Sulphoxidation of ethyl methyl sulphide, 4-chlorophenyl methyl sulphide and diphenyl sulphide by purified pig liver flavin-containing monooxygenase.

1. The biotransformation of ethyl methyl sulphide (EMS), 4-chlorophenyl methyl sulphide (CPMS) and diphenyl sulphide (DPS) to their corresponding sulphoxides by purified flavin-containing monooxygenase (FMO) is described. 2. Purified pig liver flavin-containing monooxygenase catalysed the sulphoxidation of EMS, CPMS and DPS to their corresponding sulphoxides and the reactions followed single enzyme Michelis-Menten kinetics. 3. The apparent K(m) and V(max) for the sulphoxidation of EMS were 1.38+/-0.05 mM and 78.74+/-3.9 nmoles mg(-1) protein min(-1), respectively. The apparent K(m) and V(max) for the sulphoxidation of CPMS were 0.185+/-0.03 mM and 103+/-5.0 nmoles mg(-1) protein min(-1), respectively. The apparent K(m) and V(max) for the sulphoxidation of DPS were 0.068+/-0.002 mM and 49.26+/-2.05 nmoles mg(-1) protein min(-1), respectively. 4. A significant reduction of the sulphoxidation of these simple sulphides was observed with addition of 1-naphthylthiourea in the incubation medium. On the other hand, incorporation of catalase and superoxide dismutase into the incubation media produced no appreciable inhibition of the observed sulphoxidation of the sulphides. 5. These results suggest that FMO is responsible, at least in part, for the sulphoxidation of nucleophilic sulphides as well as for the oxidation of sulphur atoms that reside within or adjacent to aromatic systems.

Pharmacokinetics of trimethylamine in rats, including the effects of a synthetic diet.

1. The pharmacokinetic profile of trimethylamine (TMA) was examined in the male Wistar rat and the effects of a synthetic diet on TMA pharmacokinetics were also evaluated. 2. The concentrations of TMA and its N-oxide in blood were analysed by a sensitive headspace gas chromatographic assay. 3. The pharmacokinetics of TMA were essentially linear for intravenous (i.v.) bolus doses of 10-40 mg kg(-1). Over the range of administered i.v. doses, the concentrations of TMA in blood declined approximately monoexponentially with half-lives of 2.03-2.48 h. The Vd of TMA ranged from 3.2 to 4.39 l kg(-1) and clearance ranged from 18.78 to 23.92 ml min(-1) kg(-1). The peak concentration of TMA in blood occurred at 1 h after oral administration of a 20 mg kg(-1) dose and the bioavailability for the oral dose averaged 81%. 4. Peak concentrations of trimethylamine N-oxide (TMAO) in blood were attained at 0.73 and 1 h after i.v and oral administration of TMA (20 mg kg(-1)), respectively. 5. Feeding the male Wistar rat with a synthetic diet resulted in a twofold decrease in the clearance of TMA. Furthermore, the concentration of TMAO in blood after i.v. administration of TMA peaked at 1.25 h in rat placed on the synthetic diet as opposed to 0.75 h in rat placed on normal laboratory rat chow. The altered pharmacokinetic profile of TMA and its N-oxide suggest a diminished drug-elimination capacity in rat placed on the synthetic diet. 6. Dietary modulation of flavin-containing monooxygenase (FMO) activity may explain the effects of diet on the pharmacokinetics of TMA and its N-oxide.

The effects of a synthetic diet on the pharmacokinetics of ethyl methyl sulphide and its sulphoxide and sulphone metabolites in rats.

Ethyl methyl sulphide (EMS) is a simple dialkyl sulphide, which occurs naturally and forms part structures of more complex drug molecules. EMS is oxidized to the corresponding sulphoxide (EMSO) and sulphone (EMSO2) derivatives both in vitro and in vivo. Two distinct enzymatic pathways appear to be involved in this sulphoxidation process; the flavin-containing monooxygenase (FMO) is largely responsible for the S-oxidation of EMS to its sulphoxide while both cytochrome P450 and FMO are involved in the further oxidation of the sulphoxide to the sulphone. The pharmacokinetics of EMS and its sulphoxide and sulphone metabolites were examined in male wistar rats placed on normal rat chow and those placed on a synthetic diet. Blood levels of EMS were analysed by a sensitive headspace gas chromatographic assay. A separate gas chromatographic assay was developed to monitor the blood levels of EMSO and EMSO2. The pharmacokinetics of EMS in control rats were linear from 10 to 40 mg/kg dose range. The blood concentration-time profile of EMS declined monoexponentially. EMS was rapidly eliminated from rat blood with a terminal half-life of 0.14 h and was not dytectable 1 h after administration. Following intravenous administration of EMSO (5 mg/kg), the blood concentration-time profile of EMSO declined with a terminal half-life (t 1/2) of 1.46 h, about ten times longer than that of the parent sulphide. After administration of EMSO2 (15 mg/kg), the sulphone was metabolically stable and was eliminated very slowly from the blood. The in vivo disposition of EMS and EMSO were clearly altered in rats maintained on a synthetic diet following administration of EMS or EMSO. The pharmacokinetic data were consistent with a diminished drug oxidising capacity in rats placed on the synthetic diet and could serve as a useful probe for monitoring the regulation of FMO in animals.

Metabolism of ethyl methyl sulphide and sulphoxide in rat microsomal fractions.

1. Gas chromatographic (GC) methods for the analysis of ethyl methyl sulphide (EMS) and its corresponding sulphoxide (EMSO) and sulphone (EMSO2) in rat microsomes and aspects of the in vitro metabolism of EMS and EMSO are described. 2. EMS and the internal standard (dimethyl sulphide) were extracted by a headspace procedure and separated satisfactorily using a column packed with 4% Carbowax 20 M/0.8% KOH on Carbopack B. EMSO, EMSO2 and the internal standard n-propyl sulphone were separated satisfactorily using a 2-m column packed with 10% Carbowax 20 M on Chromosorb W. 3. Under the optimum conditions (incubation of 10 min and microsomal protein content of approximately 4 mg/ml), 10% of the initial EMS concentration (2.5 mM) was converted to the corresponding sulphoxide in rat liver microsomal incubations. However, < 0.1% of the sulphone was detected when rat liver microsomes were incubated with EMS. Similarly, 2.5% of the initial EMSO concentration (2.5 mM) was converted to the corresponding sulphone by rat liver microsomes (approximately 4 mg/ml protein) during an incubation of 30 min. However, no EMS was detected after incubation with EMSO under these conditions. 4. The estimated apparent Vmax and Km for the sulphoxidation of EMS were 3.8+/-0.02 nmol/mg protein/min and 1.9+/-0.10 mM respectively. Vmax1, Vmax2 and Km1 and Km2 for the S-oxidation of EMSO were 0.5+/-0.01 and 0.2+/-0.01 nmol/mg protein/min and 0.7+/-0.02 and 0.1+/-0.00 mM respectively. 5. Studies with selective inducers and inhibitors of microsomal monooxygenases indicated that the sulphoxidation of EMS is mediated mainly by FMO, whereas both FMO and cytochrome P450 are involved in the S-oxidation of EMSO.

Pharmacokinetics of 2-methoxyphenylmetyrapone and 2-bromophenylmetyrapone in rats.

The pharmacokinetics of two 2-substituted phenylmetyrapone analogues, 2-methoxyphenylmetyrapone (2-MPMP) and 2-bromophenylmetyrapone (2-BrPMP), developed as potential adrenal imaging agents, were investigated in conscious male rats following an intravenous dose of 25 mg/kg. Arterial blood samples (0.25 ml) were collected at various intervals for up to 7 h after dose and subjected to reversed-phase HPLC analysis. Blood concentrations versus time profile for each compound was determined and the pharmacokinetic parameters calculated using the model-independent approach. Blood concentrations of 2-MPMP declined biexponentially with mean initial (t1/2alpha) and terminal (t1/2beta) half-lives of 3.6 and 23.1 min, respectively. The corresponding area under the curve (AUC(0-infinity)) was 159.3 microg x min/ml, the total blood clearance (CI) was 158.3 ml/min and the volume of distribution (Vd) was 5.2 l. Two metabolites of 2-MPMP, namely 2-hydroxyphenylmetyrapone (2-OHPMP) and 2-methoxyphenylmetyrapone N-oxide (2-MPMP-NO), were detected in the blood and their elimination from blood was almost parallel to that of the parent compound. The maximum blood concentrations (Cmax) of 2-OHPMP and 2-MPMP-NO were approximately 0.9 and 1.7 microg/ml, respectively. Blood concentrations of 2-BrPMP declined monoexponentially with a mean t1/2beta of 12.0 min. The pharmacokinetic parameters for 2-BrPMP were: AUC(0-infinity), 193.7 microg x min/ml; Cl, 131.7 ml/min and Vd, 2.3 l. 2-Bromophenylmetyrapone N-oxide was the only one metabolite detected in the blood, its Cmax and AUC0-infinity were 10.1 microg/ml and 1690.0 microg x min/ml, respectively.

High performance liquid chromatographic analysis of a novel aminoalkylpyridine anticonvulsant 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethane.

A simple, rapid and specific high performance liquid chromatographic (HPLC) method for the quantitation of 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethane (AAP-Cl) and identification of its putative metabolite, 2-(4-chlorophenyl)amino-2-(4-pyridyl)ethanol (beta-AA) in rat blood and urine has been developed. AAP-Cl, beta-AA and an appropriate internal standard were extracted from rat biofluids by a solid phase extraction technique using C18 cartridges prior to the HPLC analysis. The extractibility was 92% for AAP-Cl and 98% for beta-AA. The HPLC analysis employed a symmetrical or standard reversed-phase HPLC column (Apex ODS, 5 microm, 25 cm x 0.46 cm) for blood or urine analysis, a mobile phase of water methanol acetonitrile (40:30:30) containing 20 microl 100 ml(-1) diethylamine at a flow rate of 1 ml min(-1), and UV detection at 254 nm. The limit of detection was 100 ng ml(-1) for both analytes in both blood and urine. The calibration curves for AAP-Cl in rat biofluids were shown to be linear in both low and high concentration ranges (blood: 0-1 and 1-10 microg ml(-1); urine: 0-10 and 10-100 microg ml(-1)) with intra- and inter-day coefficients of variation of no more than 18% for blood and 14% for urine. The method developed was successfully applied to a preliminary analysis of intact AAP-Cl in both blood and urine obtained from rats dosed with AAP-Cl.

Efficient high-performance liquid chromatographic assay for the simultaneous determination of metoprolol and two main metabolites in human urine by solid-phase extraction and fluorescence detection.

An improved, more efficient method for the determination of metoprolol and its two metabolites in human urine is reported. The simultaneous analysis of the zwitterionic metoprolol acidic metabolite (III, H117/04) with the basic metabolites alpha-hydroxymetoprolol (II, H119/66), metoprolol (I) and guanoxan (IV, internal standard) was achieved employing solid-phase extraction and isocratic reversed-phase HPLC. The analytes were extracted from urine (100 microliters) using C18 solid-phase extraction cartridges (100 mg), and eluted with aqueous acetic acid (0.1%, v/v)-methanol mixture (40:60, v/v, 1.2 ml). The eluents were concentrated (250 microliters) under vacuum, and aliquots (100 microliters) were analysed by HPLC with fluorescence detection at 229 nm (excitation) and 309 nm (emission) using simple isocratic reversed-phase HPLC (Novapak C18 radial compression cartridge, 4 microns, 100 x 5 mm I.D.). Acetonitrile-methanol-TEA/phosphate buffer pH 3.0 (9:1:90, v/v) was employed as the eluent (1.4 ml/min). All components were fully resolved within 18 min, and the calibration curves for the individual analytes were linear (r2 > or = 0.996) within the concentration range of 0.25-40.0 mg/ml. Recoveries for all four analytes were greater than 76% (n = 4). The assay method was validated with intra-day and inter-day variations less than 2.5%.

Spectral and chromatographic properties of 2-methoxyphenylmetyrapone and its potential metabolites.

In the search for new metyrapone derivatives as radioligands for the functional diagnosis of adrenal pathology, 2-methoxyphenylmetyrapone [2-MPMP, 1-(2-methoxyphenyl)-2-methyl-2-(3-pyridyl)-1-propanone] (1), and related 2-substituted phenylmetyrapone derivatives, have been separated as potent inhibitors of adrenal 11 beta-hydroxylase, with high affinity for adrenal mitochondrial binding sites. Surprisingly, 2-[11C]MPMP showed a rapid loss of the radioactive label, which prompted investigation of its metabolism. Synthetic 2-MPMP (1) and its seven potential metabolites (2-8) have been identified spectroscopically (1H- and 13C-NMR and mass spectrometry) and further characterised by chromatography (TLC and gradient reversed-phase HPLC). Chromatographic and mass analysis of urinary extracts from rats dosed with 2-MPMP have confirmed the major metabolites as 2-hydroxyphenylmetyrapone (2-OHPMP), 2) and its N-oxide (2-OHPMP-NO, 6), which are present predominantly as conjugates.

Simultaneous analysis of 2-methoxyphenylmetyrapone and its seven potential metabolites by high-performance liquid chromatography.

A sensitive and specific high-performance liquid chromatographic (HPLC) assay has been developed for the quantification of 2-methoxyphenylmetyrapone (2-MPMP) and its seven potential metabolites in rat urine and whole blood. 2-MPMP, 2-hydroxyphenylmetyrapone and their N-oxides, together with 2-methoxyphenylmetyrapol, 2-hydroxyphenylmetyrapol and their N-oxides were separated on an Isco Spherisorb ODS-2 reversed-phase column (250 x 4.6 mm, I.D., 5 microm), with an Isco Spherisorb ODS-2 guard cartridge (10 x 4.6 mm I.D.). A gradient elution was employed using solvent system A (acetonitrile-water-triethylamine-acetic acid, 27.3:69.1:0.9:2.7%, v/v) and solvent system B (methanol), the gradient program being as follows: initial 0-4 min A:B=74:26; 4-10 min linear change to A:B=50:50; 10-16 min maintain A:B=50:50; 16 min return to initial conditions (A:B=74:26). Flow-rate was maintained at 1.25 ml/min, and the eluent monitored using a diode array multiple wavelength UV detector set at 260 nm. Most of the analytes were baseline resolved, and analysis of samples recovered from blood or urine (pH 12, 3 x 5 ml of dichloromethane, recovery approximately 20-95%) revealed no interference from any co-extracted endogenous compounds in the biological matrices, except for 2-hydroxyphenylmetyrapol N-oxide (2-OHPMPOL-NO) at low concentrations. The calibrations (n=6) were linear (r > or = 0.996) for all analytes (approximately 0.5-100 microg/ml), with acceptable inter- and intra-day variability. Subsequent validation of the assay revealed acceptable precision, as measured by coefficient of variation (C.V.) at the low (0.5 mg/ml), medium (50 microg/ml) and high (100 microg/ml) concentrations. The limits of detection for 2-MPMP and their available potential metabolites, except 2-OHPMPOL-NO, in rat urine and blood were both 0.5 microg/ml, respectively.

Identification of lactams as in vitro metabolites of piperidine-type phenothiazine antipsychotic drugs.

The metabolism of the piperidine-type phenothiazine antipsychotic agents thioridazine, mesoridazine and sulforidazine was studied in vitro with 10,000 g liver supernatants obtained from rats and dogs. After incubations at 37 degrees C for different time intervals, the incubates were extracted with dichloromethane and the isolated compounds analyzed by HPLC, direct probe MS and on-line HPLC-MS. Five lactam metabolites of these three drugs were unequivocally identified in the rat in vitro system, but none was found in dog preparations; at least one lactam metabolite was identified for each drug in the rat. The lactams of thioridazine and thioridazine ring sulfoxide were characterized as metabolites of thioridazine for the first time in any system. The other three lactam metabolites, namely the lactams of mesoridazine, sulforidazine and mesoridazine ring sulfoxide, were found in vitro for the first time, although they have been previously reported as in vivo metabolites of these drugs. The results indicate that rat would be a more suitable animal model than dog for further studies on the formation of lactam metabolites of these drugs.

Triazolines--XXVII. delta2-1,2,3-triazoline anticonvulsants: novel 'built-in' heterocyclic prodrugs with a unique 'dual-action' mechanism for impairing excitatory amino acid L-glutamate neurotransmission.

The delta2-1,2,3-triazoline anticonvulsants (1) may be considered as representing a unique class of 'built-in' heterocyclic prodrugs where the active 'structure element' is an integral part of the ring system and can be identified only by a knowledge of their chemical reactivity and metabolism. Investigations on the metabolism and pharmacology of a lead triazoline, ADD17014 (1a), suggest that the triazolines function as 'prodrugs' and exert their anticonvulsant activity by impairing excitatory amino acid (EAA) L-glutamate (L-Glu) neurotransmission via a unique 'dual-action' mechanism. While an active beta-amino alcohol metabolite, 2a, from the parent prodrug acts as an N-methyl-D-aspartate (NMDA)/MK-801 receptor antagonist, the parent triazoline impairs the presynaptic release of L-Glu. Various pieces of theoretical reasoning and experimental evidence led to the elucidation of the dual-action mechanism. Based on the unique chemistry of the triazolines, the potential metabolic pathways and biotransformation products of 1a were predicted to be the beta-amino alcohols 2a and 2a', the alpha-amino acid 3a, the triazole 4a, the aziridine 5a, and the ketimine 6a. In vivo and in vitro pharmacological studies of 1a and potential metabolities, along with a full quantitative urinary metabolic profiling of 1a, indicated the beta-amino alcohol 2a as the active species. It was the only compound that inhibited the specific binding of [3H]MK-801 to the MK-801 site, 56% at 10 microM drug concentration, but itself had no anticonvulsant activity, suggesting 1a acted as a prodrug. Three metabolites were identified; 2a was the most predominant, with lesser amounts of 2a', and very minor amounts of aziridine 5a. Since only 5a can yield 2a', its formation indicated that the biotransformation of 1a occurred, at least in part, through 5a. No amino acid metabolite 3a was detected, which implied that no in vivo oxidation of 2a or oxidative biotransformation of 1a or 5a by hydroxylation at the methylene group occurred. While triazoline 1a significantly decreased Ca2(+)-dependent, K(+)-evoked L-Glu release (83% at 100 microM drug concentration), triazolines 1a-1c showed an augmentation of 50-63%, in the Cl- channel activity, a useful membrane action that reduces the excessive L-Glu release that occurs during epileptic seizures. The high anticonvulsant activity of 1a may be due to its unique dual-action mechanism whereby 1a and 2a together effectively impair both pre- and postsynaptic aspects of EAA neurotransmission, and has clinical potential in complex partial epilepsy which is refractory to currently available drugs.

Urinary metabolic profile in rat of 1-(2-methoxyphenyl)-2-methyl-2-(3-pyridyl)-1-propanone: a potential radioligand for functional diagnosis of adrenal pathology.

1. The metabolism of 1-(2-methoxyphenyl)-2-methyl-2-(3-pyridyl)-1-propanone (2-MPMP) was studied in the male Sprague-Dawley rat after 50 mg/kg, i.v. dose. 2. Organic solvent extracts of urine samples were directly analysed by reversed-phase gradient hplc. The identified metabolites were also isolated by preparative tlc, and analyzed by direct probe mass spectrometry. In the case of conjugated metabolites, the urine samples were deconjugated by enzyme hydrolysis prior to extraction. The structures of metabolites were confirmed by comparison of their chromatographic behaviours, UV spectra, and mass spectra with those of authentic standards. 3. The metabolites identified in the 0-24-h urine samples were 2-hydroxyphenyl-metyrapone (2-OHPMP) and 2-hydroyphenylmetyrapone N-oxide (2-OHPMP-NO), which were present predominantly as their glucuronide and/or sulphate conjugates. 4. 2-MPMP and four of its metabolites present in the 0-24-h urine samples were quantified by a reversed-phase hplc method. The mean total urinary excretion was 75.4% of the administered dose. The major metabolites present in the urine were conjugates of 2-OHPMP-NO (54.4%) and of 2-OHPMP (18.6%). The excretion of the unchanged drug, unconjugated 2-OHPMP and 2-OHPMP-NO accounted for 1.1, 1.1 and 0.2% of the dose respectively.

Species differences in the stereoselectivity of N-oxygenation of N-ethyl-N-methylaniline in vitro.

The prochiral tertiary amine N-ethyl-N-methylaniline (EMA) is known to be stereoselectively N-oxygenated in the presence of hepatic microsomal preparations. This biotransformation is thought to be mediated predominantly by the flavin-containing monooxygenase (FMO) enzyme system. In order to characterise this reaction further, the in vitro metabolism of EMA in the presence of hepatic microsomal preparations derived from a number of laboratory species has been examined. EMA N-oxide formation was stereoselective with respect to the (-)-S-enantiomer in the presence of microsomal preparations from all species examined, with the degree of selectivity decreasing in the order of rabbit > rat approximately LACA mouse approximately DBA/2Ha mouse > guinea-pig > dog. The enantiomeric composition of the metabolically derived EMA N-oxide appeared to be determined solely by the differential rate of formation of the two enantiomers as opposed to any differences in affinities for the substrate in its pro-R and pro-S conformations. The use of enzyme inhibitors, activators and inducers indicated that EMA N-oxide formation was predominantly mediated by FMO in the presence of rabbit hepatic microsomes and that these agents did not generally affect the stereochemical outcome of the biotransformation.

The assessment of flavin-containing monooxygenase activity in intact animals.

A large number of drug metabolising enzymes with different substrate specificities and induction and inhibition characteristics have been described, suggesting that specific test drugs, i.e. probes, should be used for assessing the activity of distinct metabolising enzymes. The flavin-containing monooxygenase (FMO) and cytochrome P-450 (P-450) are the two main microsomal enzyme systems involved in the oxidation of xenobiotics. FMO is present in liver and other tissues of most vertebrates. It catalyses the oxidation of a wide range of xenobiotics, especially soft nucleophiles bearing nitrogen and sulphur centres. There is substantial information on both in vitro and in vivo probes for cytochrome P-450. For example antipyrine has been widely used for assessing the activity of P-450 in vivo by utilising pharmacokinetic parameters as indices of enzyme activity. In more recent years, isozyme specific probes have also been developed for some of the P-450s. Whereas a number of substrates are available for measuring FMO activity in vitro (e.g. N,N-dimethylaniline), probes for assessing FMO activity in vivo are limited. In this review a background to the use of in vitro and in vivo probes for assessing the activity of FMO is presented, and approaches and criteria for development of potential pharmacokinetic probes for FMO are described. Preliminary data on the development of ethyl methyl sulphide (EMS) and trimethylamine (TMA) as potential pharmacokinetic probes for assessing FMO activity in rats are discussed in detail. Clinical implications of modulation of FMO activity are discussed, and arguments presented as to why the development of FMO probes for use in man will be useful additions to the range of other compounds available for assessment of liver metabolic function.

HPLC determination of 1,2-diethyl-3-hydroxypyridin-4-one (CP94), its iron complex [Fe(III) (CP94)3] and glucuronide conjugate [CP94-GLUC] in serum and urine of thalassaemic patients.

Sensitive and selective high performance liquid chromatographic (HPLC) methods for the quantification of 1,2-diethyl-3-hydroxypyridin-4-one (CP94), its iron complex [Fe(III) (CP94)3] and glucuronide metabolite (CP94-GLUC) in urine and serum of thalassaemic patients are described. Three separate analyses are involved. The first assay quantifies both CP94 and its iron complex. This procedure requires the conversion of the iron complex to the free ligand and is carried out using diethylenetriaminepentaacetic acid (DTPA). CP94 and the internal standard, 1-propyl-2-ethyl-3-hydroxypyridin-4-one (CP95) present in either serum or urine are then extracted at pH 7.0 with dichloromethane. Extraction efficiency is 96.0 +/- 5.6% and 100 +/- 7.1% for CP94 and CP95, respectively, and 31.2 +/- 2.1% at 30 microM and 53.2 +/- 4.2% at 300 microM for the corresponding iron complex. In the second assay, samples are incubated (16 h) with beta-glucuronidase and processed as before. In this assay, the drug, its iron complex and glucuronide conjugate are measured. In the third assay the iron complex of CP94, [Fe(III) (CP94)3] is quantified. From the three separate analyses it is possible to calculate the individual concentrations of the three separate components present in serum and urine of thalassaemic patients. Calibration for both components, i.e. CP94 (assays 1 and 2) and its iron complex (assay 3) are linear with correlation coefficients > 0.99 and are reproducible over the required concentration range of 0-500 microM for the free ligand and 0-100 microM for the iron complex. The minimum quantifiable level is 0.5 microM for the free ligand and 1.0 microM for the iron complex.

Species variability in the stereoselective N-oxidation of pargyline.

The monoamine oxidase inhibitor pargyline (N-benzyl-N-methyl-2-propynylamine) is known to undergo extensive in vitro microsomal N-oxidation, thought to be mediated predominantly by the flavin-containing monooxygenase (FMO) enzyme system. Formation of the pargyline N-oxide (PNO) metabolite creates a chiral nitrogen centre and thus asymmetric oxidation is possible. This study describes a reverse-phase high-performance liquid chromatographic (HPLC) method for the quantitation of PNO and a chiral-phase HPLC method for the determination of the enantiomeric ratio of PNO. In vitro microsomal N-oxidation of pargyline was found to be highly stereoselective in a number of species, with the (+)-enantiomer being formed preferentially. This metabolic transformation was stereospecific when purified porcine hepatic FMO was used as the enzyme source.

Asymmetric metabolic N-oxidation of N-ethyl-N-methylaniline by purified flavin-containing monooxygenase.

The prochiral tertiary amine N-ethyl-N-methylaniline (EMA) is known to be metabolically N-oxygenated in vitro with microsomal preparations. This biotransformation is thought to be mediated predominantly by the flavin-containing monooxygenase (FMO) enzyme system. Microsomal N-oxygenation of EMA is known to be stereoselective and varies between species. In order to further characterise this metabolic transformation, we have examined the in vitro metabolism of EMA using purified porcine hepatic FMO. Following incubation of EMA with purified FMO, EMA N-oxide, the only metabolic detected, was found to be produced stereoselectively [ratio (-)-(S):(+)-(R), ca. 4:1]. The enantiomeric ratio of the N-oxide product did not change markedly with respect to time, enzyme or substrate concentration. Determination of the kinetics of formation of the N-oxide indicated a single affinity for the prochiral substrate with differential rates of formation of the enantiomers. The extent of EMA N-oxide formation was shown to be affected by activators and inhibitors of FMO and pH, but its stereoselectively was unaltered.