N-demethylation Pathway Research Articles

New insight into biodegradation mechanism of phenylurea herbicides by cytochrome P450 enzymes: Successive N-demethylation mechanism

Phenylurea herbicides (PUHs) present one of the most important herbicides, which have cause serious effects on ecological environment and humans. Nowadays enzyme strategy shows great advantages in degradation of PUHs. Here density functional theory (DFT), quantitative structure − activity relationship (QSAR) and quantum mechanics/molecular mechanics (QM/MM) approaches are used to investigate the degradation mechanism of PUHs catalyzed by P450 enzymes. Two successive N-demethylation pathways are identified and two hydrogen abstraction (H-abstraction) reaction pathways are identified as the rate-determining step through high-throughput DFT calculations. The Boltzmann-weighted average energy barrier of the second H-abstraction pathway (19.95 kcal/mol) is higher than that of the first H-abstraction pathway (16.80 kcal/mol). Two QSAR models are established to predict the energy barriers of the two H-abstraction pathways based on the quantum chemical descriptors and mordred molecular descriptors. The determination coefficient (R2) values of QSAR models are > 0.9, which reveal that the established QSAR models have great predictive capability. QM/MM calculations indicate that human P450 enzymes are more efficient in degradation of PUHs than crop and weed P450 enzymes. Correlations between energy barriers and key structural/charge parameters are revealed and key parameters that have influence on degradation efficiency of PUHs are identified. This study provides lateral insights into the biodegradation strategy and removal method of PUHs and valuable information for designing or engineering of highly efficient degradation enzymes and genetically modified crops.

Caffeine Synthesis and Its Mechanism and Application by Microbial Degradation, A Review.

Caffeine is a metabolite derived from purine nucleotides, typically accounting for 2-5% of the dry weight of tea and 1-2% of the dry weight of coffee. In the tea and coffee plants, the main synthesis pathway of caffeine is a four-step sequence consisting of three methylation reactions and one nucleosidase reaction using xanthine as a precursor. In bacteria, caffeine degradation occurs mainly through the pathways of N-demethylation and C-8 oxidation. However, a study fully and systematically summarizing the metabolism and application of caffeine in microorganisms has not been established elsewhere. In the present study, we provide a review of the biosynthesis, microbial degradation, gene expression, and application of caffeine microbial degradation. The present review aims to further elaborate the mechanism of caffeine metabolism by microorganisms and explore the development prospects in this field.

Environmental biotransformation mechanisms by flavin-dependent monooxygenase: A computational study

The enzyme-catalyzed metabolic biotransformation of xenobiotics plays a significant role in toxicology evolution and subsequently environmental health risk assessment. Recent studies noted that the phase I human flavin-dependent monooxygenase (e.g., FMO3) can catalyze xenobiotics into more toxic metabolites. However, details of the metabolic mechanisms are insufficient. To fill the mechanism in the gaps, the systemic density functional theory calculations were performed to elucidate diverse FMO-catalyzed oxidation reactions toward environmental pollutants, including denitrification (e.g., nitrophenol), N-oxidation (e.g., nicotine), desulfurization (e.g., fonofos), and dehalogenation (e.g., pentachlorophenol). Similar to the active center compound 0 of cytochrome P450, FMO mainly catalyzed reactions with the structure of the tricyclic isoalloxazine C-4a-hydroperoxide (FADHOOH). As will be shown, FMO-catalyzed pathways are more favorable with a concerted than stepwise mechanism; Deprotonation is necessary to initiate the oxidation reactions for phenolic substrates; The regioselectivity of nicotine by FMO prefers the N-oxidation other than N-demethylation pathway; Formation of the P–S–O triangle ring is the key step for desulfurization of fonofos by FMO. We envision that these fundamental mechanisms catalyzed by FMO with a computational method can be extended to other xenobiotics of similar structures, which may aid the high-throughput screening and provide theoretical predictions in the future.

Efficient degradation and decolorization of triphenylmethane dyes by Serratia sp. WKD under extreme environmental conditions and the mechanism

Microbial degradation of dyes is fundamental and essential for understanding their environmental fate. In this study, a new bacterial strain with efficient degrading capability of triphenylmethane (TPM) dyes named WKD was isolated and identified as the genus Serratia sp. by 16S rDNA analysis. The decolorization of TPM dyes by the strain especially under extreme environmental conditions were studied. The results showed that the strain could completely decolorize Malachite Green (MG) and Crystal Violet (CV) at pH values ranging from 3.0 to 10.0 and at temperatures ranging from 20 °C to 45 °C in a 12 h of incubation. The strain still showed an excellent decolorizing capability even at low (5 °C) and high (60 °C) temperatures. Moreover, the enzymatic analysis indicated that MnP, NADH-DCIP reductase, MG reductase, and CV reductase might be involved in TPM dyes biodegradation. Besides, the biodegradation pathways were proposed based on UV–visible, FTIR, and LC-Q-TOF-MS analyses. Results indicated that degradation of MG by the strain was mainly regulated by N-demethylation and hydroxylation pathways, while degradation of CV was only by the N-demethylation pathway. Therefore, Serratia sp. strain WKD is expected to have a broad application prospect for the bioremediation of TPM dyes, especially in extreme environments.

Photocatalytic degradation of ketamine using a reusable TiO2/SiO2@Fe3O4 magnetic photocatalyst under simulated solar light

TiO2 heterogeneous photocatalytic processes can carry out efficient abatement of various organic pollutants; however, the difficult recyclability of catalysts largely hinders their application. This study aims to develop a TiO2/SiO2@Fe3O4 core-shell structure magnetic photocatalyst for the degradation of ketamine, a recalcitrant pharmaceutical pollutant commonly found in aquatic environments. The structure and morphology of the TiO2/SiO2@Fe3O4 photocatalyst were comprehensively characterized. Compared with commercial P25 TiO2, the fabricated TiO2/SiO2@Fe3O4 photocatalyst exhibited a higher ketamine degradation efficiency under simulated solar irradiation, with a pseudo-first-order rate constant of 0.1768 min–1 (conditions: [ketamine]0 = 0.3 μM, [TiO2/SiO2@Fe3O4] = 100 mg/L and pH = 7). Additionally, the magnetic TiO2/SiO2@Fe3O4 was easily recovered and showed stable performance, with the ketamine degradation rate only slightly decreasing from 100% to 91% within six cycles of use. The mechanistic investigation demonstrated the participation of •OH, h+ and •O2– in ketamine photocatalytic degradation, with •O2– playing the dominant role. During the photocatalytic pathways of ring opening, oxygen addition, N-demethylation and hydroxylation, ketamine was decomposed into byproducts and further achieved efficient mineralization and detoxification (performed by the Microtox® acute toxicity test), showing the promising potential of TiO2/SiO2@Fe3O4 for use in water purification.

The regioselectivity of the interaction between dextromethorphan and CYP2D6.

CYP2D6 is an important enzyme of the cytochrome P450 superfamily, and catalyzes nearly 25% of the drugs sold in the market. For decades, the interactions and metabolism between CYP2D6 and substrates have been a hot topic. However, the key factors of the catalytic regioselectivity for CYP2D6 still remain controversial. Here, we construct four systems to explore the interaction between dextromethorphan (DM) and CYP2D6. A new binding mode of CYP2D6 is defined, and two key residues (residue Asp301 and residue Glu216) are discovered working simultaneously to stabilize the DM at the reactive site by forming water bridge hydrogen bonds when CYP2D6 binds DM. Our results also indicate that the substrate concentration could mediate the binding mode between the substrate and CYP2D6 by decreasing the volume of the catalytic pocket, which is not conducive to the O-demethylation of DM but benefits the N-demethylation of DM. These results could shed light on the process of CYP2D6 binding to the substrate, and help to better understand the regioselectivity of CYP2D6 catalyzing the substrates.

Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates

The AlkB family of nonheme Fe(II)/2-oxoglutarate-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6-methyladenosine (m6A) but furnish different major products, N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a direct m6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for nonredundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems.

Draft Genome Sequence of Pseudomonas sp. Strain CES, Containing the Entire Alkylxanthine Gene Cluster for Caffeine Breakdown.

Pseudomonas strain CES was isolated from caffeine-enriched soil and found to possess the N-demethylation pathway for caffeine breakdown. We report the nucleotide sequence of the draft genome with 5,827,822 bp, 62.6% G+C content, and 5,427 protein-coding regions.

Novel caffeine degradation gene cluster is mega-plasmid encoded in Paraburkholderia caffeinilytica CF1.

The widespread use of caffeine in food and drug industries has caused great environmental pollution. Herein, an efficient caffeine-degrading strain Paraburkholderia caffeinilytica CF1 isolated from a tea garden in China can utilize caffeine as its sole carbon and nitrogen source. Combination of chromatographic and spectrophotometric techniques confirmed that strain CF1 adopts N-demethylation pathway for caffeine degradation. Whole genome sequencing of strain CF1 reveals that it has two chromosomes with sizes 3.62Mb and 4.53Mb, and a 174-kb mega-plasmid. The plasmid P1 specifically harbors the genes essential for caffeine metabolism. By analyzing the sequence alignment and quantitative real-time PCR data, the redundant gene cluster of caffeine degradation was elucidated. Genes related to catalyzing the N1-demethylation of caffeine to theobromine, the first step of caffeine degradation were heterologously expressed, and methylxanthine N1-demethylase was purified and characterized. Above all, this study systematically unravels the molecular mechanism of caffeine degradation by Paraburkholderia. KEY POINTS: • Caffeine degradation cluster in Paraburkholderia caffeinilytica CF1 was located in mega-plasmid P1. • The whole genome and the caffeine degrading pathway of P. caffeinilytica CF1 were sequenced and elucidated, respectively. • This study succeeded in heterologous expression of methylxanthine N1-demethylase (CdnA) and Rieske oxygenase reductase (CdnD) and illuminated the roles of CdnA and CdnD in caffeine degradation of P. caffeinilytica CF1.

Bioassay for Determining the Concentrations of Caffeine and Individual Methylxanthines in Complex Samples.

Caffeine and other methylxanthines are stimulant molecules found in formulated beverages, including sodas and energy drinks, and in brewed beverages, such as coffee and teas. Previously, we developed a bioassay for caffeine that involves monitoring the growth of a ΔguaB mutant of Escherichia coli defective in de novo guanine biosynthesis. When supplemented with a plasmid expressing the genes for an N-demethylation pathway from Pseudomonas putida CBB5, these bacteria demethylate caffeine (1,3,7-trimethylxanthine) and other methylxanthines into xanthine, which is then converted into guanine to support cell growth. A major limitation of this bioassay was that it could only measure the total concentration of all methylxanthines in a mixture. Therefore, it could not be used to measure the caffeine content of beverages like teas, which contain substantial quantities of multiple methylxanthines. To overcome this limitation, we created seven new plasmids containing all subsets of the three demethylase genes (ndmA, ndmB, and ndmC). We show that strains of ΔguaBE. coli containing each plasmid are able to demethylate specific subsets of methylxanthines and that they can be used to determine the concentrations of individual methylxanthines in complex mixtures containing multiple methylxanthines, including coffee doped with an additional methylxanthine. While validating this assay, we also discovered an unexpected demethylation event at the 1-methyl position when NdmB and NdmC were expressed in the absence of NdmA. The improved cell-based bioassay is inexpensive, is easy to use, and gives results comparable to standard high-performance liquid chromatography methods for measuring methylxanthine concentrations.IMPORTANCE Caffeine (1,3,7-trimethylxanthine) is the dominant neurostimulant found in coffee, teas, sodas, and energy drinks. Measuring the amount of caffeine and other methylxanthines in these beverages is important for quality assurance and safety in food science. Methylxanthines are also used in medicine and as performance-enhancing drugs, two contexts in which accurately determining their concentrations in bodily fluids is important. Liquid chromatography is the standard method for measuring methylxanthine concentrations in a sample, but it requires specialized equipment and expertise. We improved a previous bioassay that links E. coli growth to methylxanthine demethylation so that it can now be used to determine the amounts of individual methylxanthines in complex mixtures or beverages, such as coffee.

A Primer of Deuterium in Drug Design

A Primer of Deuterium in Drug Design

Gender Dependency in Streoselective Pharmacokinetics of Tramadol and Its Phase I Metabolites in Relation to CYP2D6 Phenotype in Iranian Population.

The stereoselective pharmacokinetic of Tramadol (T) and its main metabolites concerning the influence of CYP2D6 phenotype and gender on the phase I metabolism of this compound was studied after administration of 100 mg single oral dose of racemic T to 24 male and female subjects. The pharmacokinetic parameters were estimated from plasma concentrations of the analytes enantiomers. The metabolic ratio of T enantiomers was used for CYP2D6 phenotype determination. The plasma concentrations of both tramadol enantiomers were considerably higher in Poor metabolizers (PM) than in extensive metabolizers (EM), resulting in 43% and 37% increase in AUC values of (+)-T and (-)-T respectively. The plasma concentrations of the (+)- and (-)-M1 enantiomers in EMs were significantly higher than the respective concentrations in PMs. The N-demethylation pathway was indirectly affected by CYP2D6 phenotypic differences. The plasma concentration of both enantiomers of M2 in PMs was higher than Ems. Although the concentration profiles and most of the calculated pharmacokinetic parameters of T and its main metabolites appears to be different in EMs and PMs, only the stereoselectivity of M1 enantiomers was significantly different in relation to CYP2D6 subgroups. No significant gender-related difference in the pharmacokinetics of T and its metabolites was observed.

Theoretical study on the N-demethylation mechanism of theobromine catalyzed by P450 isoenzyme 1A2

Theobromine, a widely consumed pharmacological active substance, can cause undesirable muscle stiffness, nausea and anorexia in high doses ingestion. The main N-demethylation metabolic mechanism of theobromine catalyzed by P450 isoenzyme 1A2 (CYP1A2) has been explored in this work using the unrestricted hybrid density functional method UB3LYP in conjunction with the LACVP(Fe)/6-31G (H, C, N, O, S, Cl) basis set. Single-point calculations including empirical dispersion corrections were carried out at the higher 6-311++G** basis set. Two N-demethylation pathways were characterized, i.e., 3-N and 7-N demethylations, which involve the initial N-methyl hydroxylation to form carbinolamines and the subsequent carbinolamines decomposition to yield monomethylxanthines and formaldehydes. Our results have shown that the rate-limiting N-methyl hydroxylation occurs via a hydrogen atom transfer (HAT) mechanism, which proceeds in a spin-selective mechanism (SSM) in the gas phase. The carbinolamines generated are prone to decomposition via the contiguous heteroatom-assisted proton-transfer. Strikingly, 3-N demethylation is more favorable than 7-N demethylation due to its lower free energy barrier and 7-methylxanthine therefore is the optimum product reported for the demethylation of theobromine catalyzed by CYP1A2, which are in good agreement with the experimental observation. This work has first revealed the detail N-demethylation mechanisms of theobromine at the theoretical level. It can offer more significant information for the metabolism of purine alkaloid.

Genetic characterization of caffeine degradation by bacteria and its potential applications.

The ability of bacteria to grow on caffeine as sole carbon and nitrogen source has been known for over 40 years. Extensive research into this subject has revealed two distinct pathways, N-demethylation and C-8 oxidation, for bacterial caffeine degradation. However, the enzymological and genetic basis for bacterial caffeine degradation has only recently been discovered. This review article discusses the recent discoveries of the genes responsible for both N-demethylation and C-8 oxidation. All of the genes for the N-demethylation pathway, encoding enzymes in the Rieske oxygenase family, reside on 13.2-kb genomic DNA fragment found in Pseudomonas putida CBB5. A nearly identical DNA fragment, with homologous genes in similar orientation, is found in Pseudomonas sp. CES. Similarly, genes for C-8 oxidation of caffeine have been located on a 25.2-kb genomic DNA fragment of Pseudomonas sp. CBB1. The C-8 oxidation genes encode enzymes similar to those found in the uric acid metabolic pathway of Klebsiella pneumoniae. Various biotechnological applications of these genes responsible for bacterial caffeine degradation, including bio-decaffeination, remediation of caffeine-contaminated environments, production of chemical and fuels and development of diagnostic tests have also been demonstrated.

Optimized on-line enantioselective capillary electrophoretic method for kinetic and inhibition studies of drug metabolism mediated by cytochrome P450 enzymes.

Pharmacokinetic and pharmacodynamic properties of a chiral drug can significantly differ between application of the racemate and single enantiomers. During drug development, the characteristics of candidate compounds have to be assessed prior to clinical testing. Since biotransformation significantly influences drug actions in an organism, metabolism studies represent a crucial part of such tests. Hence, an optimized and economical capillary electrophoretic method for on-line studies of the enantioselective drug metabolism mediated by cytochrome P450 enzymes was developed. It comprises a diffusion-based procedure, which enables mixing of the enzyme with virtually any compound inside the nanoliter-scale capillary reactor and without the need of additional optimization of mixing conditions. For CYP3A4, ketamine as probe substrate and highly sulfated γ-cyclodextrin as chiral selector, improved separation conditions for ketamine and norketamine enantiomers compared to a previously published electrophoretically mediated microanalysis method were elucidated. The new approach was thoroughly validated for the CYP3A4-mediated N-demethylation pathway of ketamine and applied to the determination of its kinetic parameters and the inhibition characteristics in presence of ketoconazole and dexmedetomidine. The determined parameters were found to be comparable to literature data obtained with different techniques. The presented method constitutes a miniaturized and cost-effective tool, which should be suitable for the assessment of the stereoselective aspects of kinetic and inhibition studies of cytochrome P450-mediated metabolic steps within early stages of the development of a new drug.

New genetic insights to consider coffee waste as feedstock for fuel, feed, and chemicals

Abstract Caffeine is a natural plant product found in many drinks, including coffee, tea, soft and energy drinks. Due to caffeine’s presence in the environment, microorganisms have evolved two different mechanisms to live on caffeine. The genetic maps of the caffeine N-demethylation pathway and C-8 oxidation pathway have been discovered in Pseudomonas putida CBB5 and Pseudomonas sp. CBB1, respectively. These genes are the only characterized bacterial caffeine-degrading genes, and may be of great value in producing fine chemicals, biofuels, and animal feed from coffee and tea waste. Here, we present preliminary results for production of theobromine and 7-methylxanthine from caffeine and theobromine, respectively, by two strains of metabolically engineered E. coli. We also demonstrate complete decaffeination of tea extract by an immobilized mixed culture of Klebsiella and Rhodococcus cells. These processes provide a first level demonstration of biotechnological utilization of coffee and tea waste.

Theophylline metabolism by human, rabbit and rat liver microsomes and by purified forms of cytochrome P450.

The capacity of human, rabbit and rat liver microsomes and purified isozymes of cytochrome P450 to metabolize theophylline has been assessed. In all three species the 8-hydroxylation of theophylline to 1,3-dimethyluric acid (1,3-DMU) was the major pathway. In human, control rabbit and rat liver microsomes this metabolite accounted for 59, 77 and 94%, respectively, of the total metabolites formed. In both human and control rabbit liver microsomes the N-demethylation of theophylline to 1-methylxanthine (1-MX) accounted for 20% of the total metabolites formed. N-demethylation of theophylline to 3-methylxanthine (3-MX) accounted for 21% of theophylline metabolism in human microsomes but was a minor pathway in control rabbit and rat microsomes. Acetone and phenobarbitone pretreatment markedly increased the formation of 1,3-DMU by rabbit liver microsomes. Rifampicin and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) administration caused a slight but significant increase in this pathway. In general the N-demethylation pathways in rabbit liver microsomes were refractory to induction. In the rat, the metabolism of theophylline to 1-MX, 3-MX and 1,3-DMU were all significantly increased in Aroclor 1254, dexamethasone, phenobarbitone and 3-methylcholanthrene-treated microsomes. In reconstitution experiments the polycyclic hydrocarbon inducible rabbit cytochrome P450 Forms 4 and 6 and the constitutive Form 3b all metabolized theophylline to its three metabolites. In human liver microsomes from four subjects anti-rabbit cytochrome P450 Form 4 IgG inhibited the metabolism of theophylline to 1-MX, 3-MX and 1,3-DMU by approximately 30%. These data indicate that theophylline is metabolized by multiple forms of cytochrome P450 in human, rabbit and rat liver microsomes.

Two Distinct Pathways for Metabolism of Theophylline and Caffeine Are Coexpressed in Pseudomonas putida CBB5

Pseudomonas putida CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1- and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1- and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1- and 3-methylxanthines to 1,3-dimethyluric acid and 1- and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methylxanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria.

Expression, Purification, Biochemical Characterization, and Comparative Function of Human Cytochrome P450 2D6.1, 2D6.2, 2D6.10, and 2D6.17 Allelic Isoforms

Polymorphism at the cytochrome P450 2D6 (CYP2D6) locus is one of the most widely known causes of pharmacogenetic variability in humans. Our goal is to investigate the intrinsic enzymatic differences that exist among active CYP2D6 isoforms to test the hypothesis that these enzymatic differences are substrate-dependent. Active CYP2D6.1, 2, 10, and 17 holo-enzymes were expressed in vitro and purified to a high degree of homogeneity as confirmed with SDS-polyacrylamide gel electrophoresis, CO-difference spectroscopy, and mass spectral analysis. Purified enzyme was reconstituted with lipid and cytochrome P450 reductase in a 2:1 ratio before kinetic analysis. The reaction rate for dextromethorphan (DXM) O-demethylation, DXM N-demethylation, codeine O-demethylation, and fluoxetine N-demethylation catalyzed by each of the variants was determined. The CYP2D6.10 enzyme was the most impaired, exhibiting an estimated enzyme efficiency (as V(max)/K(m)) 50-fold lower for DXM O-demethylation and 100-fold lower for fluoxetine N-demethylation when compared with CYP2D6.1, whereas no measurable catalytic activity was observed for this variant toward codeine. The atypical DXM N-demethylation pathway catalyzed by this variant decreased only 2-fold in comparison. In the case of CYPD6.17, estimated clearances for each metabolite were decreased 6 to 33%. Likewise, the intrinsic clearance of CYP2D6.2 enzyme was consistently decreased for each reaction examined, indicating that the ultra-rapid metabolizer phenotype sometimes associated with this genotype is not a function of the underlying amino acid substitutions. Overall enzyme efficiencies for the metabolism of each substrate therefore decreased in the order of 2D6.1 > 2D6.2 > 2D6.17 > 2D6.10.

Identification of the cytochrome P450 enzymes involved in the N-demethylation of sildenafil.

To characterize the cytochrome P450 (CYP) enzymes responsible for the N-demethylation of sildenafil to its main metabolite, UK-103 320, to investigate the potential inhibitory effects of sildenafil on CYP enzymes and to evaluate the potential of selected drugs to affect sildenafil metabolism. The metabolic pathways of sildenafil N-demethylation were studied using human liver microsomes, as well as microsomes expressing individual human CYP enzymes. Further studies to identify the individual enzymes were performed at 2.5 and 250 microM sildenafil, and employed a combination of chemical inhibition, correlation analysis, and metabolism by expressed recombinant CYP enzymes. In addition, the effect of sildenafil on the activity of the six major drug metabolizing enzymes was investigated. Sildenafil conversion was found to be mediated by at least two CYP enzymes, for which the mean kinetic parameters were Km1 = 6(+/-3 microM), Km2 = 81(+/-45 microM), Vmax1 = 22(+/-9 pmol) and Vmax2 = 138(+/-77 pmol) UK-103 320 formed min(-1) mg(-1). At 250 microM sildenafil, N-demethylation was primarily mediated through the low-affinity, high-Km enzyme (approximately 83%), whilst at 2.5 microM there was a greater role for the high-affinity, low-Km enzyme (approximately 61%). Ketoconazole strongly inhibited metabolism at both sildenafil concentrations and was the only significant inhibitor at 250 microM sildenafil. At the lower sildenafil concentration, sulphaphenazole and quinidine also inhibited formation of UK-103 320. Overall, 75% or more of the N-demethylation of sildenafil at any concentration is probably attributable to CYP3A4. These results were supported by experiments using expressed human CYP enzymes, in which only CYP3A4 and CYP2C9 exhibited substantial sildenafil N-demethylase activity (respective Km values of 221 microM and 27 microM). Sildenafil metabolism was inhibited by potent CYP3A4 inhibitors which are used clinically, but was found to be only a weak inhibitor of drug metabolizing enzymes itself, the strongest inhibition occurring against CYP2C9 (Ki = 80 microM). Evidence is provided for CYP3A4 and to a lesser extent CYP2C9-mediated metabolism of sildenafil. There is the possibility that elevated plasma concentrations of sildenafil could occur with coadministration of known inhibitors of CYP2C9 or CYP3A4. Since peak plasma concentrations of clinical doses of sildenafil are only 200 ng ml(-1) ( approximately 0.4 microM) it is very unlikely that sildenafil will significantly alter the plasma concentration of other compounds metabolized by cytochrome P450 enzymes.