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J S Walsh

Publications and source records attributed to J S Walsh.

At least 37 records · Page 2Linked to original sources
PubMed1987

Structural alterations that differentially affect the mutagenic and antitrichomonal activities of 5-nitroimidazoles.

Two approaches have been used to develop nonmutagenic 5-nitroimidazoles. Both approaches are based on knowledge of the likely mechanisms by which this class of compounds cause mutagenicity. The first approach involved incorporating readily oxidizable gallate derivatives into the molecule. In one case, a very weakly mutagenic active antitrichomonal agent was obtained. The second approach involved incorporating a substituent at the C4 position of the ring. This generally resulted in a large reduction in mutagenicity and a lowering of antitrichomonal activity in vitro. In certain cases, however, mutagenicity was dramatically reduced while moderate antitrichomonal activity was retained. For example, 1,2-dimethyl-4-(2-hydroxyethyl)-5-nitroimidazole (5) showed good antitrichomonal activity in vitro (ED50 = 2 micrograms/kg) while possessing only 4% of the mutagenicity of metronidazole.

Animals↗
PubMed1986

Studies on the mechanism of activation and the mutagenicity of ronidazole, a 5-nitroimidazole.

Substantial evidence implicates the obligatory nucleophilic attack by water at C4 for the elimination of the carbamate and subsequent immobilization by electrophilic attack on protein thiols. Consequently, the strong correlation between the structural requirements for protein alkylation and for mutagenicity in TA100 suggests a possible role of nucleophilic addition at C4 or at the 2-methylene carbon for the expression of mutagenicity. Further studies directed at evaluating this possibility are currently in progress.

Alkylation↗
PubMed1984

The mechanism of the cytochrome P-448 mediated 6-hydroxylation of 7-ethoxycoumarin.

Deuterium labeling of 7-ethoxycoumarin on the carbon undergoing oxidation by cytochrome P-450 results in a redirection of metabolism from O-deethylation to ring hydroxylation at C6 (Harada, N. et al (1984) J Biol Chem 259, 3005-3010). Consequently, this substrate has been specifically labeled with deuterium at C6 aromatic in order to determine the mechanism of this ring hydroxylation reaction. Although product yield occurred over a 20-fold range when catalyzed by purified and microsomal preparations of cytochrome P-450, deuterium migration to C5 was always observed. Moreover, cumene hydroperoxide-supported reactions also proceeded with this NIH-shift. These data demonstrate that the 6-hydroxylation of 7-ethoxycoumarin occurs through a 5,6-oxide intermediate.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1984

Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions. The oxidative O-dealkylation of 7-ethoxycoumarin.

The primary deuterium and tritium isotope effects on Vm/Km and on Vm have been measured for the O-deethylation of 7-ethoxycoumarin catalyzed by two purified isozymes of cytochrome P-450. From these data the intrinsic isotope effects have been calculated as described by D. B. Northrop (Biochemistry (1975) 14, 2644-2651). The observed deuterium isotope effects on Vm/Km are 3.79 and 1.90 for the isozymes isolated from the livers of rats induced by phenobarbital and 3-methylcholanthrene, respectively. The calculated intrinsic isotope effects, however, are similar and much larger (kH/kD = 12.8 to 14.0) than the observed isotope effects on Vm/Km for the two enzymes. This demonstrates that the intrinsic isotope effects are attenuated by various steps preceding the isotopically sensitive C-H bond cleavage step resulting in the low values for the observed isotope effects. Thus, the observed isotope effects do not accurately reflect the magnitude of the intrinsic isotope effect associated with this reaction. No incorporation of 18O into the 7-hydroxycoumarin product was observed in studies employing H218O or 18O2 demonstrating that the phenolic oxygen arises exclusively from the substrate. Taken together, these data provide compelling evidence that both cytochrome P-450 isozymes catalyze the O-dealkylation of this substrate by an identical radical recombination mechanism during the obligatory formation of a hemiacetal intermediate.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1984

Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions. Evidence for the irreversible formation of an activated oxygen intermediate of cytochrome P-448.

Substitution of deuterium for hydrogen on the alpha-carbon of 7-ethoxycoumarin results in an intrinsic isotope effect of approximately 14 during the cytochrome P-448-catalyzed O-deethylation of this substrate (G. T. Miwa, J. S. Walsh, and A. Y. H. Lu (1984) J. Biol. Chem. 259, 3000-3004). This dramatic decrease in the C-H bond cleavage rate does not, however, alter the rate of 7-ethoxycoumarin disappearance or the rates of NADPH and oxygen consumption indicating that the catalytic turnover of this enzyme is unaffected. Moreover, hydrogen peroxide formation and the concentrations of an oxycytochrome P-448 complex (lambda max = 440 nm) are also unchanged demonstrating that the steady state concentrations of various oxy intermediates of cytochrome P-448 are also unchanged. An inescapable conclusion from these data is that an irreversible step exists between the formation of these intermediates and the oxidation of the substrate. These data are in agreement with the view that an irreversible cleavage of the dioxygen bond precedes substrate oxidation. Moreover, the oxidatively competent form of the cytochrome must then be committed to substrate oxidation. The latter conclusion is substantiated from high performance liquid chromatography studies which demonstrate the formation of a second metabolite, 6-hydroxy-7-ethoxycoumarin, from the deuterated substrate arising from the metabolic switching away from the O-ethyl group to the aromatic ring of this substrate.

Animals↗
PubMed1984

Drug residue formation from ronidazole, a 5-nitroimidazole. VII. Comparison of protein-bound products formed in vitro and in vivo.

In vivo experiments were conducted with ronidazole radiolabelled in the 2-14CH2-, 4,5-14C-, N-14CH3- and 4-3H-positions. The hepatic protein-bound residues, assessed by the radioactivity of exhaustively washed protein samples, were independent of the radiolabel position and occurred with 4-3H loss (greater than 80%) in excellent agreement to previous results obtained in vitro with anaerobic incubations of liver microsomes (Miwa et al., Chem. Biol. Interact., 41 (1982) 297). HPLC analysis of acid hydrolyzed in vivo protein-bound residues, obtained from [2-14CH2] ronidazole, produced a radiochromatographic profile which was virtually identical to that obtained from a similarly treated in vitro sample. Moreover, almost quantitative (76-96%) liberation of radiolabelled methylamine was obtained from hydrolysates of in vivo and in vitro residue samples formed from [N-14CH3] ronidazole. With 4,5-ring labeled ronidazole the distribution of total radioactivity of the protein hydrolysate on cation exchange resin and the fraction of the residue recovered as oxalic acid were nearly identical for the in vivo and in vitro products. We interpret these data to indicate that ronidazole alkylates proteins with retention of most of the carbon framework of the molecule, in vivo. It is also concluded that the in vitro model, previously used to examine the mechanism of protein alkylation, accurately reflects the salient process initially occurring in the intact animal during the formation of protein-bound residues of this drug.

Alkylation↗
PubMed1983

The use of intramolecular isotope effects to distinguish between deprotonation and hydrogen atom abstraction mechanisms in cytochrome P-450- and peroxidase-catalyzed N-demethylation reactions.

Intramolecular isotope effects were determined for the N-demethylation of N-methyl-N-trideuteriomethylaniline catalyzed by two isozymes of cytochrome P-450 and several peroxidases in order to differentiate between deprotonation and hydrogen atom abstraction steps. Lactoperoxidase, hemoglobin, myoglobin, and two isozymes of horseradish peroxidase catalyzed the hydroperoxide-dependent N-demethylation at initial rates ranging from 20 to 1700 min-1. These hemeproteins exhibited large and comparable intramolecular isotope effects (kH/kD = 8.6 to 10.1). In contrast, two isozymes of cytochrome P-450 as well as chloroperoxidase (v = 1.5 to 1700 min-1) gave low isotope effects (kH/kD = 1.7 to 3.1) under identical conditions. Catalase exhibited an intermediate intramolecular isotope effect (kH/kD = 5.4). These results have been interpreted to indicate that most of the hemeproteins investigated catalyze N-demethylation reactions via alpha-carbon hydrogen atom abstraction, while the reactions catalyzed by cytochrome P-450 and chloroperoxidase proceed via alpha-carbon deprotonation.

Animals↗
PubMed1982

Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro.

Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation ara cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbonyl-14C-labeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this compound is activated by microsomal enzymes. However, only about 5% of this metabolite can be accounted for as protein-bound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole.

Alkylation↗
PubMedDate not supplied

Oxidation of carbovir, a carbocyclic nucleoside, by rat liver cytosolic enzymes. Enantioselectivity and enantiomeric inhibition.

Previous metabolism studies of (-)-cis-carbovir (1'R-cis-2-amino-1,9-dihydro-9-[4'S-hydroxymethyl-2-cyclopenten-1- yl]-6H- purin-6-one), an antiviral agent, have shown that the major route of metabolism of carbovir in the rat is oxidation of the methylene hydroxyl group of the cyclopentadiene ring to form the corresponding 4'-carboxylic acid metabolite. We have determined that rat hepatic alcohol dehydrogenase and aldehyde dehydrogenase are responsible for this biotransformation through sequential oxidation of the alcohol through the aldehyde to the carboxylic acid. The results of incubations of racemic (+/-)-cis-carbovir with rat liver cytosol showed that this oxidation occurs enantioselectively favoring the (+)-enantiomer by a factor of 6- to 7-fold. We have proven that alcohol dehydrogenase contributes to the enantioselectivity of the overall oxidation process, but were unable to determine whether or not any contribution is made by aldehyde dehydrogenase. Parallel incubations conducted with the separate enantiomers revealed that the concentration required to achieve a half-maximal rate for the oxidation of the (+)-enantiomer (0.27 mM) was one-fifth that required for the (-)-enantiomer (1.36 mM). Results from enantiomeric inhibition studies showed that (+)-carbovir inhibited the oxidation of (-)-carbovir. In contrast, (-)-carbovir did not inhibit the oxidation of (+)-carbovir.

Animals↗
PubMedDate not supplied

The pharmacokinetics of (-)-carbovir in rats. Evidence for nonlinear elimination.

This study evaluated the pharmacokinetics and bioavailability of (-)-carbovir in rats following iv and po administration at doses of 10 to 120 mg/kg. The systemic clearance decreased 5-fold as the iv dose was increased from 10 to 120 mg/kg. At lower doses, (-)-carbovir was eliminated primarily by the renal route. The renal component of clearance became saturated as the dose was increased, leading to nonlinearity in the pharmacokinetics of (-)-carbovir. There were no changes in the metabolic clearance or the formation clearance of the major metabolite over the dose range of 10 to 60 mg/kg. The clearance value estimated following iv bolus administration was a "concentration averaged" value, and was not predictive of the steady-state concentrations. IV infusion studies indicated that at plasma concentrations less than 2500 ng/ml, the pharmacokinetics of (-)-carbovir were linear. The bioavailability was calculated for each treatment level and ranged from 43% at 10 mg/kg (iv and po) to 3% at 120 mg/kg (iv and po). The nonlinearity in the pharmacokinetics of (-)-carbovir must be taken into account when determining the bioavailability. At doses lower than 10 mg/kg, where the serum concentrations after iv administration would always remain in the linear range, the bioavailability may approach 50 to 60%. Poor bioavailability at high doses in the rat may not be reflective of the clinical situation, since the anticipated doses will be much lower than those administered in the rat.

Administration, Oral↗
PubMedDate not supplied

The metabolism and excretion of carbovir, a carbocyclic nucleoside, in the rat.

The metabolism and disposition of carbovir [(1R-cis)-2-amino 1,9-(4-(hydroxymethyl)-2-cyclopenten-1-yl)-6H-purin-6-one], an antiviral agent, was examined in rats using an isolated perfused liver, and in vivo following iv and po administration at two dosing levels. HPLC analysis of perfusate and bile after perfusion of the isolated liver with racemic (+/-)[8-3H]carbovir showed conversion to two major metabolites. The major component in the bile was shown to be a glucuronide conjugate of carbovir. The perfusate contained a single major component that was isolated and identified as the 4'-carboxylic acid derivative. In vivo excretion balance studies were conducted with [8-14C](-)-carbovir using four animals in each dosing group. Following iv administration at 10 mg/kg doses, the majority of the dose (77%) was excreted in the urine. At 60 mg/kg iv dosing, this value dropped to 42% (the remainder appearing in the feces). With po administration at both doses, the bulk of the dose (41-61%) was excreted in the feces. HPLC profiling of the urine showed that in all cases, most of the radioactivity was accounted for as carbovir and the 4'-acid metabolite. This metabolite accounted for up to 25% of the administered dose following 60 mg/kg iv administration, and less than 3% following 10 mg/kg po dosing. A second urinary metabolite accounting for up to 5% of the dose also was seen in all samples. This was isolated and identified as the trans-diastereomer of the 4'-acid (resulting from epimerization at the 4' position).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Metabolism of a nitrogen-containing steroid by rat hepatocytes and hepatic subcellular fractions.

Freshly isolated rat hepatocytes metabolized the nitrogen-containing steroid N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta- carboxamide (I) to six products analyzed by reverse phase HPLC. The major metabolite, which could account for greater than 50% of the total I-related material, exhibited chromatographic, NMR, and mass spectral properties identical to those of the authentic 4-carbinolamide of I. Thus, the major biotransformation pathway in hepatocytes was hydroxylation of the N-methyl group of I to form a stable carbinolamide intermediate of N-demethylation. Desmethyl-I was observed as a minor metabolite. Another metabolite which accounted for approximately 10% of the total I-related material had chromatographic and spectral properties identical to those of the authentic monoethyl analog of I. The other three metabolites were formed in variable quantities and were unstable when isolated. Mass spectral data suggested that one metabolite was the carbinolamide intermediate of N-deethylation. Treatment of rats with phenobarbital or dexamethasone increased the formation of the monoethyl metabolite of I in hepatocytes but had no effect upon the formation of the 4-carbinolamide metabolite. Rat hepatic microsomes catalyzed the NADPH-dependent metabolism of I to the same metabolites in the same relative amounts as observed with intact hepatocytes. Studies with alternative substrates and inhibitors demonstrated that microsomal cytochrome P-450 was responsible for the metabolism of I. Dog and human hepatic microsomes metabolized I to the same products as rat hepatic microsomes, but monoethyl I was the major metabolite.

Animals↗
PubMedDate not supplied

The effects of indomethacin on resorption and ankylosis in replanted teeth.

Indomethacin, a nonsteroidal, antiinflammatory analgesic, interferes with the action of prostaglandin synthetase such that osteoclastic activity is reduced. Encouraging results were obtained, pointing toward an improved long-term prognosis for replanted teeth, and as a possible solution to excessive root resorption.

Animals↗