Enantioselective capillary electrophoresis for the assessment of CYP3A4-mediated ketamine demethylation and inhibition in vitro (original) (raw)
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Analytical and Bioanalytical Chemistry
It is just the right point in time to publish a special issue on cytochrome P450 (CYP), because this year we can celebrate the 50th anniversary of the discovery of the CYP proteins. This unique enzyme system was found by Klingenberg and colleagues in 1958 [1], on the basis of its distinct spectroscopic properties which later led to its designation as P450 [2], a pigment with characteristic absorption at 450 nm under particular conditions. Since then, cytochrome P450 has been shown to be more complex than initially suspected. By and by, dissection of the enzyme revealed more and more individual enzymes and the functional significance of the enzymes for toxicology, pharmacology, and cancer research became obvious. In consequence, interest in cytochrome P450 is still high and increasing but appears to be reaching a plateau phase judging from the number of papers published per year on cytochromes P450 . Retrospectively, the average annual number of publications on P450 amounted to about 1600 in recent years. Interest in cytochromes P450 is also underlined by a brand new monograph dealing with toxicological and metabolism aspects of cytochromes P450 [3]. However, methodical aspects of CYP research are not dealt with in this book so that this special issue on cytochrome P450 is, again, the right complement at the right time.
Functional High Level Expression of Cytochrome P450 CYP2D6 Using Baculoviral Expression Systems
Archives of Biochemistry and Biophysics, 1996
Cytochrome P-450 CYP2D6 plays a central role in metabolism. the metabolism of many widely used therapeutic drugs including b-adrenergic antagonists, antiarrhythmics, and tricyclic antidepressants. Recombinant baculoviruses have been constructed containing the full-length human CYP2D6 cDNA and Cytochrome P450's belong to a superfamily of enused to express CYP2D6 in Spodoptera frugiperda zymes which catalyze the hydroxylation of a broad (Sf 9) cells. High levels of recombinant protein have spectrum of xenobiotic chemicals and procarcinogens been produced using either polyhedrin or basic pro-(1, 2). Cytochrome P450 CYP2D6 plays a crucial role tein promoters (0.05 -0.20 nmol/mg cell protein; 0.05in the metabolism of several widely used therapeutic 0.15 nmol/liter). The enzyme is catalytically active toagents including cardiovascular drugs, b-adrenergic ward CYP2D6 substrates such as bufuralol and metoblocking agents, tricyclic antidepressents, and miscelprolol. In order to optimize catalytic activity human laneous compounds such as methoxy amphetamine, coreductase was coexpressed with CYP2D6 in Sf 9 cells; deine, and dextromethorphan (3). In addition, CYP2D6 reductase activity was in the region of 1000 -1500 is highly polymorphic, particularly among Caucasians units per mg cell protein, while spectrally active where 5-10% of the populace are deficient in this en-CY2D6 was in the range 10 -20 pmol/mg cell protein.
Expression and Functional Analysis of CYP2D6.24, CYP2D6.26, CYP2D6.27, and CYP2D7 Isozymes
Drug Metabolism and Disposition, 2008
The objectives of this study were to compare the drug-metabolizing activity of human CYP2D6.24 (I297L), CYP2D6.26 (I369T), CYP2D6.27 (E410K) allelic isoforms with wild-type CYP2D6.1, and to express the CYP2D7 protein derived from an indel polymorphism (CYP2D7 138delT) and investigate its possible codeine O-demethylase activity. Successful creation of individual cDNAs corresponding to CYP2D6*24 (2853 A>C), CYP2D6*26 (3277 T>C), CYP2D6*27 (3853 G>A) allelic variants and CYP2D7 was achieved via molecular cloning. The corresponding proteins, CYP2D6.24, CYP2D6.26, CYP2D6.27 and CYP2D7, were expressed in insect cells using a baculovirus-mediated expression system. All CYP2D proteins showed the empirical carbon monoxide difference spectra. Surprisingly, CYP2D7 protein was detected mainly in mitochondrial fraction, whereas all CYP2D6 allelic isoforms were present in microsomal fraction. Furthermore, CYP2D7 did not produce any morphine from codeine. In contrast, CYP2D6.24, CYP2D6.26 and CYP2D6.27 allelic isoforms all showed active drugmetabolizing activities towards both codeine and dextromethorphan O-demethylation. While CYP2D6.24 exhibited the highest intrinsic clearance in dextromethorphan O-demethylation (~6fold higher than that by CYP2D6.1), it had the lowest enzyme efficiency in codeine Odemethylation (~50% lower than that by CYP2D6.1). Overall, the enzymatic consequences of CYP2D6 allelic isozymes are substrate dependent. These data would help preclinical and clinical assessment of the metabolic elimination of drugs that are mediated by human CYP2D enzyme.
Enzyme Kinetics for Clinically Relevant CYP Inhibition
Current Drug Metabolism, 2005
In vitro cytochrome P450 (CYP)-associated metabolic studies have been considered cost-effective for predicting the potential clinical drug-drug interactions (DDIs), one of the major attritions in drug development. The breakthroughs during the past decade in understanding the biochemistry of CYP-mediated biotransformation and molecular biology of CYP gene regulation in humans have provided the scientific bases for such endeavors in early drug development. In this review, the enzyme kinetics of CYP inhibitions is described, with the primary focus on the ones proven with clinical relevance, namely the competitive inhibition and mechanism-based inactivation (MBI). Competitive CYP inhibition, the most often detected reversible inhibition, is well understood and has been studied extensively both in vitro and in clinical setting. Recently, MBI has received increasing attention. It has been recognized that MBI could occur more often than anticipated, due in part to the redox cycling-allied enzymatic action of CYPs. As commonly as an irreversible inhibition, MBI would inactivate the target proteins, and thus would be generally considered of high potential for causing clinical DDI. Moreover, the reversible inhibitions other than the competitive, namely noncompetitive, uncompetitive and mixed, were also documented for the important drug-metabolizing CYP members, particularly CYP1A2 and CYP2C9. Finally, the unusual kinetic interactions, which did not follow the Michaelis-Menten (M-M) kinetics, were detected in vitro for the majority of drug-metabolizing CYP members, and manifested for CYP3A4. However, the clinical relevance of the interactions involving the unusual CYP kinetics has not yet been fully understood. Nonetheless, the reversibility and inhibitory potency should be considered as the major determinants of the clinical relevance, particularly in combination with the therapeutic exposure levels. With rapid expansion of knowledge and technology, the evaluation of the clinically relevant CYP-associated DDIs in vitro is not only desirable but also achievable.
Induction of CYP1A1 and CYP2E1 in rat liver by histamine: binding and kinetic studies
Archives of Toxicology, 2007
Histamine (HA) may bind to cytochrome P450 (CYP450) in rat liver microsomes. The CYP450-HA complex seems to regulate some cellular processes such as proliferation. In the present work, it is shown that HA increases the activity and protein level of CYP1A1 and CYP2E1, in vivo. CYP1A1 is associated with polycyclic aromatic hydrocarbon-mediated carcinogenesis and CYP2E1 with liver damage by oxidative stress. Studies of enzyme kinetics and binding with rat liver microsomes and supersomes ® were carried out to determine whether HA is a substrate of CYP1A1 and/or CYP2E1. The lack of NADPH oxidation in the presence of HA showed that it is not a substrate for CYP1A1. Activity measurements using the O-dealkylation of ethoxyresoruWn indicated that HA is a mixed-type inhibitor of CYP1A1 in both microsomes and supersomes. On the other hand, HA induced a signiWcant NADPH oxidation catalyzed by CYP2E1 supersomes ® , strongly suggesting that HA is a substrate for this isoform. Furthermore, HA is consumed in the presence of CYP2E1-induced microsomes and supersomes, as determined by o-phtalaldehyde complexes with HA by HPLC. The present Wndings may contribute to understand better the physiological function of CYP450 in relation with inXammation and other physiological processes in which HA may have a relevant role.
Pharmacogenetics, 2001
CYP2D6 is involved in the metabolism of several classes of drugs, including tricyclic antidepressants, selective serotonin reuptake inhibitors and various amphetamines. CYP2D6 à 10 is an allelic variant, producing an enzyme with Pro 34 Ser and Ser 486 Thr amino acid substitutions. Approximately 75% of Asians possess the à 10 allele. We sought to further characterize CYP2D6.10 catalytically in vitro in a baculovirus expression system using various substrates and inhibitors, in comparison to CYP2D6.1 (wild-type). Using dextromethorphan (DEX), P-methoxyamphetamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and (6)3,4-methylenedioxymethamphetamine (MDMA), the ratios of intrinsic clearance (V max /K m ) of à 1 to à 10 were 50, 34, 22 and 123, respectively. The CYP2D6 substrates amitriptyline, and ( ) and (2) methamphetamine (MAMP) are both p-hydroxylated and Ndemethylated (NDM). The intrinsic clearance à 1/ à 10 ratios were 42, 30 and 67 for the phydroxylation; and 60, 120 and 157 for the NDM, respectively, illustrating chemical pathway and enantiomeric selectivity for MAMP. It was apparent that ( ) and (2) MAMP NDM and MDMA demethylenation were most signi®cantly different in CYP2D6.10. Using DEX as the substrate, the ratios of K i ( à 10)/K i ( à 1) for inhibitors were: budipine (1.3), sparteine (1.6), debrisoquine (8.1),¯uoxetine (16), nor¯uoxetine (30), paroxetine (14), MDMA (21) and MMDA-2 (7.1), indicating that CYP2D6.10 shows drug-speci®c altered susceptibility to inhibition. Taken together, these data suggest that CYP2D6 à 10/ à 10 individuals may be expected to require different drug doses; and show altered susceptibility to toxicity, interaction risk and, in the case of the amphetamines, drug dependence and toxicity compared to CYP2D6 à 1/ à 1 individuals.