Neutrophil function and glutathione-peroxidase (GSH-px) activity in healthy individuals after treatment with N-acetyl-L-cysteine.

The objective of this study was to evaluate the effect of N-acetyl-L-cysteine (NAC) on neutrophilic functions and as an antioxidant. NAC, 600 mg daily, given orally to healthy individuals for a period of 2 weeks, affected some functions of human neutrophilic granulocytes when tested in vitro. NAC treatment caused a decrease in the production of superoxide anions by stimulated neutrophils and the improvement of their phagocytic capacity although it did not affect their random or chemotactic migration. The level of glutathione peroxidase (GSH-px) in thrombocytes of the NAC-treated individuals was increased in comparison with the activity before treatment. These results suggest that NAC might act as a scavenger of oxygen-derived free radicals released by stimulated neutrophils and thereby protect the tissue against the radical caused injury as well as optimize phagocytosis.

Effect of N-acetylcysteine(NAC) treatment on HIV-1 infection: a double-blind placebo-controlled trial.

OBJECTIVE: In a double-blind placebo-controlled trial, human immunodeficiency virus (HIV)-seropositive patients with a CD4 lymphocyte cell count of more than 200 x 10(6) . l-1 were randomised to receive either 800 mg N-acetylcysteine (NAC) or placebo for 4 months. Before treatment low plasma cysteine levels, high free radical activity in neutrophils in the presence of autologous plasma-measured by the nitroblue tetrazolium (NBT) test- and increased tumor necrosis factor (TNF)-alpha levels were found in the HIV positive patients. RESULTS: After treatment the low plasma cysteine level in the NAC group increased to normal, and the decline of the CD4+ lymphocyte count before the study start, was less steep in the NAC group than in the placebo group after treatment. There was also a reduction in TNF-alpha level. However, NAC had no effect on the radical production by neutrophils, and although it did not increase the CD4+ cell count, it may have decreased the decline in CD4+ cells. CONCLUSION: Further controlled trials with NAC are needed to determine whether it has a beneficial effect in the treatment of asymptomatic HIV-infected individuals.

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. XIII. Enzyme interactions with a series of beta-alkyl-substituted 2-phenylethanamines and corresponding N-hydroxylamines.

The formation of Metabolic Intermediate (MI) complexes from a series of beta-alkylsubstituted 2-phenylethanamines and corresponding N-hydroxylamines is investigated during NADPH-dependent metabolism in liver microsomes from phenobarbital pretreated rats. The beta-alkyl substituents are methyl, dimethyl, ethyl, di-ethyl, n-propyl, di-n-propyl and i-propyl groups. The amines are synthesized by LiAlH4-reduction of the corresponding nitriles, which are prepared through alkylation of the enolate anion of phenylacetonitrile. The hydroxylamines are prepared either by oxidation of the corresponding benzylimines with m-chloroperbenzoic acid and subsequent hydrolysis of the initially formed 3-phenyloxaziridines, or by H2O2-mediated oxidation of the corresponding amines in the presence of catalytic amounts of sodium tungstate, followed by reduction with cyanoborohydride. The amines are found to be completely devoid of complexing activity, while the hydroxylamines form the MI complex at high rates. Complex formation from these substrates thus parallels the known behaviour of 2-phenylethanamine and its corresponding N-hydroxylamine. Since N-oxygenation is known to be a prerequisite for MI complex formation from amines our results suggest that the beta-alkylated 2-phenylethanamines are metabolized exclusively through other pathways. In accordance with this hypothesis, capillary GC-analysis of the incubation mixture of 2-phenylpropanamine shows no formation of N-hydroxylated metabolites; only 2-phenylpropanol, a metabolite formed through the deamination pathway, is found.

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. XII. Enantioselectivity and temperature dependence in microsomes and reconstituted cytochrome P-450 systems from rat liver.

Formation of metabolic intermediate (MI) complexes was studied with the enantiomers of amphetamine, 1-phenyl-2-pentanamine, N-hydroxyamphetamine, and 2-nitroso-1-phenylpropane (the C-nitroso analogue of amphetamine). Three different enzyme systems were used; liver microsomes from phenobarbital pretreated rats and two reconstituted systems containing the P450 2B1 and P450 2C11 forms of cytochrome P-450. Enantioselective complex formation in microsomes was shown for the amines and the nitroso compound, but not for the hydroxylamine. The highly purified P450 2B1 system formed the MI complex with all substrates tested, and the enantioselectivity observed with the microsomal system was reproduced. In the P450 2C11 system the nitroso compounds were completely inactive, whereas the enantiomers of N-hydroxyamphetamine still produced the complex at a high rate. Changes in temperature were shown to affect (R)-2-nitroso-1-phenylpropane more than its enantiomer. Both enantiomers showed biphasic Arrhenius plots for MI complex formation in microsomes (breaks around 22 degrees C), but the activation energies of the (R)-isomer were about five times higher than those of the (S)-isomer. A theory is presented which suggests different modes of interaction with the active site of P-450 to account for the different behaviour of the various substrates.

Cellular effects of some metabolic oxidation products pertinent to 4-ethoxyaniline.

The toxicity of some metabolic products pertinent to 4-ethoxyaniline in isolated hepatocytes were investigated. The compounds investigated were 4-ethoxynitrosobenzene (1), 4-ethoxy-4'-nitrosodiphenylamine (2), 3,6-bis(4-ethoxy-phenylimino)-4-ethoxy-1,4-cyclohexadienylamine (3), 4-(4-ethoxyphenylimino)-2,3-dimethyl-2,5-cyclohexadiene-1-one (4) and 4-(4-ethoxyphenylimino)-2,6-dimethyl-2,5-cyclohexadiene-1-one (5). Of these, 1, 2 and 3 are oxidation products of 4-ethoxyaniline. Compounds 4 and 5 are dimethyl analogues of previously investigated oxidation product 4-(4-ethoxyphenylimino(-2,5-cyclohexadiene-1-one (NEPBQI). Among the investigated compounds, 1 and 2 were the most toxic towards isolated hepatocytes. In hepatocytes treated with compounds 1, 2 and 4, loss of cell viability was also accompanied by surface bleb formation. All compounds except 3 reacted with GSH resulting in depletion of cellular GSH. No formation of GSSG was observed, however. Thus, the GSH depletion was apparently due to conjugate formation rather than oxidation. No superoxide dismutase inhibitable reduction of acetylated cytochrome c was observed, thus none of the compounds undergoes measurable redox cycling.

On the chemistry of the reaction between N-acetylcysteine and 4-[(4-ethoxyphenyl)imino]-2,5-cyclohexadien-1-one, a 4-ethoxyaniline metabolite formed during peroxidase reactions.

4-Ethoxyaniline (p-phenetidine) is oxidized by peroxidases to form several products, one of which is 4-[(4-ethoxyphenyl)imino]-2,5-cyclohexadien-1-one (1). This compound reacts with N-acetylcysteine (NAC) in methanol-phosphate buffers, generating at least four different products. Four major products, 4-[(4-ethoxyphenyl)amino]phenol (2), 3-(N-acetylcystein-S-yl)-4-[(4-ethoxyphenyl)amino]phenol (3), 2,5-bis(N-acetylcystein-S-yl)-4-[(4-ethoxyphenyl)-amino]phenol (4), and 2,5-bis(N-acetylcystein-S-yl)-4-[(4-ethoxyphenyl)imino]-2,5- cyclohexadien-1-one (5), were isolated and identified by NMR spectroscopy and mass spectrometry. The relative ratio between the formed products depends on the pH, the concentration of NAC, and the reaction time. Compound 2, which is the reduced form of 1, was the dominating product when the reaction took place at pH 3, whereas formation of the mono conjugate (3) was more extensive at a neutral pH. Under alkaline conditions 2 and 3 were oxidized by 1 or O2. The oxidized form of 3 was subsequently attacked by a second molecule of NAC, generating the bis conjugate (4). Unless an excess of NAC was present, compound 4 underwent rapid oxidation to 5. Quinone imines, like 1, generating mono conjugates, which are more reactive than the quinone imines per se, are likely to inflict an increased toxic potential and an increased stress on the endogenous thiol pool, resulting in an overall greater toxicity.

Absorption of a pharmacological dose of vitamin D3 from two different lipid vehicles in man: comparison of peanut oil and a medium chain triglyceride.

The absorption of a pharmacological dose of vitamin D3 from two different lipid vehicles, peanut oil, containing long chain fatty acids, and a medium chain triglyceride was compared. Serial measurements of the serum concentration of vitamin D3 after dosage were made. The serum levels of 25-hydroxyvitamin D3, the major circulating vitamin D3 metabolite, were also determined. The analytical methods used were based on HPLC. In the fasting state, the serum levels of vitamin D3 were significantly higher after administration in peanut oil than after administration in the medium chain triglyceride. When the vitamin D3 dose was ingested together with food no difference between the two formulations was observed. Only small inter-formulation differences in serum 25-hydroxyvitamin D3 levels were detected. The results indicate that the presence of long chain fatty acids facilitates the absorption of vitamin D3.

The protective effect of sulfite on menadione- and diquat-induced cytotoxicity in isolated rat hepatocytes.

Menadione and diquat cause toxicity in isolated hepatocytes. The toxicities of both menadione and diquat are primarily due to redox cycling and consequent oxidative stress. Menadione toxicity, however, has another component as the compound also possesses alkylating and oxidating properties allowing it to interact directly with cellular nucleophiles. Sulfite afforded considerable protection of isolated rat hepatocytes against the toxicity of menadione. This protective effect of sulfite may have several components. Sulfite effectively competed with glutathione (GSH) for conjugation with menadione, sparing intracellular GSH which may continue to detoxify reactive oxygen species formed through menadione redox cycling. The menadione sulfite conjugate undergoes much slower redox cycling than both menadione and the menadione glutathione conjugate. Sulfite also showed some degree of protection of hepatocytes from the toxicity of diquat. Diquat is a "pure" redox cycling agent and the protective effect of sulfite may involve the liberation of GSH from GSSG by sulfitolysis. This would again bolster intracellular GSH levels allowing further GSH-dependent detoxification of reactive oxygen species through cellular GSH peroxidases. In conclusion, our data illustrate the potential of inorganic sulfite to support the intracellular detoxification function of GSH, both against reactive electrophilic metabolites and against agents undergoing redox cycling.

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. XI. Peroxygenase versus monooxygenase function of cytochrome P-450 in rat liver microsomes.

Cytochrome P-455 nm complex formation in phenobarbital induced rat liver microsomes was investigated using both an NADPH/O2-dependent monooxygenase system and a peroxygenase/peroxidase system where hydrogen peroxide was substituted for NADPH. The substrates tested were the enantiomers of four 1-alkyl-substituted 2-phenylethanamines (unbranched 1-alkyl substituents, comprising one to four carbons), S(+)- and R(-)-N-hydroxyamphetamine and racemic mixtures of N-hydroxy-1-phenyl-2-butanamine and N-hydroxy-3-methyl-1-phenyl-2-butanamine. During NADPH/O2-dependent metabolism the amines showed a positive correlation between extent of complex formation and lipophilicity; furthermore the S(+)-isomers gave rise to larger amounts of complex than the corresponding R(-)-analogues. With the hydroxylamines the ability to form complexes was greater than with any of the amines but no definite difference was seen among the hydroxylamines. In the peroxygenase system the hydroxylamines still gave larger amounts of complex than the amines but the differences seen within the homologous series of chiral amines when using the monooxygenase system were no longer observed. Although the quantitative trends in complex formation seen in the monooxygenase system were non-existent when H2O2 was substituted for NADPH, mere qualitative rules still seemed to apply; substrates which failed to give the complex during NADPH-dependent metabolism (2-phenylethanamine, phentermine, N-hydroxyphentermine and phenylacetone oxime) were inactive also in the peroxygenase system. The results substantiate the notion that the monooxygenase and peroxygenase reaction mechanisms of cyt. P-450 follow similar but not identical pathways.

The metabolism of sulfite in liver. Stimulation of sulfate conjugation and effects on paracetamol and allyl alcohol toxicity.

Sulfite is rapidly oxidized to sulfate in the liver. This was shown both in isolated rat hepatocytes and isolated perfused liver. In addition sulfite treatment resulted in release of GSH originating probably from low molecular disulfides such as GSSG and/or mixed disulfides between GSH and protein sulfhydryl groups. Sulfite was demonstrated to be an efficient precursor for sulfate conjugation. This was demonstrated using paracetamol as a substrate. Sulfite was even more efficient in supplying sulfate for sulfate conjugation than inorganic sulfate. Sulfite was furthermore shown to be protective against the toxicity of both N-acetyl-p-benzoquinone imine (NAPQI), the reactive paracetamol metabolite, and acrolein, a reactive aldehyde which is a metabolite of allyl alcohol. This protection is most likely due to direct reaction between sulfite and these reactive metabolites in a manner similar to that occurring with GSH and other thiols. When NAPQI and acrolein were generated intracellularly in isolated hepatocytes from paracetamol and allyl alcohol, respectively, toxicity was also expressed. In this case sulfite only protected against allyl alcohol induced toxicity and not against paracetamol induced toxicity. The reason for this discrepancy is not clear but may depend on factors such as site of generation of the reactive metabolite or the reactivity of the reactive metabolite.

Biotransformation of terodiline. V. Stereoselectivity in hydroxylation by human liver microsomes.

The stereoselective hydroxylation of N-tert-butyl-4,4-diphenyl-2-butylamine (Terodiline) was studied in human liver microsomes. Formation of the two main metabolites, N-tert-butyl-4(4-hydroxyphenyl)-4-phenyl-2-butylamine (II) and N-(2-hydroxymethyl-2-propyl)-4,4-diphenyl-2-butylamine (VI), was found to be stereoselective. R-Terodiline was preferentially transformed by phenolic hydroxylation to the 2R,4S-II and 2R,4R-II forms with a pronounced selectivity for the former. The formation rate ratio 2R,4S-II/2R,4R-II was about 6, obtained from two liver preparations. S-Terodiline was mainly hydroxylated to the alcohol 2S-VI although phenolic hydroxylation to the 2S,4S-II and 2S,4R-II also occurred, yielding about equal amounts of the two phenols.

Single-dose pharmacokinetics of terodiline, including a stable isotope technique for improvement of statistical evaluations.

A bioequivalence study with terodiline (Mictrol) was performed in 8 healthy volunteers given a 25 mg oral dose of either of two solid dosage forms together with a water solution of the deuterated drug. The solid dosage forms were found to be bioequivalent. Moreover, their pharmacokinetic profiles were the same as for the water solution. The basic pharmacokinetic parameters (means +/- SE) of terodiline were calculated to: biological half-life in serum 60 +/- 4 h, maximum serum concentration 79 +/- 4 micrograms l-1 and the corresponding time 4 +/- 1 h, oral serum clearance 75 +/- 7 ml min-1, urinary excretion 15.3 +/- 1.5 per cent of dose, and renal serum clearance 10.9 +/- 2.2 ml min-1. The within-subject variability (serum-derived parameters) was about 8 per cent (CV per cent) and the between-subject variation 2-4 times higher. A single parameter estimate in subjects of a comparative population can be expected to show a 3-fold variation (95 per cent confidence). The deuterated drug could be used as a covariate to increase the power/precision in the statistical evaluation of the bioequivalence. In that way the 95 per cent confidence interval for the difference between the formulations, as well as the difference that could be detected with 80 per cent power, was reduced 2- to 5-fold. The covariate method was thus in this respect extremely efficient. In bioequivalence studies with drugs where a large number of subjects would be needed using conventional statistical analyses, this method also offers a possibility to considerably reduce the size of the panel, while retaining sufficient power and precision in the estimates.

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. IX. A comparative study with enantiomeric 2-nitroso-1-phenylpropane in microsomes and a reconstituted cytochrome P-450 system from rat liver.

Formation of cytochrome P-455 nm complexes was investigated with enantiomeric 2-nitroso-1-phenylpropane--the C-nitroso analogue of amphetamine--and optically active N-hydroxyamphetamine, in the presence of NADPH. For comparative reasons, three different drug-metabolizing enzyme systems were used, namely microsomes from control and phenobarbital-treated rats, and a reconstituted system containing the main phenobarbital-induced form of cytochrome P-450 from rat liver. In microsomes obtained from phenobarbital-treated rats, pronounced differences in the kinetics of complex formation were observed between the enantiomeric C-nitroso compounds, but not between the isomers of N-hydroxyamphetamine. In the reconstituted enzyme system the S-nitroso compound formed the P-455 nm chromophore at the highest initial rate, while the R analogue was devoid of complexing activity. The rates of complex formation from the N-hydroxylamine enantiomers were high and equal.

Biotransformation of terodiline. III. Opposed stereoselectivity in the benzylic and aromatic hydroxylations in rat liver microsomes.

1. Terodiline (N-tert-butyl-4,4-diphenyl-2-butylamine) is a racemic drug with anticholinergic and/or calcium antagonistic activity, which is subject to renewed interest as a potential remedy for urinary incontinence. As part of the current investigations on terodiline, the metabolism of its enantiomers is being investigated. 2. The metabolism of the enantiomers of terodiline in rat liver microsomes is slow, as for the racemate, though the S-enantiomer is metabolized more rapidly than its optical antipode. Phenobarbitone pretreatment of the rats enhances the metabolism with a marked increase in the conversion of the S-enantiomer. 3. While aromatic p-hydroxylation greatly exceeds benzylic oxidation in the metabolism of R-terodiline, this situation is reversed in the metabolism of S-terodiline. Moreover, the rate of aromatic p-hydroxylation of racemic terodiline follows that of R-terodiline, while the rate of benzylic hydroxylation of racemic terodiline follows that of S-terodiline. Phenobarbital pretreatment of the rats had little or no effect on aromatic p-hydroxylation but markedly increased benzylic oxidation. 4. Separation of the mixture of p-hydroxylated metabolites into diastereomeric pairs showed that their composition is highly dependent on which form of terodiline is used as substrate. 5. The results from the study are compatible with the participation of multiple forms of cytochrome P-450 enzymes.

Cellular effects of N(4-ethoxyphenyl)p-benzoquinone imine, a p-phenetidine metabolite formed during peroxidase reactions.

The interaction of N-(4-ethoxyphenyl)p-benzoquinone imine (NEPBQI), a metabolite formed during peroxidase catalyzed metabolism of p-phenetidine, with GSH and its effects in isolated rat hepatocytes were investigated. When reacted with GSH NEPBQI formed both a mono- and a diglutathione conjugate as well as GSSG. Formation of glutathione conjugates and GSSG also occurred when NEPBQI was added to isolated hepatocytes. The formation of GSSG was, however, only detectable if the hepatocytes had been pretreated with the GSSG reductase inhibitor BCNU (1,3-bis-(2-chloroethyl-1-nitrosourea). Similarly, NEPBQI caused a rapid decrease in cellular free protein thiols when added to hepatocytes, however this was expressed at higher concentrations than for effects on GSH. The protein thiol decrease was correlated with protein binding of NEPBQI. NEPBQI was also shown to be toxic to isolated hepatocytes. At a concentration of 400 microM extensive bleb formation was followed by loss of cell membrane integrity and cell death. To assess further the subcellular metabolism of NEPBQI microsomes and cytosol was used. NEPBQI was found to be preferentially reduced by cytochrome P-450 reductase in the microsomes whereas DT-diaphorase catalyzed its reduction in cytosol. NEPBQI did not undergo significant redox cycling since no formation of O2 was observed. Thus, the cytotoxic effect of NEPBQI appears to be due to protein arylation rather than redox cycling.

Peroxidative N-oxidation and N-dealkylation reactions of pargyline.

The hydrogen peroxide-supported oxidation of pargyline in rat-liver microsomes was investigated and compared to that promoted by cytochrome P-450 in the presence of an NADPH-generating system. The metabolic conversions promoted by hydrogen peroxide and cytochrome P-450 comprised N-demethylation, N-depropargylation, N-debenzylation and N-oxidation. For the hydrogen peroxide-cytochrome P-450-promoted oxidation, cyanide, but not carbon monoxide, was an effective inhibitor of all the reactions. Similarly, 2,4-dichloro-6-phenyl phenoxyethylamine (DPEA) inhibited all reactions, particularly N-demethylation and N-oxidation more extensively than the NADPH-dependent microsomal oxidation. Using microsomes from rats pretreated with phenobarbital caused no increase in the metabolites above the levels seen with microsomes from untreated animals. Various other peroxidase systems which were investigated were essentially unable to promote oxidation of pargyline.

The metabolic fate of pargyline in rat liver microsomes.

The availability of a sensitive analytical assay for the simultaneous quantitation of pargyline (PARG) and four of its major metabolites have made possible a detailed study on the metabolism of the drug in rat liver microsomes with emphasis put on comparisons between optional N-dealkylation reactions and N-oxide formation. Pargyline is a lipophilic amine with a low pKa-value of 6.6 and undergoes extensive metabolism. The conversion of the substrate is rapid and comprizes three N-dealkylation and one N-oxidation reactions, yielding N-benzylpropargylamine (BPA), N-methyl-propargylamine (MPA), N-benzylmethylamine (BMA) and pargyline N-oxide (PNO), respectively. Phenobarbital (PB) pretreatment of the rats causes a pronounced increase in the metabolism with about 90% of the substrate being consumed within the first minute of incubation at 100 microM substrate concentration. At this substrate concentration the most pronounced induction is seen in the formation of BPA and also in its further metabolism, while levels of BMA and MPA remain fairly constant. Pargyline N-oxide is the most abundant metabolite in microsomes from untreated rats and its formation is not increased by PB induction. Moreover, the inhibition of PNO formation by typical cytochrome P-450 inhibitors is marginal, while that of BPA, BMA and MPA formation is not. N-Debenzylation, yielding MPA, is the least important of the N-dealkylation reactions and the effect of PB induction on this reaction becomes noticeable only at high substrate concentrations. The studies suggest that various cytochrome P-450 enzymes are involved in the N-dealkylation reactions of PARG while N-oxidation appears to occur mainly by a cytochrome P-450-independent pathway. As propiolaldehyde, a potential hepatotoxin, is formed concomitant to BMA, and as PNO, under certain conditions, can decompose to acrolein, another well-known hepatotoxin, both these quantitatively important metabolic routes have to be considered in evaluating the toxicity of pargyline.

The oxidation of p-phenetidine by horseradish peroxidase and prostaglandin synthase and the fate of glutathione during such oxidations.

The oxidation of p-phenetidine by horseradish peroxidase and prostaglandin synthase was investigated. The existence of a free radical intermediate formed during enzymatic oxidation was supported by a ratio of hydrogen peroxide: p-phenetidine consumed of 1:2 in the horseradish peroxidase system. Furthermore in both enzyme systems a rapid oxidation of added glutathione was observed and in the presence of the thiol there was a decreased removal of p-phenetidine. This suggests the reduction of a p-phenetidine radical by glutathione generating p-phenetidine and a thiyl radical. The latter react with oxygen and a rapid oxygen uptake was observed during enzymic oxidation in the presence of thiols. That p-phenetidine radicals were produced during horseradish peroxidase catalyzed oxidation of p-phenetidine was supported by experiments using the spin probe OXANOH. This was oxidized to its stable free radical form (OXANO.) in an enzyme- and substrate-dependent reaction and the EPR signal obtained was not decreased by SOD (80 micrograms/ml) or benzoate (10-100 mM). TLC characteristics of the products of the oxidation of p-phenetidine by both enzymes were almost identical inferring a similar mechanism of oxidation. Two of the metabolites were characterized by mass spectrometry and by comparison with reference compounds prepared by chemical oxidation. One metabolite was identified as 4,4'-diethoxyazobenzene, which further supports a radical mechanism, and the other was a p-phenetidine trimer which could exist in both oxidized and reduced forms. On the basis of these observations a mechanism for the oxidation of p-phenetidine and the fate of glutathione during such oxidations is proposed.