J. W. Gorrod

Dehydration is the first step in the bioactivation of haloperidol to its pyridinium metabolite

Haloperidol was found to have a similar metabolic pathway to that of the neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice microsomal preparations. The 1,2,3,6-tetrahydropyridine derivative of haloperidol was detected in NADPH-fortified metabolic incubation mixtures of haloperidol. Incubation of this dehydrated haloperidol produced the pyridinium metabolite. These metabolites were confirmed by comparison with synthesised compounds using HPLC and HPLC-MS. Dehydration of an alcohol to a double bond represents a novel metabolic pathway. This novel metabolic pathway indicates a MPTP-like mechanism for the Parkinsonism observed with haloperidol in clinical use.

The metabolism of alicyclic amines to reactive iminium ion intermediates

SummaryThe evidence implicating the formation of iminium ions as reactive intermediates in the metabolism of alicyclic amines has been reviewed. The mechanism of formation of iminium ions and their conversion to α-carbonyl compounds or demethylated amines is discussed. The use of a simple cyanide trapping technique for iminium ions has been demonstrated to monitor a large number of alicyclic drugs for iminium ion formation. The possible role of iminium ions in the pharmacology and toxicology of alicyclic amines is considered.

Investigation of the in vitro metabolism of the H2-antagonist mifentidine by on-line capillary electrophoresis—mass spectrometry using non-aqueous separation conditions

The in vitro metabolism of mifentidine, a prototype second-generation histamine H2-antagonist, is investigated using on-line capillary electrophoresis-mass spectrometry (CE-MS) by analysis of hepatic microsomal incubates. Consideration of the hydrophobicity of this drug and putative metabolites led to the development of a non-aqueous CE separation medium consisting of 5 mM NH4OAc in methanol containing 100 mM acetic acid. Benefits of non-aqueous media in CE-MS studies of small hydrophobic molecules are discussed. In addition, we elucidate both chemical transformations and the in vitro metabolism of mifentidine using guinea pig hepatic microsomes.

Investigation of the metabolism of the neuroleptic drug haloperidol by capillary electrophoresis

Free solution capillary electrophoresis (FSCE) conditions were previously reported to be of limited use for the separation of pharmaceuticals, since many of these compounds are neutral. We show that by consideration of compound hydrophobicity and ionisable functional groups, FSCE conditions can be developed to effect the separation of a drug and its phase I metabolites. This is brought about by adding a suitable organic modifier to aid solubility, and modifying pH to effect a change in the mass to charge ratio of the metabolites present. Furthermore, we show that in this drug metabolism study, FSCE presents an advantage over both reversed-phase HPLC and micellar electrokinetic chromatography. We also demonstrate the use of FSCE for investigation of the phase I metabolites produced by the in vitro incubation of haloperidol (a neuroleptic agent) with both mouse and guinea pig hepatic microsomes and show that such an approach can be used to detect both qualitative and quantitative differences in species metabolism.

High-performance liquid chromatographic method for the detection and quantitation of haloperidol and seven of its metabolites in microsomal preparations

An isocratic high-performance liquid chromatographic (HPLC) system was developed to analyze haloperidol and its potential metabolites. These compounds included 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), haloperidol N-oxide (HNO), reduced haloperidol (RHAL), the 1,2,3,6-tetrahydropyridine analogue and its N-oxide, and the pyridinium ion from haloperidol (HP+). The HPLC system comprised a Hypersil CPS5 column with a mobile phase of acetonitrile (67%) and ammonium acetate (final concentration 10 mM) which was adjusted to pH 5.4 by acetic acid. The solvent was delivered at 1 ml/min. RHAL and CPHP were determined by an ultraviolet detector at 220 nm with a detection limit of 1 nmol/ml. All other compounds were determined at 245 nm and had a detection limit of 0.3 nmol/ml. This system was used to analyze a microsomal metabolic mixture of haloperidol. It was found that all above compounds except HNO were metabolites of haloperidol. In addition, two other metabolites were also well separated in this HPLC system which are proposed to be oxygenated haloperidol and the pyridone analogue of haloperidol. The HPLC system was used to carry out quantitative metabolic studies of haloperidol. It was found that the metabolism of haloperidol exhibits large inter-species differences. The apparent enzyme kinetic parameters were also determined using mice microsomes.

Metabolism of (−)-(S)-nicotine by guinea pig and rat brain: identification of cotinine

SummarySince the brain is the major site of pharmacological activity of nicotine, it was of interest to investigate the metabolism of nicotine by this organ. We now report our findings using guinea pig and rat brain as the enzyme source. Whole brains were removed and washed with isotonic KCl, blotted dry and cut into small pieces. The tissue was weighed and homogenized in pH 7.4 Tris-KCl buffer, 2 ml/g tissue. Incubations were carried out using 0.5 ml of brain homogenate and 0.1–1 μmol of nicotine at 37°C. The reactions were terminated by freezing at −80°C. The samples were extracted and analyzed by capillary GC with nitrogen-phosphorus detection. Cotinine was detected as the major metabolite and its identity confirmed by GC-MS. Cotinine formation may contribute to the detoxication pathway of nicotine and may be important in controlling nicotine levels in the brain. Furthermore, the conversion of nicotine to cotinine involves the intermediacy of nicotine-Δ1′(5′)-iminium ion, which is an alkylating agent. This finding supports the concept that reactive intermediates may play a role in the pharmacology and toxicology of nicotine.

Investigation of the neuroleptic drug haloperidol and its metabolites using tandem mass spectrometry

Abstract The in vitro metabolism of haloperidol, a clinically utilized neuroleptic drug, was investigated using guinea pig derived hepatic microsomal incubates. By employing a combination of reversed phase HPLC and tandem mass spectrometry, it was revealed that haloperidol was metabolized to at least eight different compounds, including the proposed dopaminergic toxin 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4- oxobutyl]-pyridinium species and an intermediate metabolite 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4- oxobutyl]- 1,2,3,6-tetrahydropyridine.

Conformational Analysis of 9‐Substituted Adenines in Relation to Their Microsomal N1‐Oxidation

Metabolic TV‐oxidation of adenine, 9‐methyladenine, 9‐benzyladenine, 9‐benzhydryladenine and 9‐trityladenine has been investigated using hepatic microsomes from hamster, guinea‐pig, rabbit, mouse, rat, and dog. N1‐Oxide formation occurs with 9‐benzyladenine and 9‐benzhydryladenine using liver preparations of all species examined, although to different extents. The N‐oxidase activity was found, amongst rodents, in the order hamster > mouse > rabbit > rat > guinea‐pig. Microsomal preparations from dog liver contained a small quantity of P‐450 and yet produced a relatively large amount of the N‐oxides, possibly indicating that other cytochromes in addition to P‐450 may be involved in the TV‐oxidation of these compounds. The most favourable conformations of these 9‐substituted analogues have been established using computer graphics modelling and 1H NMR techniques. Results obtained confirmed the importance of the stereochemical properties of these compounds in relation to N1‐oxidation. These observations substantiate and extend our previous findings on the electronic, lipophilic, and stereochemical factors affecting the TV‐oxidation of adenine derivatives.

Capillary electrophoresis and capillary electrophoresis-mass spectrometry in drug and metabolite analysis.

SummaryThe structural diversity of modern therapeutic agents can lead to labour intensive method development for each drug and the structural characterization of their metabolites. In this work, we show the benefits of the high resolution capabilities of capillary electrophoresis (CE) and demonstrate that nonaqueous CE and on-line CE-mass spectrometry (CE-MS) leads to enhanced resolution and recovery of mixtures containing the prototype H2-antagonist, mifentidine, and putative metabolites. Furthermore, the usefulness of CE-tandem MS (CE-MS/MS) is also demonstrated by the structural characterization of the novel N2-hydroxylamine metabolite of mifentidine.

Biological N-oxidation of adenine and 9-alkyl derivatives

SummaryIn vitro metabolism of adenine, 9-methyladenine, and 9-benzyladenine using hepatic microsomes of hamster, mouse and rat was investigated. The results indicated that adenine was apparently not susceptible to microsomal N-oxidation. N-oxidation of 9-methyladenine was also not detected, whereas N-demethylation was observed with hepatic microsomes derived from hamster and rat but not from mouse. With 9-benzyladenine, both 1-N-oxide formation and N-debenzylation occurred with microsomes of all species in various amounts. N-Hydroxylation of the 6-amino group was not observed with any substrate in any species. Metabolic results are discussed in relation to chemical structure, electronic, lipophilic and steric factors.