L.A. Damani

The effect of various potential inhibitors, activators and inducers on the N-oxidation of 3-substituted pyridines in vitro

1. The N-oxidation of pyridine, 3-methylpyridine and 3-chloropyridine was inhibited by SKF525A and DPEA. The C-oxidation of 3-methylpyridine was also inhibited by these compounds. 2. The N-oxidation of these pyridines was also inhibited by various other nitrogenous substrates including n-octylamine. 3. Incubation in an atmosphere of carbon monoxide resulted in inhibition of both C- and N-oxidation of 3-methylpyridine. 4. Any treatment of microsomes which resulted in a reduction of cytochrome P-450 also produced a concomitant fall in N-oxidation of the pyridines. 5. Pretreatment of animals with phenobarbitone resulted in an increase in the N-oxidation of the pyridines. Pretreatment with 3-methylcholanthrene had no appreciable effect on the N-oxidation of the pyridines in vitro.

Species differences in the metabolic C- and N-oxidation, and N-methylation of [14C]pyridine in vivo

1. The metabolism of [2,6-14C]pyridine in vivo has been investigated in the rat, hamster, mouse, gerbil, rabbit, guinea-pig, cat and man, and the quantitative determination of the various urinary metabolites carried out by radiochromatographic analysis. 2. Unchanged pyridine and its N-methylated metabolite, N-methylpyridinium ion, were determined using a Partisil-10 SCX cation-exchange h.p.l.c. column, whereas the C- and N-oxidation products were assayed by reverse-phase chromatography, using a Partisil-10 ODS column. 3. Most of the species studied produce pyridine N-oxide, N-methylpyridinium ion, 2-pyridone, 3-hydroxypyridine and 4-pyridone as metabolites, but the proportion of the dose excreted as each of these metabolites is species-dependent.

Enantioselective metabolism during continuous administration of S-(−)- and R-(+)-nicotine isomers to guinea-pigs

Abstract— The S‐(−)‐ and R‐(+)‐nicotine isomers were administered subcutaneously via Alzet osmotic pumps to male Hartley guinea‐pigs (n = 5 with each isomer) over a 23‐day period. Estimated dosage rate throughout the experiment was 0.6 mg−1. Urine samples were collected over this time and the levels of urinary oxidative and N‐methylated nicotine metabolites were measured by cation‐exchange HPLC analysis. S‐(−)‐Nicotine formed only oxidative metabolites, whereas the R‐(+)‐isomer formed both oxidative and N‐methylated metabolites. 3′‐Hydroxycotinine and nicotine‐1′‐oxide were major metabolites of both enantiomers; cotinine and nornicotine were only minor metabolites. The major N‐methylated metabolite of R‐(+)‐nicotine was N‐methylnicotinium ion; N‐methylcotininium ion and N‐methylnornicotinium ion were also identified as metabolites of this nicotine isomer. Total N‐methylated quaternary ammonium metabolites accounted for 15 to 20% of the administered dose of R‐(+)‐nicotine. An interesting enantioselective reduction in the percent of oxidative urinary metabolites formed from S‐(−)‐nicotine was observed over 23 days. This may indicate the enantioselective induction of an uncharacterized metabolic pathway for this nicotine isomer.

HPLC analysis of 4-chlorophenyl methyl sulphide and diphenyl sulphide and their corresponding sulphoxides and sulphones in rat liver microsomes

Simple high performance liquid chromatography (HPLC) methods for the analysis of 4-chlorophenyl methyl sulphide (CPMS), diphenyl sulphide (DPS) and their corresponding sulphoxide and sulphone metabolites in rat liver microsomes are described. The assay methods are based on a reversed phase HPLC column (Spherisorb(R) 5 ODS, 15 x 0.46 cm) using a mixture of water and tetrahydrofuran (THF) as mobile phase at a flow rate of 0.5 ml/min and ultraviolet detection at 260 nm. The compounds were extracted into diethyl ether (2 x 5 ml) from rat liver microsomal incubation mixture (2 ml) and the recoveries were more than 80%. The calibration curves for determining the sulphoxide and sulphone of CPMS or DPS were linear (r > or =0.995) in the range of 0-50 microg/ml and the assays were reproducible with low inter- and intra-assay variation of less than 13.5%. The lower limit of quantitation (LOQ) was 0.1 microg/ml for CPMSO and 0.025 microg/ml for CPMSO(2), diphenyl sulphoxide (DPSO) and diphenyl sulphone (DPSO(2)). The HPLC methods were successfully applied to measure enzymically formed CPMSO, CPMSO(2), DPSO and DPSO(2) in rat liver microsomes and to characterise the Michaelis-Menten kinetics associated with the metabolism of CPMS and DPS and their corresponding sulphoxides. About 20% of the initial CPMS (0.5 mM) concentration in the incubation was converted to the sulphoxide although the sulphone was not detected under these optimum incubation conditions. Similarly, about 15-20% of DPS was converted to the sulphoxide while less than 0.1% of DPS was converted to DPSO(2). Eadie-Hofstee plot of CPMS sulphoxidation was biphasic. This suggests that the sulphoxidation of CPMS is a consequence of at least two enzyme systems, one characterized by low affinity and high capacity (K(m)=0.1 mM; V(max)=2.1 nmoles/mg protein/min) and the other by high affinity and low capacity (K(m)=0.05 mM; V(max)=1.5 nmoles/mg protein/min). On the other hand, the Eadie-Hofstee plot of DPS sulphoxidation was monophasic with an apparent V(max) and K(m) of 1.8 nmoles/mg protein/min and 0.036 mM, respectively.

Synthesis of N-oxide derivatives of metyrapone and their detection as in vitro metabolites.

A variety of possible N‐oxidation products of 2‐methyl‐1, 2‐bis(3‐pyridyl)propan‐1‐one (metyrapone) have been synthesized by peracid oxidation, and characterized using various spectroscopic techniques. Specific and sensitive chromatographic techniques have been developed for the separation and identification of its in vitro metabolites. Incubation of metyrapone with rat or mouse hepatic microsomes, in the presence of a NADPH‐regenerating system, leads to the formation of metyrapol (keto‐reduction), and two mono‐N‐oxides.

The origin of 1-(3-pyridyl-N-oxide)ethanol as a metabolite of 3-acetylpyridine

1. 3-Acetylpyridine was metabolized extensively to 1-(3-pyridyl)ethanol when the hepatic enzyme source was the soluble fraction (140,000 g supernatant). 3-Acetylpyridine-N-oxide was identified as a metabolite using the 10,000 g or the microsomal fraction. 2. 1-(3-Pyridyl-N-oxide)ethanol was not detected as a metabolite of 1-(3-pyridyl)ethanol using tissue preparations. However, 3-acetylpyridine was formed in trace amounts when the alcohol was incubated with the 10,000 g or the microsomal fraction. 3. Incubation of 3-acetylpyridine-N-oxide with the soluble or 10,000 g fraction resulted in the formation of 1-(3-pyridyl-N-oxide)ethanol (keto-reduction) as the major metabolite. 3-Acetylpyridine was formed in trace amounts (N-oxide reduction) with the 10,000 g and the microsomal fractions.

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.

The ability of amine N‐methyltransferases from rabbit liver to N‐methylate azaheterocycles

The substrate specificity of two homogeneous amine N‐methyltransferases from rabbit liver has been demonstrated to extend to the azaheterocycles pyridine, R‐(+)‐nicotine and S‐(−)‐nicotine. Both enzymes methylate R‐(+)‐nicotine at the pyridyl nitrogen to afford the N‐methylnicotinium salt, whereas S‐(−)‐nicotine does not act as a substrate for either enzyme. Surprisingly, R‐(+)‐nicotine is methylated at either the pyridyl nitrogen, or the pyrrolidine nitrogen, to afford the two isomeric monomethylate nicotinium ions when an enzymic preparation containing both methyl transferase activities was used. Under similar conditions S‐(−)‐nicotine was methylated only at the pyridyl nitrogen. The production of charged metabolites in‐vivo, from the large number of pyridine‐compounds that are used as drugs, or are present in the environment, may be of toxicological significance, in view of the reported toxicities of several such quaternary ammonium compounds.

Stereoselective microsomal N-oxidation of N-ethyl-N-methylaniline

The stereoselectivity of metabolic N-oxidation of N-ethyl-N-methylaniline (EMA) was investigated in vitro following incubation of the compound (1mM) with fortified hepatic microsomal preparations of both male Wistar rats and New Zealand White (NZW) rabbits. The major metabolites in both species were found to be N-ethylaniline, N-methylaniline and EMA N-oxide. Chromatographic resolution of the N-oxide enantiomers was achieved using a Chiralcel OD stationary-phase with a mobile-phase of hexane:ethanol (98:2, v/v). Examination of the enantiomeric composition of the N-oxide metabolites indicated a predominance of the (-)-(S)-N-oxide from both species with enantiomeric excesses of 52 +/- 2.5% and 65 +/- 2.1% (n = 3) in rat and rabbit tissue respectively. These preliminary observations indicate that the N-oxidation of EMA shows product stereoselectivity, the extent of which varies between species.

The metabolism of N,N-dimethylaniline by isolated rat hepatocytes: identification of a novel N-conjugate.

1. Incubation of N,N-dimethylaniline (DMA) with isolated rat hepatocytes resulted in the production of N-methylaniline, aniline, N,N-dimethylaniline N-oxide (DMA N-Oxide) and a highly water-soluble metabolic tentatively identified as N-methylaniline N-glucuronide. 2. After the removal of aniline, N-methylaniline and DMA, treatment of the media with either strong acid or beta-glucuronidase, resulted in the release of N-methylaniline, identified by chromatography and mass spectrometry. 3. Pre-incubation of rat hepatocytes with 2 mM D-galactosamine, which decreased 7-hydroxycoumarin conjugate formation by 40%, selectively decreased the formation of this highly water-soluble metabolite from DMA by 70%. DMA N-demethylase and N-oxidase activities remained unchanged. 4. Incubation of rat hepatocytes with N-methylaniline resulted in the production of the novel metabolite, the formation of which was proportional to cell number, incubation time, and N-methylaniline (substrate) concentration. 5. The N-glucuronidation of the secondary N-alkylarylamine, N-methylaniline, by rat hepatocytes represents a quantitatively important and previously uncharacterized route of metabolism in these cells. Further studies are, however, required to identify this metabolite unequivocally as the N-glucuronide of N-methylaniline.