Responsible for the metabolism of many drugs and environmental chemicals that it oxidizes. It is involved in the metabolism of drugs such as antiarrhythmics, adrenoceptor antagonists, and tricyclic antidepressants.
Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.
Interacting selectively and non-covalently with a drug, any naturally occurring or synthetic substance, other than a nutrient, that, when administered or applied to an organism, affects the structure or functioning of the organism; in particular, any such substance used in the diagnosis, prevention, or treatment of disease.
Cytochrome P450 (P450) protein-protein interactions have been observed with various in vitro systems. It is interesting to note that these interactions seem to be isoform-dependent, with some combinations producing no effect and others producing increased or decreased catalytic activity. With some exceptions, most of the work to date has involved P450s from rabbit, rat, and other animal species, with few studies including human P450s. In the studies presented herein, the interactions of two key drug-metabolizing enzymes, CYP2C9 and CYP2D6, were analyzed in a purified, reconstituted enzyme system for changes in both substrate-binding affinity and rates of catalysis. In addition, an extensive study was conducted as to the "order of mixing" for the reconstituted enzyme system and the impact on the observations. CYP2D6 coincubation inhibited CYP2C9-mediated (S)-flurbiprofen metabolism in a protein concentration-dependent manner. V(max) values were reduced by up to 50%, but no appreciable effect on K(m) was observed. Spectral binding studies revealed a 20-fold increase in the K(S) of CYP2C9 toward (S)-flurbiprofen in the presence of CYP2D6. CYP2C9 coincubation had no effect on CYP2D6-mediated dextromethorphan O-demethylation. The order of combination of the proteins (CYP2C9, CYP2D6, and cytochrome P450 reductase) influenced the magnitude of catalysis inhibition as well as the ability of increased cytochrome P450 reductase to attenuate the change in activity. A simple model, congruent with current results and those of others, is proposed to explain oligomer formation. In summary, CYP2C9-CYP2D6 interactions can alter catalytic activity and, thus, influence in vitro-in vivo correlation predictions.
Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.
Members of the cytochrome P450 (P450) enzyme families CYP1, CYP2, and CYP3 are responsible for the metabolism of approximately 75% of all clinically relevant drugs. With the increased prevalence of nonalcoholic fatty liver disease (NAFLD), it is likely that patients with this disease represent an emerging population at significant risk for alterations in these important drug-metabolizing enzymes. The purpose of this study was to determine whether three progressive stages of human NALFD alter hepatic P450 expression and activity. Microsomes isolated from human liver samples diagnosed as normal, n = 20; steatosis, n = 11; nonalcoholic steatohepatitis (NASH) (fatty liver), n = 10; and NASH (no longer fatty), n = 11 were analyzed for P450 mRNA, protein, and enzyme activity. Microsomal CYP1A2, CYP2D6, and CYP2E1 mRNA levels were decreased with NAFLD progression, whereas CYP2A6, CYP2B6, and CYP2C9 mRNA expression increased. Microsomal protein expression of CYP1A2, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 tended to decrease with NAFLD progression. Likewise, functional activity assays revealed decreasing trends in CYP1A2 (p = 0.001) and CYP2C19 (p = 0.05) enzymatic activity with increasing NAFLD severity. In contrast, activity of CYP2A6 (p = 0.001) and CYP2C9 (diclofenac, p = 0.0001; tolbutamide, p = 0.004) was significantly increased with NAFLD progression. Increased expression of proinflammatory cytokines tumor necrosis factor alpha and interleukin 1beta was observed and may be responsible for observed decreases in respective P450 activity. Furthermore, elevated CYP2C9 activity during NAFLD progression correlated with elevated hypoxia-induced factor 1alpha expression in the later stages of NAFLD. These results suggest that significant and novel changes occur in hepatic P450 activity during progressive stages of NAFLD.
AIMS: To confirm the identity of the major metabolites of domperidone and to characterize the cytochrome P450s (CYPs) involved in their formation. METHODS: Human liver microsomes (HLMs) were used to characterize the kinetics of domperidone metabolism and liquid chromatography-mass spectrometry to identify the products. Isoform-specific chemical inhibitors, correlation analysis and expressed human CYP genes were used to identify the CYPs involved in domperidone oxidation. RESULTS: In HLMs, domperidone underwent hydroxylation to form 5-hydroxydomperidone (MIII) and N-dealkylation to form 2,3-dihydro-2-oxo-1H-benzimidazole-1-propionic acid (MI) and 5-chloro-4-piperidinyl-1,3-dihydro-benzimidazol-2-one (MII). The formation of all three metabolites (n = 4 HLMs) followed apparent Michaelis-Menten kinetics. The mean Km values for MI, MII and MIII formation were 12.4, 11.9, and 12.6 micro m, respectively. In a panel of HLMs (n = 10), the rate of domperidone (5 microm and 50 microm) metabolism correlated with the activity of CYP3A (r > 0.94; P < 0.0001). Only ketoconazole (1 microm) (by 87%) and troleandomycin (50 microm) (by 64%) inhibited domperidone (5 microm) metabolism in HLMs. Domperidone (5 and 50 microm) hydroxylation and N-dealkylation was catalyzed by expressed CYP3A4 at a higher rate than the other CYPs. CYP1A2, 2B6, 2C8 and 2D6 also hydroxylated domperidone CONCLUSIONS: CYP3A-catalyzed N-dealkylation and aromatic hydroxylation are the major routes for domperidone metabolism. The drug would be expected to demonstrate highly variable bioavailability due to hepatic, and possibly intestinal first-pass metabolism after oral administration. Increased risk of adverse effects might be anticipated during concomitant administration with CYP3A inhibitors, as well as decreased efficacy with inducers of this enzyme.
Catalysis of an oxidation-reduction (redox) reaction, a reversible chemical reaction in which the oxidation state of an atom or atoms within a molecule is altered. One substrate acts as a hydrogen or electron donor and becomes oxidized, while the other acts as hydrogen or electron acceptor and becomes reduced.
Acetaminophen is a widely used analgesic antipyretic agent. When used at low doses, it is a safe drug, but at higher doses it can cause acute hepatic necrosis in humans and experimental animals. The key mechanism in the hepatotoxicity is cytochrome P450 (CYP)-catalysed formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) that is capable of binding to cellular macromolecules and in that way an LC/MS liquid chromatography/mass spectrometry (LC/MS) method was developed to measure NAPQI formation by trapping it to reduced glutathione. This method was used to determine the bioactivation of acetaminophen at two concentrations: 50 microM therapeutic and 1 mM toxic by using nine human recombinant CYP enzymes: CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4; and with different microsomes from experimental animals. At the toxic concentration the formation of NAPQI-glutathione was highest with CYP3A4 followed by CYP2E1, CYP1A2, and CYP2D6. At the therapeutic concentration, CYP3A4 had also the highest bioactivation capacity. In a comparison of the enzyme kinetics, CYP3A4 was the most efficient CYP with the lowest K(m) value 130 microM (95% confidence interval = 63-210 microM). Dexamethasone-induced rat liver microsomes had the most effective bioactivation capacity at therapeutic and toxic acetaminophen concentrations. This study suggests that CYP3A4 is the major CYP enzyme form catalysing acetaminophen oxidation to NAPQI in human liver.
Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.
Oxycodone undergoes N-demethylation to noroxycodone and O-demethylation to oxymorphone. The cytochrome P450 (P450) isoforms capable of mediating the oxidation of oxycodone to oxymorphone and noroxycodone were identified using a panel of recombinant human P450s. CYP3A4 and CYP3A5 displayed the highest activity for oxycodone N-demethylation; intrinsic clearance for CYP3A5 was slightly higher than that for CYP3A4. CYP2D6 had the highest activity for O-demethylation. Multienzyme, Michaelis-Menten kinetics were observed for both oxidative reactions in microsomes prepared from five human livers. Inhibition with ketoconazole showed that CYP3A is the high affinity enzyme for oxycodone N-demethylation; ketoconazole inhibited >90% of noroxycodone formation at low substrate concentrations. CYP3A-mediated noroxycodone formation exhibited a mean K(m) of 600 +/- 119 microM and a V(max) that ranged from 716 to 14523 pmol/mg/min. Contribution from the low affinity enzyme(s) did not exceed 8% of total intrinsic clearance for N-demethylation. Quinidine inhibition showed that CYP2D6 is the high affinity enzyme for O-demethylation with a mean K(m) of 130 +/- 33 microM and a V(max) that ranged from 89 to 356 pmol/mg/min. Activity of the low affinity enzyme(s) accounted for 10 to 26% of total intrinsic clearance for O-demethylation. On average, the total intrinsic clearance for noroxycodone formation was 8 times greater than that for oxymorphone formation across the five liver microsomal preparations (10.5 microl/min/mg versus 1.5 microl/min/mg). Experiments with human intestinal mucosal microsomes indicated lower N-demethylation activity (20-50%) compared with liver microsomes and negligible O-demethylation activity, which predict a minimal contribution of intestinal mucosa in the first-pass oxidative metabolism of oxycodone.
The chemical reactions and pathways resulting in the breakdown of alkaloids, nitrogen containing natural products not otherwise classified as peptides, nonprotein amino acids, amines, cyanogenic glycosides, glucosinolates, cofactors, phytohormones or primary metabolites (such as purine or pyrimidine bases).
Oxycodone undergoes N-demethylation to noroxycodone and O-demethylation to oxymorphone. The cytochrome P450 (P450) isoforms capable of mediating the oxidation of oxycodone to oxymorphone and noroxycodone were identified using a panel of recombinant human P450s. CYP3A4 and CYP3A5 displayed the highest activity for oxycodone N-demethylation; intrinsic clearance for CYP3A5 was slightly higher than that for CYP3A4. CYP2D6 had the highest activity for O-demethylation. Multienzyme, Michaelis-Menten kinetics were observed for both oxidative reactions in microsomes prepared from five human livers. Inhibition with ketoconazole showed that CYP3A is the high affinity enzyme for oxycodone N-demethylation; ketoconazole inhibited >90% of noroxycodone formation at low substrate concentrations. CYP3A-mediated noroxycodone formation exhibited a mean K(m) of 600 +/- 119 microM and a V(max) that ranged from 716 to 14523 pmol/mg/min. Contribution from the low affinity enzyme(s) did not exceed 8% of total intrinsic clearance for N-demethylation. Quinidine inhibition showed that CYP2D6 is the high affinity enzyme for O-demethylation with a mean K(m) of 130 +/- 33 microM and a V(max) that ranged from 89 to 356 pmol/mg/min. Activity of the low affinity enzyme(s) accounted for 10 to 26% of total intrinsic clearance for O-demethylation. On average, the total intrinsic clearance for noroxycodone formation was 8 times greater than that for oxymorphone formation across the five liver microsomal preparations (10.5 microl/min/mg versus 1.5 microl/min/mg). Experiments with human intestinal mucosal microsomes indicated lower N-demethylation activity (20-50%) compared with liver microsomes and negligible O-demethylation activity, which predict a minimal contribution of intestinal mucosa in the first-pass oxidative metabolism of oxycodone.
The chemical reactions and pathways involving alkaloids, nitrogen containing natural products which are not otherwise classified as peptides, nonprotein amino acids, amines, cyanogenic glycosides, glucosinolates, cofactors, phytohormones or primary metabolites (such as purine or pyrimidine bases).
Members of the cytochrome P450 (P450) enzyme families CYP1, CYP2, and CYP3 are responsible for the metabolism of approximately 75% of all clinically relevant drugs. With the increased prevalence of nonalcoholic fatty liver disease (NAFLD), it is likely that patients with this disease represent an emerging population at significant risk for alterations in these important drug-metabolizing enzymes. The purpose of this study was to determine whether three progressive stages of human NALFD alter hepatic P450 expression and activity. Microsomes isolated from human liver samples diagnosed as normal, n = 20; steatosis, n = 11; nonalcoholic steatohepatitis (NASH) (fatty liver), n = 10; and NASH (no longer fatty), n = 11 were analyzed for P450 mRNA, protein, and enzyme activity. Microsomal CYP1A2, CYP2D6, and CYP2E1 mRNA levels were decreased with NAFLD progression, whereas CYP2A6, CYP2B6, and CYP2C9 mRNA expression increased. Microsomal protein expression of CYP1A2, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 tended to decrease with NAFLD progression. Likewise, functional activity assays revealed decreasing trends in CYP1A2 (p = 0.001) and CYP2C19 (p = 0.05) enzymatic activity with increasing NAFLD severity. In contrast, activity of CYP2A6 (p = 0.001) and CYP2C9 (diclofenac, p = 0.0001; tolbutamide, p = 0.004) was significantly increased with NAFLD progression. Increased expression of proinflammatory cytokines tumor necrosis factor alpha and interleukin 1beta was observed and may be responsible for observed decreases in respective P450 activity. Furthermore, elevated CYP2C9 activity during NAFLD progression correlated with elevated hypoxia-induced factor 1alpha expression in the later stages of NAFLD. These results suggest that significant and novel changes occur in hepatic P450 activity during progressive stages of NAFLD.
Constitutively expressed human cytochrome P450 2D6 (CYP2D6; EC 1.14.14.1) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however, we have identified this enzyme in the mitochondria of human liver samples and found that extensive inter-individual variability exists with respect to the level of the mitochondrial enzyme. Metabolic assays using 7-methoxy-4-aminomethylcoumarin as a substrate show that the human liver mitochondrial enzyme is capable of oxidizing this substrate and that the catalytic activity is supported by mitochondrial electron transfer proteins. In the present study, we show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the endoplasmic reticulum and mitochondria. In vitro mitochondrial import studies using both N-terminal deletions and point mutations suggest that the mitochondrial targeting signal is localized between residues 23-33 and that the positively-charged residues at positions 24, 25, 26, 28 and 32 are required for mitochondrial targeting. The importance of the positively-charged residues was confirmed by transient transfection of a CYP2D6 mitochondrial targeting signal mutant in COS-7 cells. Both the mitochondria and the microsomes from a CYP2D6 stable expression cell line contain the enzyme and both fractions exhibit bufuralol 1'-hydroxylation activity, which is completely inhibited by CYP2D6 inhibitory antibody. Overall, these results suggest that the targeting of CYP2D6 to mitochondria could be an important physiological process that has significance in xenobiotic metabolism.
Oxycodone undergoes N-demethylation to noroxycodone and O-demethylation to oxymorphone. The cytochrome P450 (P450) isoforms capable of mediating the oxidation of oxycodone to oxymorphone and noroxycodone were identified using a panel of recombinant human P450s. CYP3A4 and CYP3A5 displayed the highest activity for oxycodone N-demethylation; intrinsic clearance for CYP3A5 was slightly higher than that for CYP3A4. CYP2D6 had the highest activity for O-demethylation. Multienzyme, Michaelis-Menten kinetics were observed for both oxidative reactions in microsomes prepared from five human livers. Inhibition with ketoconazole showed that CYP3A is the high affinity enzyme for oxycodone N-demethylation; ketoconazole inhibited >90% of noroxycodone formation at low substrate concentrations. CYP3A-mediated noroxycodone formation exhibited a mean K(m) of 600 +/- 119 microM and a V(max) that ranged from 716 to 14523 pmol/mg/min. Contribution from the low affinity enzyme(s) did not exceed 8% of total intrinsic clearance for N-demethylation. Quinidine inhibition showed that CYP2D6 is the high affinity enzyme for O-demethylation with a mean K(m) of 130 +/- 33 microM and a V(max) that ranged from 89 to 356 pmol/mg/min. Activity of the low affinity enzyme(s) accounted for 10 to 26% of total intrinsic clearance for O-demethylation. On average, the total intrinsic clearance for noroxycodone formation was 8 times greater than that for oxymorphone formation across the five liver microsomal preparations (10.5 microl/min/mg versus 1.5 microl/min/mg). Experiments with human intestinal mucosal microsomes indicated lower N-demethylation activity (20-50%) compared with liver microsomes and negligible O-demethylation activity, which predict a minimal contribution of intestinal mucosa in the first-pass oxidative metabolism of oxycodone.
The chemical reactions and pathways involving a drug, a substance used in the diagnosis, treatment or prevention of a disease; as used here antibiotic substances (see antibiotic metabolism) are considered to be drugs, even if not used in medical or veterinary practice.
This study examined the cytochrome P450 (CYP) enzyme selectivity of in vitro bioactivation of lynestrenol to norethindrone and the further metabolism of norethindrone. Screening with well-established chemical inhibitors showed that the formation of norethindrone was potently inhibited by CYP3A4 inhibitor ketoconazole (IC(50)=0.02 microM) and with CYP2C9 inhibitor sulphaphenazole (IC(50)=2.13 microM); the further biotransformation of norethindrone was strongly inhibited by ketoconazole (IC(50)=0.09 microM). Fluconazole modestly inhibited both lynestrenol bioactivation and norethindrone biotransformation. Lynestrenol bioactivation was mainly catalysed by recombinant human CYP2C9, CYP2C19 and CYP3A4; rCYP3A4 was responsible for the hydroxylation of norethindrone. A significant correlation was observed between norethindrone formation and tolbutamide hydroxylation, a CYP2C9-selective activity (r=0.63; p=0.01). Norethindrone hydroxylation correlated significantly with model reactions of CYP2C19 and CYP3A4. The greatest immunoinhibition of lynestrenol bioactivation was seen in incubations with CYP2C-Ab. The CYP3A4-Ab reduced norethindrone hydroxylation by 96%. Both lynestrenol and norethindrone were weak inhibitors of CYP2C9 (IC(50) of 32 microM and 46 microM for tolbutamide hydroxylation, respectively). In conclusion, CYP2C9, CYP2C19 and CYP3A4 are the primary cytochromes in the bioactivation of lynestrenol in vitro, while CYP3A4 catalyses the further metabolism of norethindrone.
Acetaminophen is a widely used analgesic antipyretic agent. When used at low doses, it is a safe drug, but at higher doses it can cause acute hepatic necrosis in humans and experimental animals. The key mechanism in the hepatotoxicity is cytochrome P450 (CYP)-catalysed formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) that is capable of binding to cellular macromolecules and in that way an LC/MS liquid chromatography/mass spectrometry (LC/MS) method was developed to measure NAPQI formation by trapping it to reduced glutathione. This method was used to determine the bioactivation of acetaminophen at two concentrations: 50 microM therapeutic and 1 mM toxic by using nine human recombinant CYP enzymes: CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4; and with different microsomes from experimental animals. At the toxic concentration the formation of NAPQI-glutathione was highest with CYP3A4 followed by CYP2E1, CYP1A2, and CYP2D6. At the therapeutic concentration, CYP3A4 had also the highest bioactivation capacity. In a comparison of the enzyme kinetics, CYP3A4 was the most efficient CYP with the lowest K(m) value 130 microM (95% confidence interval = 63-210 microM). Dexamethasone-induced rat liver microsomes had the most effective bioactivation capacity at therapeutic and toxic acetaminophen concentrations. This study suggests that CYP3A4 is the major CYP enzyme form catalysing acetaminophen oxidation to NAPQI in human liver.
The chemical reactions and pathways involving heterocyclic compounds, those with a cyclic molecular structure and at least two different atoms in the ring (or rings).
AIMS: To confirm the identity of the major metabolites of domperidone and to characterize the cytochrome P450s (CYPs) involved in their formation. METHODS: Human liver microsomes (HLMs) were used to characterize the kinetics of domperidone metabolism and liquid chromatography-mass spectrometry to identify the products. Isoform-specific chemical inhibitors, correlation analysis and expressed human CYP genes were used to identify the CYPs involved in domperidone oxidation. RESULTS: In HLMs, domperidone underwent hydroxylation to form 5-hydroxydomperidone (MIII) and N-dealkylation to form 2,3-dihydro-2-oxo-1H-benzimidazole-1-propionic acid (MI) and 5-chloro-4-piperidinyl-1,3-dihydro-benzimidazol-2-one (MII). The formation of all three metabolites (n = 4 HLMs) followed apparent Michaelis-Menten kinetics. The mean Km values for MI, MII and MIII formation were 12.4, 11.9, and 12.6 micro m, respectively. In a panel of HLMs (n = 10), the rate of domperidone (5 microm and 50 microm) metabolism correlated with the activity of CYP3A (r > 0.94; P < 0.0001). Only ketoconazole (1 microm) (by 87%) and troleandomycin (50 microm) (by 64%) inhibited domperidone (5 microm) metabolism in HLMs. Domperidone (5 and 50 microm) hydroxylation and N-dealkylation was catalyzed by expressed CYP3A4 at a higher rate than the other CYPs. CYP1A2, 2B6, 2C8 and 2D6 also hydroxylated domperidone CONCLUSIONS: CYP3A-catalyzed N-dealkylation and aromatic hydroxylation are the major routes for domperidone metabolism. The drug would be expected to demonstrate highly variable bioavailability due to hepatic, and possibly intestinal first-pass metabolism after oral administration. Increased risk of adverse effects might be anticipated during concomitant administration with CYP3A inhibitors, as well as decreased efficacy with inducers of this enzyme.
Members of the cytochrome P450 (P450) enzyme families CYP1, CYP2, and CYP3 are responsible for the metabolism of approximately 75% of all clinically relevant drugs. With the increased prevalence of nonalcoholic fatty liver disease (NAFLD), it is likely that patients with this disease represent an emerging population at significant risk for alterations in these important drug-metabolizing enzymes. The purpose of this study was to determine whether three progressive stages of human NALFD alter hepatic P450 expression and activity. Microsomes isolated from human liver samples diagnosed as normal, n = 20; steatosis, n = 11; nonalcoholic steatohepatitis (NASH) (fatty liver), n = 10; and NASH (no longer fatty), n = 11 were analyzed for P450 mRNA, protein, and enzyme activity. Microsomal CYP1A2, CYP2D6, and CYP2E1 mRNA levels were decreased with NAFLD progression, whereas CYP2A6, CYP2B6, and CYP2C9 mRNA expression increased. Microsomal protein expression of CYP1A2, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 tended to decrease with NAFLD progression. Likewise, functional activity assays revealed decreasing trends in CYP1A2 (p = 0.001) and CYP2C19 (p = 0.05) enzymatic activity with increasing NAFLD severity. In contrast, activity of CYP2A6 (p = 0.001) and CYP2C9 (diclofenac, p = 0.0001; tolbutamide, p = 0.004) was significantly increased with NAFLD progression. Increased expression of proinflammatory cytokines tumor necrosis factor alpha and interleukin 1beta was observed and may be responsible for observed decreases in respective P450 activity. Furthermore, elevated CYP2C9 activity during NAFLD progression correlated with elevated hypoxia-induced factor 1alpha expression in the later stages of NAFLD. These results suggest that significant and novel changes occur in hepatic P450 activity during progressive stages of NAFLD.
The chemical reactions and pathways involving isoquinoline alkaloids, alkaloid compounds that contain bicyclic N-containing aromatic rings and are derived from a 3,4-dihydroxytyramine (dopamine) precursor that undergoes a Schiff base addition with aldehydes of different origin.
Cytochrome P450 (P450) protein-protein interactions have been observed with various in vitro systems. It is interesting to note that these interactions seem to be isoform-dependent, with some combinations producing no effect and others producing increased or decreased catalytic activity. With some exceptions, most of the work to date has involved P450s from rabbit, rat, and other animal species, with few studies including human P450s. In the studies presented herein, the interactions of two key drug-metabolizing enzymes, CYP2C9 and CYP2D6, were analyzed in a purified, reconstituted enzyme system for changes in both substrate-binding affinity and rates of catalysis. In addition, an extensive study was conducted as to the "order of mixing" for the reconstituted enzyme system and the impact on the observations. CYP2D6 coincubation inhibited CYP2C9-mediated (S)-flurbiprofen metabolism in a protein concentration-dependent manner. V(max) values were reduced by up to 50%, but no appreciable effect on K(m) was observed. Spectral binding studies revealed a 20-fold increase in the K(S) of CYP2C9 toward (S)-flurbiprofen in the presence of CYP2D6. CYP2C9 coincubation had no effect on CYP2D6-mediated dextromethorphan O-demethylation. The order of combination of the proteins (CYP2C9, CYP2D6, and cytochrome P450 reductase) influenced the magnitude of catalysis inhibition as well as the ability of increased cytochrome P450 reductase to attenuate the change in activity. A simple model, congruent with current results and those of others, is proposed to explain oligomer formation. In summary, CYP2C9-CYP2D6 interactions can alter catalytic activity and, thus, influence in vitro-in vivo correlation predictions.
Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.
Any process that stops or reduces the rate or extent of binding, the selective interaction of a molecule with one or more specific sites on another molecule.
Cytochrome P450 (P450) protein-protein interactions have been observed with various in vitro systems. It is interesting to note that these interactions seem to be isoform-dependent, with some combinations producing no effect and others producing increased or decreased catalytic activity. With some exceptions, most of the work to date has involved P450s from rabbit, rat, and other animal species, with few studies including human P450s. In the studies presented herein, the interactions of two key drug-metabolizing enzymes, CYP2C9 and CYP2D6, were analyzed in a purified, reconstituted enzyme system for changes in both substrate-binding affinity and rates of catalysis. In addition, an extensive study was conducted as to the "order of mixing" for the reconstituted enzyme system and the impact on the observations. CYP2D6 coincubation inhibited CYP2C9-mediated (S)-flurbiprofen metabolism in a protein concentration-dependent manner. V(max) values were reduced by up to 50%, but no appreciable effect on K(m) was observed. Spectral binding studies revealed a 20-fold increase in the K(S) of CYP2C9 toward (S)-flurbiprofen in the presence of CYP2D6. CYP2C9 coincubation had no effect on CYP2D6-mediated dextromethorphan O-demethylation. The order of combination of the proteins (CYP2C9, CYP2D6, and cytochrome P450 reductase) influenced the magnitude of catalysis inhibition as well as the ability of increased cytochrome P450 reductase to attenuate the change in activity. A simple model, congruent with current results and those of others, is proposed to explain oligomer formation. In summary, CYP2C9-CYP2D6 interactions can alter catalytic activity and, thus, influence in vitro-in vivo correlation predictions.
Any process that decreases the rate, frequency or extent of the chemical reactions and pathways involving organofluorine compounds, as carried out by individual cells.
Cytochrome P450 (P450) protein-protein interactions have been observed with various in vitro systems. It is interesting to note that these interactions seem to be isoform-dependent, with some combinations producing no effect and others producing increased or decreased catalytic activity. With some exceptions, most of the work to date has involved P450s from rabbit, rat, and other animal species, with few studies including human P450s. In the studies presented herein, the interactions of two key drug-metabolizing enzymes, CYP2C9 and CYP2D6, were analyzed in a purified, reconstituted enzyme system for changes in both substrate-binding affinity and rates of catalysis. In addition, an extensive study was conducted as to the "order of mixing" for the reconstituted enzyme system and the impact on the observations. CYP2D6 coincubation inhibited CYP2C9-mediated (S)-flurbiprofen metabolism in a protein concentration-dependent manner. V(max) values were reduced by up to 50%, but no appreciable effect on K(m) was observed. Spectral binding studies revealed a 20-fold increase in the K(S) of CYP2C9 toward (S)-flurbiprofen in the presence of CYP2D6. CYP2C9 coincubation had no effect on CYP2D6-mediated dextromethorphan O-demethylation. The order of combination of the proteins (CYP2C9, CYP2D6, and cytochrome P450 reductase) influenced the magnitude of catalysis inhibition as well as the ability of increased cytochrome P450 reductase to attenuate the change in activity. A simple model, congruent with current results and those of others, is proposed to explain oligomer formation. In summary, CYP2C9-CYP2D6 interactions can alter catalytic activity and, thus, influence in vitro-in vivo correlation predictions.
A metabolic process that results in the removal or addition of one or more electrons to or from a substance, with or without the concomitant removal or addition of a proton or protons.
Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.
Acetaminophen is a widely used analgesic antipyretic agent. When used at low doses, it is a safe drug, but at higher doses it can cause acute hepatic necrosis in humans and experimental animals. The key mechanism in the hepatotoxicity is cytochrome P450 (CYP)-catalysed formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) that is capable of binding to cellular macromolecules and in that way an LC/MS liquid chromatography/mass spectrometry (LC/MS) method was developed to measure NAPQI formation by trapping it to reduced glutathione. This method was used to determine the bioactivation of acetaminophen at two concentrations: 50 microM therapeutic and 1 mM toxic by using nine human recombinant CYP enzymes: CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4; and with different microsomes from experimental animals. At the toxic concentration the formation of NAPQI-glutathione was highest with CYP3A4 followed by CYP2E1, CYP1A2, and CYP2D6. At the therapeutic concentration, CYP3A4 had also the highest bioactivation capacity. In a comparison of the enzyme kinetics, CYP3A4 was the most efficient CYP with the lowest K(m) value 130 microM (95% confidence interval = 63-210 microM). Dexamethasone-induced rat liver microsomes had the most effective bioactivation capacity at therapeutic and toxic acetaminophen concentrations. This study suggests that CYP3A4 is the major CYP enzyme form catalysing acetaminophen oxidation to NAPQI in human liver.
Oxycodone undergoes N-demethylation to noroxycodone and O-demethylation to oxymorphone. The cytochrome P450 (P450) isoforms capable of mediating the oxidation of oxycodone to oxymorphone and noroxycodone were identified using a panel of recombinant human P450s. CYP3A4 and CYP3A5 displayed the highest activity for oxycodone N-demethylation; intrinsic clearance for CYP3A5 was slightly higher than that for CYP3A4. CYP2D6 had the highest activity for O-demethylation. Multienzyme, Michaelis-Menten kinetics were observed for both oxidative reactions in microsomes prepared from five human livers. Inhibition with ketoconazole showed that CYP3A is the high affinity enzyme for oxycodone N-demethylation; ketoconazole inhibited >90% of noroxycodone formation at low substrate concentrations. CYP3A-mediated noroxycodone formation exhibited a mean K(m) of 600 +/- 119 microM and a V(max) that ranged from 716 to 14523 pmol/mg/min. Contribution from the low affinity enzyme(s) did not exceed 8% of total intrinsic clearance for N-demethylation. Quinidine inhibition showed that CYP2D6 is the high affinity enzyme for O-demethylation with a mean K(m) of 130 +/- 33 microM and a V(max) that ranged from 89 to 356 pmol/mg/min. Activity of the low affinity enzyme(s) accounted for 10 to 26% of total intrinsic clearance for O-demethylation. On average, the total intrinsic clearance for noroxycodone formation was 8 times greater than that for oxymorphone formation across the five liver microsomal preparations (10.5 microl/min/mg versus 1.5 microl/min/mg). Experiments with human intestinal mucosal microsomes indicated lower N-demethylation activity (20-50%) compared with liver microsomes and negligible O-demethylation activity, which predict a minimal contribution of intestinal mucosa in the first-pass oxidative metabolism of oxycodone.
Constitutively expressed human cytochrome P450 2D6 (CYP2D6; EC 1.14.14.1) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however, we have identified this enzyme in the mitochondria of human liver samples and found that extensive inter-individual variability exists with respect to the level of the mitochondrial enzyme. Metabolic assays using 7-methoxy-4-aminomethylcoumarin as a substrate show that the human liver mitochondrial enzyme is capable of oxidizing this substrate and that the catalytic activity is supported by mitochondrial electron transfer proteins. In the present study, we show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the endoplasmic reticulum and mitochondria. In vitro mitochondrial import studies using both N-terminal deletions and point mutations suggest that the mitochondrial targeting signal is localized between residues 23-33 and that the positively-charged residues at positions 24, 25, 26, 28 and 32 are required for mitochondrial targeting. The importance of the positively-charged residues was confirmed by transient transfection of a CYP2D6 mitochondrial targeting signal mutant in COS-7 cells. Both the mitochondria and the microsomes from a CYP2D6 stable expression cell line contain the enzyme and both fractions exhibit bufuralol 1'-hydroxylation activity, which is completely inhibited by CYP2D6 inhibitory antibody. Overall, these results suggest that the targeting of CYP2D6 to mitochondria could be an important physiological process that has significance in xenobiotic metabolism.
This study examined the cytochrome P450 (CYP) enzyme selectivity of in vitro bioactivation of lynestrenol to norethindrone and the further metabolism of norethindrone. Screening with well-established chemical inhibitors showed that the formation of norethindrone was potently inhibited by CYP3A4 inhibitor ketoconazole (IC(50)=0.02 microM) and with CYP2C9 inhibitor sulphaphenazole (IC(50)=2.13 microM); the further biotransformation of norethindrone was strongly inhibited by ketoconazole (IC(50)=0.09 microM). Fluconazole modestly inhibited both lynestrenol bioactivation and norethindrone biotransformation. Lynestrenol bioactivation was mainly catalysed by recombinant human CYP2C9, CYP2C19 and CYP3A4; rCYP3A4 was responsible for the hydroxylation of norethindrone. A significant correlation was observed between norethindrone formation and tolbutamide hydroxylation, a CYP2C9-selective activity (r=0.63; p=0.01). Norethindrone hydroxylation correlated significantly with model reactions of CYP2C19 and CYP3A4. The greatest immunoinhibition of lynestrenol bioactivation was seen in incubations with CYP2C-Ab. The CYP3A4-Ab reduced norethindrone hydroxylation by 96%. Both lynestrenol and norethindrone were weak inhibitors of CYP2C9 (IC(50) of 32 microM and 46 microM for tolbutamide hydroxylation, respectively). In conclusion, CYP2C9, CYP2C19 and CYP3A4 are the primary cytochromes in the bioactivation of lynestrenol in vitro, while CYP3A4 catalyses the further metabolism of norethindrone.
The chemical reactions and pathways involving a xenobiotic compound, a compound foreign to living organisms. Used of chemical compounds, e.g. a xenobiotic chemical, such as a pesticide.
Constitutively expressed human cytochrome P450 2D6 (CYP2D6; EC 1.14.14.1) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however, we have identified this enzyme in the mitochondria of human liver samples and found that extensive inter-individual variability exists with respect to the level of the mitochondrial enzyme. Metabolic assays using 7-methoxy-4-aminomethylcoumarin as a substrate show that the human liver mitochondrial enzyme is capable of oxidizing this substrate and that the catalytic activity is supported by mitochondrial electron transfer proteins. In the present study, we show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the endoplasmic reticulum and mitochondria. In vitro mitochondrial import studies using both N-terminal deletions and point mutations suggest that the mitochondrial targeting signal is localized between residues 23-33 and that the positively-charged residues at positions 24, 25, 26, 28 and 32 are required for mitochondrial targeting. The importance of the positively-charged residues was confirmed by transient transfection of a CYP2D6 mitochondrial targeting signal mutant in COS-7 cells. Both the mitochondria and the microsomes from a CYP2D6 stable expression cell line contain the enzyme and both fractions exhibit bufuralol 1'-hydroxylation activity, which is completely inhibited by CYP2D6 inhibitory antibody. Overall, these results suggest that the targeting of CYP2D6 to mitochondria could be an important physiological process that has significance in xenobiotic metabolism.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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