Consumption of charcoal-broiled meat as an experimental tool for discerning CYP1A2-mediated drug metabolism in vivo.

Cytochrome P450 1A2 (CYP1A2) is a major drug-metabolising enzyme. Polycyclic aromatic hydrocarbons, present in high concentrations in tobacco smoke and charcoal-broiled meat, are known to induce CYP1A2. The purpose of the present study was to validate enzyme induction by consumption of charcoal-broiled meat as an experimental tool for discerning CYP1A2-mediated drug metabolism in vivo. Twenty-four healthy, non-smoking men, all extensive metabolisers of sparteine (CYP2D6), participated in the study. All participants were genotyped for a putative CYP1A2-inducibility genotype. In the study diet period charcoal-broiled meat was served for lunch and dinner for five consecutive days. All participants were tested with probe reactions for CYP1A2 (caffeine) and CYP2C19 (proguanil) before and after consuming the study diet. Further, in three subgroups, they were tested with either the CYP1A2-substrate tacrine or probe reactions for CYP3A4 (quinidine) or CYP2C9 (tolbutamide). Neither probe reactions for CYP1A2, CYP2C9, CYP2C19 or CYP3A4 were affected by consumption of charcoal-broiled meat as practised in this study. No modifying role of the CYP1A2-inducibility genotype was evident. A number of experimental limitations are discussed, among them the lack of standardisation of exposure, the timing of phenotyping, and the choice of probe reactions. In conclusion, consumption of charcoal-broiled meat as practised in the present study appears not to be a useful experimental tool for discerning CYP1A2-mediated metabolism in vivo.

Spectrofluorimetric determination of velnacrine in human serum and urine.

A simple, sensitive and rapid spectrofluorimetric method to determine velnacrine, a cholinesterase inhibitor, has been developed and validated. The influence of pH, temperature, ionic strength, presence of excipients, metal ions and surfactants on the fluorescence intensity has been studied. The proposed method allows the determination of 5-100 ng ml(-1) of velnacrine in aqueous solution containing sodium acetate buffer (pH 5.6; 0.04 M) with lambda(excitation) 242 nm and lambda(emission) 359 nm. The detection and quantitation limits were 1.7 and 4.5 ng ml(-1) respectively. The method was successfully applied to the determination of velnacrine in human serum and urine.

Electrochemical oxidation at carbon paste electrode of tacrine and 1-hydroxytacrine and differential pulse voltammetric determination of tacrine in pharmaceuticals and human urine.

The electrochemical oxidation of tacrine and its 1-OH-metabolite, has been studied by cyclic voltammetry and differential pulse voltammetry by using carbon paste electrodes. The peak current-concentration relationship was found to be linear up to 20 micrograms ml-1 with detection limits of 0.06 microgram ml-1 for tacrine and 0.18 microgram ml-1 for 1-OH-tacrine and quantitation limits of 0.20 microgram ml-1 for tacrine and 0.37 microgram ml-1 for 1-OH-tacrine. A method for determining tacrine by differential pulse voltammetry in pharmaceuticals and human urine, in the presence of 1-OH-tacrine, has been developed.

Plasma tacrine concentrations are significantly increased by concomitant hormone replacement therapy.

BACKGROUND: In vitro results suggest that the synthetic hormones used in postmenopausal hormone replacement therapy (HRT) may be significant inhibitors of oxidative drug metabolism. Moreover, HRT has been reported to enhance response to tacrine in postmenopausal patients with Alzheimer's disease, but the mechanism of this interaction remains unclear. OBJECTIVE: To examine the effect of HRT with 2 mg estradiol valerate and 0.25 mg levonorgestrel once daily on the pharmacokinetics of tacrine. METHODS: Ten healthy female volunteers received treatment for 10 days with once-daily HRT or placebo in a randomized, double-blind crossover study. One hour after the last HRT or placebo capsule on day 10, the subjects received a single 40-mg dose of tacrine. Plasma samples were collected for 30 hours and urine samples were collected for 24 hours after tacrine intake for the measurement of tacrine and 1-hydroxytacrine concentrations. RESULTS: HRT increased the mean plasma concentration-time curve calculated from zero to infinity (AUC) of tacrine by 60% (P = .009); the greatest individual increase in the AUC was about threefold. Similarly, the mean peak concentration in plasma of tacrine was 46% (P = .031) higher in the HRT phase compared with the placebo phase. HRT reduced the mean apparent oral clearance of tacrine by 31% (P = .014), but no significant difference was found in the elimination half-life or the renal clearance of tacrine between the HRT phase and the placebo phase. The metabolic ratio (1-hydroxytacrine AUC/tacrine AUC) was significantly (mean, 26%; P < .001) reduced in all 10 subjects. CONCLUSIONS: HRT with estradiol and levonorgestrel significantly increased plasma tacrine concentrations. This interaction between tacrine and HRT involves reduced metabolic conversion of tacrine to its main metabolite 1-hydroxytacrine by CYP1A2 during the first-pass phase. The interaction may be clinically important with regard to both enhanced efficacy and increased likelihood of concentration-dependent adverse effects of tacrine in the long-term treatment of patients with Alzheimer's disease. Accordingly, smaller doses of tacrine may be appropriate when coadministered with HRT.

Determination of tacrine and its metabolites in human plasma and urine by high-performance liquid chromatography and fluorescence detection.

A new method for the simultaneous quantitation of tacrine and the three metabolites, 1-hydroxytacrine (velnacrine, maleate), 2-hydroxytacrine and 4-hydroxytacrine, in human plasma and urine has been developed. The method was based on simple one-step liquid-liquid extraction with ethyl acetate followed by isocratic, reversed-phase high-performance liquid chromatography and fluorescence detection (excitation: 330 nm and emission: 365 nm). The limit of detection in plasma was 0.5 nM for 2-hydroxytacrine and 4-hydroxytacrine, 2 nM for 1-hydroxytacrine and tacrine. In urine it was 60 nM for 2-hydroxytacrine and 4-hydroxytacrine, 30 nM for 1-hydroxytacrine and 80 nM for tacrine. The limit of quantification in plasma was 2.5 nM for 2-hydroxytacrine and 4-hydroxytacrine, 10 nM for 1-hydroxytacrine and 2 nM for tacrine. In urine it was 120 nM for all components. The overall mean recoveries ranged from 84 to 105% in plasma and from 64 to 100% in urine for all four compounds.

Influence of the CYP1A2 inhibitor fluvoxamine on tacrine pharmacokinetics in humans.

OBJECTIVE: Tacrine is extensively metabolized by cytochrome P4501A2 (CYP1A2). Fluvoxamine, a potent CYP1A2 inhibitor, may be coadministered with tacrine. The aim of this study was to examine the influence of fluvoxamine administration on the disposition kinetics of single-dose tacrine administration. METHODS: Thirteen healthy volunteers participated in this double-blind, randomized crossover study, which compared the effects of fluvoxamine (100 mg/day during 6 days) and placebo on the pharmacokinetics of a single oral dose of tacrine (40 mg). RESULTS: Fluvoxamine caused a significant increase in tacrine area under the plasma concentration versus time curve (AUC): arythmetic mean, 27 (95% confidence interval [CI], 19 to 38) ng.hr/ml versus 224 (95% CI, 166 to 302) ng. hr/ml. Fluvoxamine caused a decrease in the apparent oral clearance of tacrine from 1683 +/- 802 to 200 +/- 106 L/hr (mean +/- SD), which was explained by a decrease in its nonrenal clearance. Five subjects had gastrointestinal side effects during fluvoxamine administration. Fluvoxamine administration was associated with significant increases in the plasma AUC values of three monohydroxylated tacrine metabolites and in the total urinary recovery measurements of tacrine and its metabolites (9.1% +/- 4.6% versus 24.0% +/- 2.6% of recovery). These results may be attributable to fluvoxamine-dependent inhibition of CYP1A/, which is responsible of the biotransformation of tacrine into its monohydroxylated metabolites and further into dihydroxylated and reactive metabolites. CONCLUSION: Fluvoxamine inhibits the metabolism of tacrine. CYP1A2 may be the target of this inhibition. Fluvoxamine may modulate the hepatotoxicity of tacrine, depending on the relative contribution of tacrine and its reactive metabolites to this toxicity.

Metabolic disposition of the cognition activator tacrine in rats, dogs, and humans. Species comparisons.

The metabolic fate of tacrine [1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate (THA)] was examined in rats, dogs, and humans. After administration of single oral doses of [14C]THA to rats, dogs, and humans, drug-derived material was well absorbed, with urinary excretion being the predominant route of radiolabel elimination. Metabolic profiling of plasma and urine from rats, dogs, and humans showed THA to be extensively metabolized with marked species differences in quantitative amounts of metabolites observed. Plasma profiles were similar to respective urinary profiles in all three species. Present in profiles of urine from rats were 1-hydroxy (OH)-THA (major), 2-OH-THA, and 4-OHA-THA, and unchanged THA. Also observed were trace amounts of more polar metabolites, presumably arising from sequential metabolism. Metabolic profiling of dog urine also showed 1-OH-THA to be the major metabolite, with trace amounts of the 2-OHA-THA and 4-OH-THA regioisomers and THA excreted. In dog urine, more of the radioactivity was associated with polar metabolites, including 1,3-dihydroxy-THA and a dihydrodiol metabolite. Human urinary metabolic profiles were more similar to that in dogs than in rats, with no single metabolite constituting > 10% of urinary radioactivity. Present in human urine were phenol glucuronide metabolites, of which 7-OH-THA was identified as an aglycone. Relevance of the marked quantitative differences in THA metabolism between rats, dogs, and humans to species differences in THA hepatotoxic potential remains to be established.

Identification of a 3-hydroxylated tacrine metabolite in rat and man: metabolic profiling implications and pharmacology.

Discrepancies in urinary metabolic profiles in rats administered tacrine (1) suggested the presence of an unidentified metabolite of 1. Chromatographic methods were developed that allowed isolation of a metabolite fraction containing both 1-hydroxytacrine (2) and an unknown metabolite from rat urine. Mass spectral analysis indicated this metabolite to be a monohydroxylated derivative, which upon two dimensional COSY NMR analysis could be assigned as 3-hydroxytacrine (4). This structural assignment was confirmed by independent synthesis of 4. Compound 4 was also identified as a human urinary metabolite of 1. Biologically, 4 was found to have in vitro human red blood cell acetylcholinesterase inhibitory activity similar to that of 2 and 4-hydroxytacrine (5) and approximately 8-fold less than that of 1. These results underscore the need to conduct rigorous structural identification studies, especially in cases where isomeric metabolites are possible, in assessing the accuracy of chromatographic profiling techniques.

Stereoselective hydroxylation of tacrine in rats and humans.

An enantiospecific method was developed for assessing the stereochemistry of tacrine (9-amino-1,2,3,4-tetrahydroacridine monohydrochloride monohydrate; THA) metabolism to 1-hydroxytacrine (1-OH-THA) in humans and rats. In addition, limited metabolic studies with human liver microsomal preparations were conducted, and the stereochemistry of rac-1-OH-THA disposition was also examined. The analytical method incorporates an achiral normal phase separation and isolation of 1-OH-THA, followed by a chromatographic step using chiral normal-phase chromatography to resolve the enantiomers of 1-OH-THA. The achiral method was applied to quantitation of total 1-OH-THA in human urine specimens collected for 24 hr following administration of a single 40 mg oral dose of tacrine to 15 healthy elderly volunteers. Total 1-OH-THA accounted for approximately 5% of the administered dose. THA and 2-OH-THA were also quantitated and found to comprise < 1% and approximately 2% of the administered dose, respectively. 4-OH-THA was not detectable. The dextrorotatory (+)-isomer comprised approximately 94% of the 1-OH-THA recovered in urine. In vitro studies utilizing human liver microsomes found enantioselective formation of the (+)-isomer (approximately 90%), whereas incubations with rac-1-OH-THA showed residual substrate to be racemic. The method was also applied to determination of the enantiomeric composition of 1-OH-THA in the urine of rats given a single oral 16 mg/kg dose of THA. The percentage of 1-OH-THA excreted in urine as the (+)-isomer was 94%.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of [14C]velnacrine maleate in rats, dogs, and humans.

This study describes the disposition of [14C]velnacrine maleate in rats, dogs, and humans, and the isolation and identification of metabolites in dog urine. Following oral administration of [14C]velnacrine maleate, drug-related material was well absorbed in all three species, with the majority of the dose recovered in the urine. Fecal elimination of radioactivity accounted for the remainder of the dose. The majority of the radioactivity was eliminated within 24 hr. Pharmacokinetic parameters for the elimination of radioactivity from the plasma of rats and dogs were similar after oral dosing compared with intravenous dosing. In humans, the plasma and urinary levels of velnacrine maleate were substantially lower, and the elimination half-life shorter than for total radioactivity, indicating the presence of one or more metabolites with a longer half-life than the parent compound. Preliminary TLC analysis of urine, plasma, and feces showed that metabolism appeared to be similar in the three species investigated. Velnacrine maleate was extensively metabolized with only approximately 10%, 19%, and 33% of the dose appearing in the urine as unchanged drug in humans, dogs, and rats, respectively. Isolation and identification of dog urinary metabolites was conducted. The identity of the isolated metabolites was determined by GC/MS and proton NMR. One of the main metabolic routes was found to be via hydroxylation of the tetrahydroaminoacridine ring with other minor hydroxylated and dihydroxylated metabolites being detected. In addition two dihydrodiol metabolites were also identified. Phase II metabolism did not appear to be a significant route.

Comparison of the chromatographic characteristics of metabolites of tacrine hydrochloride in human serum and urine with those of in vitro metabolic products from hepatic microsomes.

There may be as many as five metabolites of THA in man, four corresponding to products in the rat. The study provides some evidence that one of major metabolites is 1-hydroxy-THA but lends no support to the putative oxidative deamination pathway.

Multiple dose pharmacokinetics, safety, and tolerance of velnacrine (HP 029) in healthy elderly subjects: a potential therapeutic agent for Alzheimer's disease.

The pharmacokinetics, safety, and tolerance of 1,2,3,4-tetrahydro-9-aminoacridin-1-olmaleate (HP 029) a potential therapeutic agent for Alzheimer's disease, were assessed after multiple oral doses in a randomized double-blind, placebo controlled, ascending dose study in 56 healthy elderly men (14 per dose group). The subjects in the first three groups received 25, 50, or 100 mg two times a day and a fourth group was administered 100 mg velnacrine tid for 28 days. All subjects received a final dose on day 29. Subjects were confined for continuous observation during the 36-day study period. Blood and urine samples were collected for the pharmacokinetic assessment. There were no clinically important changes in the safety variables in both age groups after any dose. There was no evidence of hepatotoxicity when elderly men were given 100 mg tid for 28 days. Nine subjects reported one or two episodes of gastrointestinal (diarrhea) side effects (6 in the 100 mg bid group and 3 in the 100 mg tid dose group) during a 29-day trial. None required treatment or were discontinued from study. These results indicate that the safety and tolerance up to 100 mg tid for 28 days in healthy elderly men are acceptable. Velnacrine was rapidly absorbed after oral administration. There were dose-related increases in Cmax, AUCs, and amount of drug excreted in urine. During multiple dosing, the Cmax increased as a function of dose. The tmax and t1/2 were not affected by dosage nor multiple dosing. Steady state levels of velnacrine were reached between days 2 and 3 with no evidence of further accumulation of velnacrine thereafter. Approximately 11-30% of the administered dose was excreted in the urine over the course of the study. The favorable pharmacokinetic characteristics and acceptable safety and tolerance of multiple dosing oral doses of velnacrine support further testing of this compound for efficacy and safety in Alzheimer's patients.

Identification of the urinary metabolites of tacrine in the rat.

Tacrine (THA) is a potent cholinesterase inhibitor being studied for the treatment of Alzheimer's disease. The metabolism and excretion of THA were studied in rats following a single oral dose of 20 mg/kg of THA. The results show THA was extensively metabolized in rats after oral administration. Three major urinary metabolites were isolated by HPLC on a semi-prep analytical phenyl column, and subsequent purification of the individual fractions on a semi-prep analytical cyano column. The major metabolic pathways involve the hydroxylation of the saturated ring at positions 1, 2, and 4. The structures of the metabolites 9-amino-1,2,3,4-tetrahydroacridin-1-ol (1-OH-THA), 9-amino-1,2,3,4-tetrahydroacridin-2-ol (2-OH-THA), and 9-amino-1,2,3,4-tetrahydroacridin-4-ol (4-OH-THA) were determined by electron impact mass spectrometry and/or 1H-NMR, and compared with synthetic references. The urinary excretion of THA and metabolites was quantitated by HPLC with UV detection. About 60% of the oral dose was eliminated as total THA, 1-OH-THA, 2-OH-THA, and 4-OH-THA over a 48-hr collection interval; and the non-conjugated THA and hydroxylated metabolites accounted for 45% of the dose.