Short communicationEstimation of chlorzoxazone hydroxylase activity in liver microsomes and of the plasma pharmacokinetics of chlorzoxazone by the same high-performance liquid chromatographic method
Introduction
Chlorzoxazone (CZX) has been used for many years as a potent long-acting central muscle relaxant [1]. The drug is rapidly absorbed from the gastro-intestinal tract and 6-hydroxychlorzoxazone (OH-CZX) is the main metabolite present in plasma as a conjugate.
Metabolic investigations of this compound were undertaken after some cases of hepatotoxicity were reported [2]. Human cytochrome P-4502 E1 (CYP2E1) was found to be the primary catalyst of chlorzoxazone 6-hydroxylation in human liver [3].
Considerable variations in the rates for this hydroxylation reaction have been measured in man and a low enzyme activity could be responsible for a hepatotoxic reaction but this remains to be determined 4, 5. Nevertheless, the use of this drug as a non-invasive probe for human CYP2E1 activity is now recognized and widely used 6, 7.
In parallel with these investigations, new high-performance liquid chromatography (HPLC) methods have been developed to measure CZX and OH-CZX concentrations in biological fluids and in microsomes 8, 9, 10, 11, 12, 13. All the published methods are based on the same preparation scheme: deconjugation of OH-CZX and liquid–liquid extraction. In most of these methods, an internal standard has been used and the compounds were separated by isocratic elution and quantified by UV detection. It must be pointed out that most of the compounds used as an internal standard are not chemically related to CZX or are synthesized by the investigators.
We find that dehalogenated CZX or 2-benzoxazolinone which is commercially available could be a convenient and stable internal standard. This paper describes a sensitive HPLC assay for CZX and OH-CZX in rat hepatic microsomes and in human plasma.
Section snippets
Chemicals and standards
CZX and 2-benzoxazolinone (internal standard) were purchased from Aldrich (Milwaukee, WI, USA). OH-CZX was obtained from Ultrafine (Manchester, UK). Acetonitrile HPLC ultra gradient grade, methanol and ethyl acetate HPLC grade were from Labscan (Dublin, Ireland). Helix Pomatia juice and nicotinamide–adenine dinucleotide phosphate, reduced (NADPH) tetrasodium salt were purchased from Boehringer (Mannheim, Germany). All other reagents were of the highest purity available.
Preparation of analytical standards
Stock solutions of CZX (1
Rat liver microsomes
CZX, its metabolite and the internal standard were well separated. Fig. 1 shows typical chromatograms obtained with rat liver microsomes before (A) and 20 min after (B) incubation with CZX. OH-CZX was eluted in 7.5 min, the I.S. in 9.5 min and CZX within 24.5 min.
Discussion
In this paper we present a simplified method, based on the paper of Peter et al. [3], to measure chlorzoxazone hydroxylase activity in rat liver microsomes. The three main modifications introduced are: the use of pure NADPH instead of a generating system, the addition of an internal standard chemically related to CZX and the direct injection of the supernatant, obtained after acid precipitation, onto the HPLC column.
We verify the experimental conditions for what concerns linearity, duration of
References (13)
- et al.
J. Chromatogr. B
(1997) - et al.
J. Pharm. Sci.
(1979) - et al.
J. Chromatogr.
(1993) - et al.
J. Chromatogr.
(1993) - et al.
J. Chromatogr. B
(1996) - et al.
J. Pharmacol. Exp. Ther.
(1960)
Cited by (31)
YSZ/MoS<inf>2</inf> modified carbon based sensor for the determination of muscle relaxant agent chlorzoxazone: A novel electroanalytical strategy
2023, Inorganic Chemistry CommunicationsNanostructured Au-graphene modified electrode for electrosensing of chlorzoxazone and its biomedical applications
2021, Materials Chemistry and PhysicsCitation Excerpt :From the last few years, chlorzoxazone (CHZ) named as 5-chloro-2-hydroxy benzoxazole, has been a powerful, long-acting central skeletal muscle relaxant [1]. It decreases the muscle stress, and dismisses the spasm and pain connected with musculoskeletal ailments [2,3]. CHZ (see Scheme 1) can quickly be absorbed from the gastrointestinal area, and 6-hydroxychlorzoxazone (OH-CHZ) is the chief metabolite presented in the plasma as a conjugate [3].
Ultrasensitive quantification of the CYP2E1 probe chlorzoxazone and its main metabolite 6-hydroxychlorzoxazone in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry after chlorzoxazone microdosing
2016, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesHepatic expression of cytochrome P450 in type 2 diabetic Goto-Kakizaki rats
2012, Chemico-Biological InteractionsCitation Excerpt :Ethoxyresorufin is a substrate sensitive to CYP1A1; CYP1A2, CYP2C6, CYP2C11 and CYP2E1 are also involved in ethoxyresorufin metabolism, while methoxyresorufin metabolism is primarily catalyzed by CYP1A1 and CYP1A2 [24,25]. CZX 6-hydroxylation is extensively mediated by the CYP2E1 isoform [23]. MDZ is predominantly metabolized to 1′-hydroxy MDZ and 4-hydroxy MDZ by CYP3A1 and CYP3A2 in rats, and it has thus been used as a selective CYP3A substrate [21,26].
Determination of brain cytochrome P450 2E1 activity in rat with the probe of chlorzoxazone by liquid chromatography-mass spectrometry
2011, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesCitation Excerpt :The SPE method was sensitive, but the cost associated with use of solid-phase extraction columns decreases the overall usability of this method [26]. For some literatures, the incubated membrane solutions were centrifuged, filtered with a filter and direct injected onto the HPLC column for analysis [24,25]; however, these methods were poor in sensitivity and could not meet the requirement of low concentration of target compounds in samples. Organic solvents, such as chloroform, ether, ethyl acetate and isopropyl ether were tested in our study for solvent extraction of OH-CLZ and IS from rat whole brain membranes.
Protective effects of thiopronin against isoniazid-induced hepatotoxicity in rats
2009, ToxicologyCitation Excerpt :Aniline hydroxylation was used to determine kinetic parameters (Km and Vmax) and the inhibition constant (Ki) of thiopronin. CZXH was assayed according to the method of Leclercq et al. (1998) with a few modifications. The reaction mixture consisted of 10 mM MgCl2, 100 mM Tris–HCl (pH 7.4), 5 mM NADPH, 0.02% chlorzoxazone (CZX) and 0.5 mg of hepatic microsomal protein for a final volume of 0.5 ml.