Relative importance of bacterial and mammalian nitroreductases for niridazole mutagenesis.

Niridazole is a nitrothiazole anthelmintic agent used to treat schistosomiasis. Its antibacterial activity was found to require the presence of the nitro group; a synthetic desnitro analog was completely inactive. Niridazole was mutagenic for Salmonella tester strains TA1538, TA98, and TA100, suggesting that it was both a frame-shift- and a base substitution-type mutagen. It was effective under both aerobic and anaerobic conditions, while similar testing of the desnitro niridazole produced consistently negative results. Addition of rat liver S-9 fraction under either aerobic or anaerobic conditions did not enhance mutagenicity. However, since bacterial killing limited the dose of niridazole to 0.33 microgram/plate in standard tester strains (1/20 Km for the mammalian liver enzymes), further studies were performed using niridazole-resistant, histidine-dependent mutants derived from strains TA98 and TA100. These mutants were found to be nitroreductase deficient and to resist the mutagenic effects of niridazole, in the presence or absence of S-9, up to concentrations of 10 microgram/plate. In addition, even at niridazole concentrations of up to 100 microgram/plate, rat liver S-9 was ineffective in enhancing the mutagenicity of niridazole. These results suggest that the mutagenicity of niridazole is dependent on its aromatic nitro group and a specific bacterial nitroreductase.

Pharmacological activity, metabolism, and pharmacokinetics of glycinexylidide.

Glycinexylidide (GX) is a metabolite of lidocaine that is frequently present in mug/ml concentrations in the plasma of patients treated with lidocaine infusions for 24 hr or more. Plasma levels of GX have 26% the antiarrhythmic activity of lidocaine in an animal model, and GX adversely affects the mental performance of normal subjects at plasma concentrations comparable to those found in patients. The total volume of GX distribution in man is similar to that of lidocaine but the plasma clearance is less, so that the 10-hr elimination phase half-life of GX is much longer than the 1 1/2 hr half-life reported in normal subjects for lidocaine. About half of an administered dose of GX is excreted unchanged in urine, roughly 15% appears in urine as conjugates of xylidine and p-OH xylidine, and the fate of the rest is unknown.

1-thiocarbamoyl-2-imidazolidinone, a metabolite of niridazole in Schistosoma mansoni.

Niridazole, a nitro heterocyclic antischistosomal drug, is extensively metabolized to unknown metabolites by Schistosoma mansoni. We report that 1-thiocarbamoyl-2-imidazolidinone was isolated by high pressure liquid chromatography and identified by high resolution electron impact mass spectroscopy as a niridazole metabolite in schistosomes. After a 20-h in vitro incubation in 30 ml of medium containing 10 micrograms ml-1 [14C]niridazole (5.2 Ci mol-1), 100 S. mansoni worm pairs contained approximately 275 ng of 1-thiocarbamoyl-2-imidazolidinone. This amount represented 4% of the total metabolized fraction of niridazole in the parasite. Incubation of schistosomes with 1-thiocarbamoyl-2-[2 14C]imidazolidinone (2.7 Ci mol-1) indicated that this metabolite was not taken up. However, schistosomes released an average of 44 ng ml-1 or 1% of the total 1-thiocarbamoyl-2-imidazolidinone found in the worm back into 1 ml of medium during incubation. No host oxidative metabolites of niridazole were found in the parasites.

Concentration time course of praziquantel in Filipinos with mild Schistosoma japonicum infection.

Despite extensive use of praziquantel, the current drug of choice for the treatment of schistosomiasis and other helminthic infections, little information is available about its pharmacokinetics in individuals living in geographic areas where such infections are endemic. We investigated the pharmacokinetics of praziquantel by determining its serum concentration-time course in four selected Filipino volunteers with mild Schistosoma japonicum infection who lived in an endemic area in the Southern Philippines. At specified intervals during a 24-hour time period after a single oral dose of praziquantel (25 mg/kg BW), intravenous samples of blood were drawn, processed and analyzed for praziquantel using reverse phase high pressure liquid chromatography. The same study was repeated one week later to assess pharmacokinetic reproducibility. A third study, simulating current field practice, consisted of dosing the patient four hours apart and analyzing for praziquantel in serial blood samples drawn at specified time intervals after the first and second dose. The following results were obtained: 1) Serum concentration-time course of praziquantel was reproducible for each patient but varied from patient to patient. 2) Praziquantel was rapidly absorbed in the gastrointestinal tract as measurable amounts appeared in the blood as early as 15 minutes after dosing. Time to peak serum concentration ranged from 1.50 to 6.00 hours with almost complete elimination from blood by 24 hours whether it was administered as a single dose (1 x 25 mg/kg BW) or as a twice a day dose (2 x 25 mg/kg BW) 4 hours apart. Half-life values ranged from 1.00 to 2.50 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Benzoyl-coenzyme A:glycine N-acyltransferase and phenylacetyl-coenzyme A:glycine N-acyltransferase from bovine liver mitochondria. Purification and characterization.

Two closely related acyl-CoA:amino acid N-acyl-transferases were purified to near-homogeneity from preparations of bovine liver mitochondria. Each enzyme consisted of a single polypeptide chain with a molecular weight near 33,000. One transferase was specific for benzoyl-CoA, salicyl-CoA, and certain short straight and branched chain fatty acyl-CoA esters as substrates while the other enzyme specifically used either phenylacetyl-CoA or indoleacetyl-CoA. Acyl-CoA substrates for one transferase inhibited the other. Glycine was the preferred acyl acceptor for both enzymes but either L-asparagine or L-glutamine also served. Peptide products for each transferase were identified by mass spectrometry. Enzymatic cleavage of acyl-CoA was stoichiometric with release of thiol and formation of peptide product. Apparent Km values were low for the preferred acyl-CoA substrates relative to the amino acid acceptors (10(-5) M range compared to greater than 10(-3) M). Both enzymes were inhibited by high nonphysiological concentrations of certain divalent cations (Mg2+, Ni2+, and Zn2+). In contrast to benzoyltransferase, phenylacetyltransferase was sensitive to inhibition by either 10(-4) M 5,5'-dithiobis(2-nitrobenzoate) or 10(-5) M p-chloromercuribenzoate; 10(-4) M phenylacetyl-CoA partially protected phenylacetyltransferase against 5,5'-dithiobis(2-nitrobenzoate) inactivation but 10(-1) M glycine did not. For activity, phenylacetyltransferase required addition of certain monovalent cations (K+, Rb+, Na+, Li+, Cs+, or (NH4)+) to the assay system but benzoyltransferase did not. Preliminary kinetic studies of both transferases were consistent with a sequential reaction mechanism in which the acyl-CoA substrate adds to the enzyme first, glycine adds before CoA leaves, and the peptide product dissociates last.

Reductive metabolism of niridazole by adult Schistosoma mansoni. Correlation with covalent drug binding to parasite macromolecules.

Niridazole, an antischistosomal nitrothiazole derivative, is metabolized by adult Schistosoma mansoni to one or more reactive intermediates, as evidenced by extensive covalent binding of [14C]niridazole to parasite macromolecules. When worm pairs were incubated for 16 hr in culture medium containing 70 microM [14C]niridazole, 26-34% of the total parasite-associated radioactivity was irreversibly bound to trichloroacetic acid-precipitable material. Drug binding was both time- and [14C]niridazole concentration-dependent. Of the bound drug fraction, 85-90% was associated with parasite proteins, 3-5% with RNA and 4-7% with DNA. When schistosomes were recovered from infected mice, treated with periodic doses of [14C]niridazole, over 40% of the total parasite-associated radioactivity was bound to macromolecules. Niridazole caused up to a 40% decrease in the concentration of total nonprotein thiols in intact schistosomes incubated with the drug over an 8-hr period. Under strictly anaerobic conditions, cell-free schistosome preparations catalyzed a reduced pyridine nucleotide-dependent reduction of niridazole's essential nitro group, as evidenced by disappearance of absorption at 400 nm. Net nitroreduction did not occur under aerobic conditions, although the drug did stimulate oxidation of the pyridine nucleotide cofactor. Covalent binding of [14C]niridazole also took place in this cell-free system, with requirements identical with those needed for enzymatic nitroreduction. Covalent drug binding, but not nitroreduction, was inhibited up to 80-85% by 2 mM L-cysteine, N-acetyl-L-cysteine, or glutathione; S-carboxymethyl-L-cysteine, which has no free sulfhydryl group, was not inhibitory. [14C]4'-Methylniridazole, a nonschistosomicidal analogue of niridazole, was taken up by intact schistosomes in vitro, but was not metabolized and did not bind covalently to parasite macromolecules. Furthermore, 4'-methylniridazole did not affect the concentration of nonprotein thiols in intact parasites and did not serve as a substrate for schistosomal nitroreductase in vitro. These results indicate a positive correlation between proximal metabolic activation of niridazole within these facultative anaerobic organisms and its antiparasitic activity.

Effect of 1-thiocarbamoyl-2-imidazolidinone on the generation of plaque forming cell responses.

Although 1-thiocarbamoyl-2-imidazolidinone (TCI) is a highly potent modulator of cellular immunity, its effects on humoral immunity have not been investigated. Given orally to mice prior to immunization with sheep red blood cells (SRBC), low TCI doses (10(-14) to 10(-10) g/kg) suppressed primary plaque forming cell (PFC) responses of spleen cells by 50-75%. TCI effects in vivo were dependent on drug dose, antigen dose and time of drug administration relative to immunization. The kinetics of this response were not appreciably altered by TCI. Higher TCI doses, immunization with higher levels of SRBC than required to produce a maximal response or administration of TCI later than 48 hours after immunization resulted in drug effects ranging from slight suppression to mild enhancement of the primary PFC response. TCI given in vivo enhanced primary PFC responses to the T-independent antigen DNP-Ficoll by greater than 700%+. TCI given before a primary immunization suppressed a secondary PFC response to SRBC elicited 28 days later. However, when TCI was given 24 h prior to a secondary immunization, doses greater than 10(-5) g/kg were necessary to suppress the PFC response. The effect of TCI on in vitro immunized spleen cell cultures was similar to that found for in vivo immunized mice. TCI at concentrations up to 10(-1) g/l did not cause a loss of lymphocyte viability or inhibit plaque production by antibody producing cells. Effects of TCI on PFC responses in vitro were reversible if cells briefly exposed to an optimal concentration of drug were washed extensively prior to immunization.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Quantitative determination of praziquantel in serum by high-performance liquid chromatography.

A simple method for the quantitation of praziquantel by high-performance liquid chromatography is described. This method has a lower limit of sensitivity of 2.5 ng of drug per ml of human serum and a relative standard deviation of 2.6% at concentrations of 5 ng/ml.

Effects of praziquantel on different developmental stages of Schistosoma mansoni in vitro and in vivo.

To discern whether stage-specific resistance of Schistosoma mansoni to praziquantel occurs in vitro, we determined minimal effective concentrations (MECs) of drug needed to increase motor activity, produce contraction and/or paralysis, and cause tegumental vesiculation of developmental stages from day 0 to day 42 of S. mansoni. Recovery of these stages from exposure to praziquantel in vitro was also evaluated. MECs of praziquantel inducing increased motor activity and muscular contraction or paralysis or both were 0.005-0.01 micrograms/ml, irrespective of the stage examined. However, day-3 lung forms were more resistant than other stages when either drug-induced tegumental vesiculation (MEC, 1 microgram/ml) or recovery from drug exposure was tested. Three-day infections with S. mansoni in CF1 mice were also less responsive to praziquantel treatment than were infections of shorter or longer duration. The concentrations of praziquantel and periods of drug exposure causing gross tegumental damage to S. mansoni in vitro correlated with the peak serum levels and time course of unchanged praziquantel associated with reduction of worm burden in vivo. Thus, stage-specific resistance of S. mansoni to praziquantel does occur in vitro and correlates better with the tegumental than the muscular action of the drug.

Further observations on the effects of 1-thiocarbamoyl-2-imidazolidinone (TCI) on cell-mediated immunity.

1-Thiocarbamoyl-2-imidazolidinone (TCI) affected several cell-mediated immune responses, including two under suppressor control. In the cutaneous delayed hypersensitivity reaction of C57BL/6J (B6) mice to 2,4-dinitro-1-fluorobenzene (DNFB). TCI produced different effects depending on when it was given relative to hapten sensitization and challenge. When given 2 days before initial sensitization, TCI (10(-9)g/kg) caused inhibition of the 24 hr ear swelling response elicited 5 days after sensitization and slight enhancement of the response elicited at 10 days. Given 2 days after sensitization, the same dose of TCI produced little effect on the day 5 elicited response but appreciable enhancement of the day 10 elicited reaction. At doses of 10(-4) to 10(-1)g/kg, TCI given to B6 mice 1 day before tolerogen decreased 2,4-dinitrobenzenesulfonic acid sodium salt (DNB SO 3NA)-induced tolerance to DNFB sensitization initiated 10 days later. In vitro, TCI at concentrations of 10(-12), 10(-12) to 10(-3), and 10(-9) to 10(-1) g/liter was demonstrated to suppress, respectively, concanavalin A (Con A)-stimulated proliferation of B6 mouse spleen cells, the one way mixed lymphocyte reaction between mitomycin-inactivated AKR mouse spleen stimulator cells and B6 mouse spleen responder cells, and the generation of cytotoxic B6 spleen cells directed against 51Cr-labeled Con A splenic lymphoblasts from AKR mice. The results indicate that TCI can affect immunological reactions under suppressor control and mimic the immunosuppressive effects of either niridazole itself in vivo or immunoactive niridazole metabolite preparations in vitro.

Suppression of cell-mediated immune responses in vivo and in vitro by 1-thiocarbamoyl-2-imidazolidinone.

Both in vivo and in vitro, the niridazole metabolite, 1-thiocarbamoyl-2-imidazolidinone (TCI), was found to suppress various manifestations of cell-mediated immunity. In vivo, doses of 10(-6) g/kg every other day delayed rejection of BALB/cJ (H--2d) skin allografts by C57BL/6J (H--2b) mice. Much lower single doses (approximately 10(-11) g/kg) markedly reduced both pulmonary granuloma formation around. Schistosoma mansoni eggs in C57BL/bJ mice and delayed footpad swelling in sensitized CF1 mice responding to S. mansoni egg antigen (SEA). Reduction of footpad swelling occurred when TCI was given 24 h before, but not 4 hr after, antigenic challenge. In vitro. SEA-dependent production of eosinophil stimulation promoter (ESP) activity by sensitized mouse spleen cells was decreased greater than 90% by 10(-10)g TCI/ml. TCI failed, however, to inhibit the action of preformed ESP lymphokine on eosinophils. The blastogenic response of sensitized lymph node cells to SEA was reduced by greater than 70% by 10(-11) to -10 (-8)g TCI/ml. The drug produced no effect on either antigen-dependent lymphokine production or blastogenesis if it was added 5 min after, instead of before, stimulating antigen.

Niridazole suppression of experimental allergic encephalomyelitis in Lewis rats.

Administration of niridazole to Lewis rats beginning 2 days before sensitization with guinea pig spinal cord in combination with immunologic adjuvants exerted a dose related suppressive influence on development of experimental allergic encephalomyelitis (EAE). A daily dose of 75 mg/kg completely prevented clinical neurologic signs as well as markedly suppressed occurrence of immunohistopathologic manifestations of this autoimmune disease. A higher daily dose level of niridazole, i.e., 100 mg/k, also inhibited EAE but was associated with neurotoxic manifestations.

The molecular weight and thiol residues of acetyl-coenzyme A synthetase from ox heart mitochondria.

1. A constant molecular weight of 57000 was obtained by gel filtration of highly purified acetyl-CoA synthetase over a 1000-fold range of enzyme concentrations. The amino acid analysis is reported. 2. With native enzyme at 20 degrees C the relatively rapid reaction of four thiol residues with p-hydroxymercuribenzoate caused an immediate inhibition reversible by either CoA or mercaptoethanol. Other substrates did not protect against this rapid inhibition. 3. The much slower reaction of the remaining four thiol residues was independent of the concentration of the mercurial, first-order with respect to enzyme, and had a large energy of activation (+136kJ/mol), suggesting that a conformation change in the protein was rate-limiting. This slow phase of the reaction was accompanied by an irreversible inactivation of the enzyme. 4. The effects of substrates on this irreversible inactivation at pH7.0 in 5 mm-MgCl(2) indicated strong binding of ATP and pyrophosphate by the enzyme (concentrations for half-maximal effects, K((1/2)), were <30mum and <10mum respectively) and weaker binding of acetyl-CoA (K((1/2)) about 1 mm), AMP (K((1/2)) about 2mm) and acetate. In the presence of acetate, MgCl(2) and p-hydroxymercuribenzoate, titration of the enzyme with ATP revealed at least two ATP binding sites/mol. 5. The experiments suggest that reaction of the thiol residues with mercurial causes loss of enzymic activity by altering the structure of the enzyme, rather than that the thiol residues play a direct role in the catalysis.

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.

Toxicogenetics of niridazole in inbred mice.

The lethal potency of the antischistosomal agent niridazole (NDZ) was compared in C57BL/6J (B6) and DBA/2J (D2) mice and in their F1 hybrid, backcross and F2 progeny. A daily i.p. dosage range was chosen so that the lethal effect, ascribed to central nervous system toxicity, did not occur before 4 to 5 days. Death was always preceded by a generalized tonic-clonic seizure which terminated in respiratory arrest. In B6 mice the LD50 was 202 mg kg-1 day-1 while in D2 mice the LD50 was 146 mg kg-1 day-1; the LD50 for NDZ in similarly treated F1 hybrid mice was found to be the arithmetic mean of the LD50 values for the parental strains (172 mg kg-1 day-1). Determination of the level of NDZ in the plasma and brains of B6 and D2 mice treated subacutely with the same daily dose of NDZ failed to reveal any strain differences. Moreover, there was no evidence of in vivo accumulation of NDZ with subacute treatment which suggests that a NDZ metabolite is responsible for the observed toxicity. An association between susceptibility to the lethal effects of NDZ and the Ah locus is suggested by experiments in backcross and F2 mice. The incidence of death observed after subacute treatment with 162 mg/kg-1 day-1 of NDZ matched that predicted on the basis of genotype, i.e., it was lethal to 72% of nonresponsive and 38% of aromatic hydrocarbon responsive mice.

Effect of niridazole and niridazole immunoregulatory factor (NIF) on cutaneous delayed hypersensitivity in mice.

It has been suggested that the suppression of cell-mediated immune phenomena following niridazole administration is most likely due to a niridazole metabolite rather than the parent drug. This hypothesis was tested using two inbred strains of mice that manifest different rates of microsomal niridazole oxidation and reduction. DBA/2J mice were found to metabolize niridazole at a rate approximately 3-fold greater than C57BL/6J mice under both aerobic and anaerobic conditions. Niridazole was found to be more potent with respect to suppression of cutaneous delayed hypersensitivity in the former than in the latter. An immunosuppressive component was isolated from the urine fraction obtained from niridazole-treated rats. This component was found to be chromatographically pure; have a simple UV absorbance spectrum containing no 360 nm absorbing material characteristic of niridazole; to show no strain difference with respect to potency or efficacy in the ear-swelling assay for cutaneous delayed hypersensitivity; and to be 10(7) times more potent than niridazole with respect to the suppression of cutaneous delayed hypersensitivity.