroreduction was proportional to the increase in the activity of NADPH-cytochrome c reductase in liver microsomes from animals pretreated with phenobarbital; (3) both of these reductase activities remained unaffected by prior treatment with 3-methylcholanthrene; (4) the nitroreductase was solubilzed by treatment of liver microsomes with steapsin; and (5) similar increases in the activities of NDZ reductase, neotetrazolium diaphorase and NADPH-cytochrome c reductase were noted after partial purification by fractionation with ammonium sulfate.

Conferences and meetings: Uses of niridazole

Conferences and meetings: Uses of niridazole