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Molecular Pharmacology Apr 2015The cytochrome P450-dependent mono-oxygenase system is responsible for the metabolism and disposition of chemopreventive agents, chemical toxins and carcinogens, and...
The cytochrome P450-dependent mono-oxygenase system is responsible for the metabolism and disposition of chemopreventive agents, chemical toxins and carcinogens, and >80% of therapeutic drugs. Cytochrome P450 (P450) activity is regulated transcriptionally and by the rate of electron transfer from P450 reductase. In vitro studies have demonstrated that cytochrome b5 (Cyb5) also modulates P450 function. We recently showed that hepatic deletion of Cyb5 in the mouse (HBN) markedly alters in vivo drug pharmacokinetics; a key outstanding question is whether Cyb5 modulates the activity of the major human P450s in drug disposition in vivo. To address this, we crossed mice humanized for CYP2D6 or CYP3A4 with mice carrying a hepatic Cyb5 deletion. In vitro triazolam 4-hydroxylation (probe reaction for CYP3A4) was reduced by >50% in hepatic microsomes from CYP3A4-HBN mice compared with controls. Similar reductions in debrisoquine 4-hydroxylation and metoprolol α-hydroxylation were observed using CYP2D6-HBN microsomes, indicating a significant role for Cyb5 in the activity of both enzymes. This effect was confirmed by the concentration-dependent restoration of CYP3A4-mediated triazolam turnover and CYP2D6-mediated bufuralol and debrisoquine turnover on addition of Escherichia coli membranes containing recombinant Cyb5. In vivo, the peak plasma concentration and area under the concentration time curve from 0 to 8 hours (AUC0-8 h) of triazolam were increased 4- and 5.7-fold, respectively, in CYP3A4-HBN mice. Similarly, the pharmacokinetics of bufuralol and debrisoquine were significantly altered in CYP2D6-HBN mice, the AUC0-8 h being increased ∼1.5-fold and clearance decreased by 40-60%. These data demonstrate that Cyb5 can be a major determinant of CYP3A4 and CYP2D6 activity in vivo, with a potential impact on the metabolism, efficacy, and side effects of numerous therapeutic drugs.
Topics: Animals; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Cytochromes b5; Debrisoquin; Ethanolamines; Female; Humans; Male; Mice, Knockout; Microsomes, Liver; Nifedipine; Sex Factors; Triazolam
PubMed: 25657337
DOI: 10.1124/mol.114.097394 -
Advances in Experimental Medicine and... 2015Dopamine (DA) is a putative neurotransmitter in the carotid body engaged in the generation of the hypoxic ventilatory response (HVR). However, the action of endogenous... (Review)
Review
Dopamine (DA) is a putative neurotransmitter in the carotid body engaged in the generation of the hypoxic ventilatory response (HVR). However, the action of endogenous DA is unsettled. This study seeks to determine the ventilatory effects of increased availability of endogenous DA caused by inhibition of DA enzymatic breakdown. The peripheral inhibitor of MAO - debrisoquine, or COMT - entacapone, or both combined were injected to conscious rats. Ventilation and its responses to acute 8 % O(2) in N(2) were investigated in a whole body plethysmograph. We found that inhibition of MAO augmented the hyperventilatory response to hypoxia. Inhibition of COMT failed to influence the hypoxic response. However, simultaneous inhibition of both enzymes, the case in which endogenous availability of DA should increase the most, reversed the hypoxic augmentation of ventilation induced by MAO-inhibition. The inference is that when MAO alone is blocked, COMT takes over DA degradation in a compensatory way, which lowers the availability of DA, resulting in a higher intensity of the HVR. We conclude that MAO is the enzyme predominantly engaged in the chemoventilatory effects of DA. Furthermore, the findings imply that endogenous DA is inhibitory, rather than stimulatory, for hypoxic ventilation.
Topics: Adaptation, Physiological; Animals; Blood Pressure; Carotid Body; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Catechols; Debrisoquin; Dopamine; Drug Synergism; Hyperventilation; Hypoxia; Male; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Nitriles; Plethysmography, Whole Body; Rats; Rats, Wistar; Respiration
PubMed: 25310955
DOI: 10.1007/5584_2014_72 -
Drug Metabolism and Disposition: the... Sep 2014Cilengitide is a stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αVβ3 and αVβ5 integrins. The candidate drug showed...
Cilengitide is a stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αVβ3 and αVβ5 integrins. The candidate drug showed unexpected inhibition of cytochrome P450 (P450) 3A4 at high concentrations, that is, a 15-mM concentration caused attenuation of P450 3A4 activity (depending on the probe substrate): 15-19% direct inhibition, 10-23% time-dependent inhibition (30-minute preincubation), and 54-60% metabolism-dependent inhibition (30-minute preincubation). The inactivation efficiency determined with human liver microsomes was 0.003 ± 0.001 min(-1) mM(-1) and was 0.04 ± 0.01 min(-1) mM(-1) with baculovirus-based microsomes containing recombinant P450 3A4. Neither heme loss nor covalent binding to apoprotein could explain the observed reductions in residual activity. Slowly forming type II difference spectra were observed, with maximum spectral changes after 2 hours. Binding to both reduced and oxidized P450 3A4 was observed, with apparent Kd values of 0.66 μM and 6 μM. The significance of the guanidine group in inhibition was demonstrated using ligand binding spectral changes and inactivation assays with guanidine analogs (debrisoquine, N-acetylarginine-O-methyl ester) and the acetylated ornithine derivative of cilengitide. The observed inhibition could be explained by direct inhibition, plus by formation of stable complexes with both ferric and ferrous forms of heme iron and to some extent by the formation of reactive species capable to react to the protein or heme. Formation of the complex required time and NADPH and is attributed to the guanidino group. Thus, the NADPH-dependent inhibition is considered to be mainly due to the formation of a stable complex rather than the formation of reactive species.
Topics: Adult; Cytochrome P-450 CYP3A; Enzyme Inhibitors; Female; Guanidine; Heme; Humans; Male; Microsomes, Liver; NADP; Oxidation-Reduction; Snake Venoms
PubMed: 24985702
DOI: 10.1124/dmd.114.059295 -
Pharmacogenomics Dec 2013The CYP2D6 -1584C>G polymorphism (rs1080985) has been identified as a major factor for CYP2D6 expression and function, with the mutant -1584G promoter type being...
BACKGROUND & AIM
The CYP2D6 -1584C>G polymorphism (rs1080985) has been identified as a major factor for CYP2D6 expression and function, with the mutant -1584G promoter type being consistently associated with significantly greater expression than -1584C. It may therefore be associated with ultrarapid metabolism. The objective of the present study was to explore the relationship between the CYP2D6 -1584C>G polymorphism and the debrisoquine metabolic ratio in healthy volunteers in order to evaluate its potential impact on the ultrarapid CYP2D6 hydroxylation capacity.
MATERIALS & METHODS
The CYP2D6 -1584C>G polymorphism was analyzed in 320 unrelated healthy individuals who were previously phenotyped for debrisoquine hydroxylation.
RESULTS
The metabolic ratio (log10 mean ± standard deviation) of individuals with the -1584G allele was lower than that of individuals with the -1584C allele for carriers of one active CYP2D6 gene (-0.13 ± 0.33 and 0.17 ± 0.52, respectively; p < 0.05) or two active CYP2D6 genes (-0.32 ± 0.39 and -0.20 ± 0.44, respectively; p < 0.05).
CONCLUSION
The presence of the -1584G allele in the promoter region of the CYP2D6 gene was related to a high CYP2D6 hydroxylation capacity.
Topics: Alleles; Cytochrome P-450 CYP2D6; Debrisoquin; Gene Expression Regulation; Genotype; Healthy Volunteers; Heterozygote; Humans; Hydroxylation; Polymorphism, Genetic; Promoter Regions, Genetic
PubMed: 24279852
DOI: 10.2217/pgs.13.181 -
The Journal of Physical Chemistry. B Feb 2013The penetration properties of drug-like molecules on human cell membranes are crucial for understanding the metabolism of xenobiotics and overall drug distribution in...
The penetration properties of drug-like molecules on human cell membranes are crucial for understanding the metabolism of xenobiotics and overall drug distribution in the human body. Here, we analyze partitioning of substrates of cytochrome P450s (caffeine, chlorzoxazone, coumarin, ibuprofen, and debrisoquine) and their metabolites (paraxanthine, 6-hydroxychlorzoxazone, 7-hydroxycoumarin, 3-hydroxyibuprofen, and 4-hydroxydebrisoquine) on two model membranes: dioleoylphosphatidylcholine (DOPC) and palmitoyloleoylphophatidylglycerol (POPG). We calculated the free energy profiles of these molecules and the distribution coefficients on the model membranes. The drugs were usually located deeper in the membrane than the corresponding metabolites and also had a higher affinity to the membranes. Moreover, the behavior of the molecules on the membranes differed, as they seemed to have a higher affinity to the DOPC membrane than to POPG, implying they have different modes of action in human (mostly PC) and bacterial (mostly PG) cells. As the xenobiotics need to pass through lipid membranes on their way through the body and the effect of some drugs might depend on their accumulation on membranes, we believe that detailed information of penetration phenomenon is important for understanding the overall metabolism of xenobiotics.
Topics: Caffeine; Cell Membrane; Chlorzoxazone; Coumarins; Cytochrome P-450 Enzyme System; Debrisoquin; Humans; Ibuprofen; Lipid Bilayers; Molecular Dynamics Simulation; Pharmaceutical Preparations; Phosphatidylcholines; Phosphatidylglycerols; Thermodynamics; Xenobiotics
PubMed: 23387302
DOI: 10.1021/jp311802x -
British Journal of Clinical Pharmacology Feb 2013Pegylated interferon-based therapy is recommended for treatment of hepatitis C virus (HCV) infection. Because interferons are known to down-regulate hepatic cytochrome...
AIM
Pegylated interferon-based therapy is recommended for treatment of hepatitis C virus (HCV) infection. Because interferons are known to down-regulate hepatic cytochrome P450 (CYP) enzymes, which are involved in drug metabolism and clearance, there is a need to investigate the effect of peginterferon (PEG-IFN) alfa-2a (40KD) on the activity of these enzymes in vivo.
METHODS
Fourteen healthy, male volunteers aged 18 to 45 years were recruited into an open label, two period, single centre study in which CYP enzyme activity was measured by administration of the selectively metabolized probe drugs theophylline (CYP1A2), tolbutamide (CYP2C9), mephenytoin (CYP2C19), debrisoquine (CYP2D6) and dapsone (CYP3A4) on day 1 of the study. PEG-IFN alfa-2a (40KD) 180 μg was given subcutaneously each week from day 15 to 36, and probe drugs were re-administered on day 37. Probe drugs and metabolites were quantified in plasma or urine samples and used to derive pharmacokinetic parameters.
RESULTS
PEG-IFN alfa-2a (40KD) significantly increased the area under the serum drug concentration vs. time curve (AUC(0,∞)) for theophylline by 24%, with a reduction in the mean oral clearance of theophylline of 20%. There were no effects on the pharmacokinetics of any of the other probe drugs. The incidence of adverse events was as expected in subjects receiving pegylated interferon.
CONCLUSION
These results suggest there may be an inhibitory effect of PEG-IFN alfa-2a (40KD) on CYP1A2. PEG-IFN alfa-2a (40KD) had no effect on CYP2C9, CYP2C19, CYP2D6 or CYP3A4 in healthy subjects.
Topics: Adolescent; Adult; Antiviral Agents; Area Under Curve; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Drug Interactions; Humans; Interferon-alpha; Male; Middle Aged; Polyethylene Glycols; Recombinant Proteins; Young Adult
PubMed: 22765278
DOI: 10.1111/j.1365-2125.2012.04373.x -
PloS One 2012The CYP2D family members are instrumental in the metabolism of 20-25% of commonly prescribed drugs. Although many CYP2D isoforms have been well characterized in other...
The CYP2D family members are instrumental in the metabolism of 20-25% of commonly prescribed drugs. Although many CYP2D isoforms have been well characterized in other animal models, research concerning the chicken CYP2Ds is limited. In this study, a cDNA encoding a novel CYP2D enzyme (CYP2D49) was cloned from the chicken liver for the first time. The CYP2D49 cDNA contained an open reading frame of 502 amino acids that shared 52%-57% identities with other CYP2Ds. The gene structure and neighboring genes of CYP2D49 are conserved and similar to those of human CYP2D6. Additionally, similar to human CYP2D6, CYP2D49 is un-inducible in the liver and expressed predominantly in the liver, kidney and small intestine, with detectable levels in several other tissues. Metabolic assays of the CYP2D49 protein heterologously expressed in E. coli and Hela cells indicated that CYP2D49 metabolized the human CYP2D6 substrate, bufuralol, but not debrisoquine. Moreover, quinidine, a potent inhibitor of human CYP2D6, only inhibited the bufuralol 1'-hydroxylation activity of CYP2D49 to a negligible degree. All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile. Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein. The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.
Topics: Amino Acid Sequence; Animals; Antibody Specificity; Chickens; Circular Dichroism; Cytochrome P-450 CYP2D6; Cytochrome P-450 Enzyme System; Ethanolamines; Female; HeLa Cells; Humans; Immune Sera; Isoenzymes; Liver; Male; Molecular Sequence Data; Multigene Family; Phylogeny; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 22675558
DOI: 10.1371/journal.pone.0038395 -
The Journal of Biological Chemistry May 2012Purification and Characterization of the Human Liver Cytochromes P-450 Involved in Debrisoquine 4-Hydroxylation and Phenacetin -Deethylation, Two Prototypes for Genetic...
Purification and Characterization of the Human Liver Cytochromes P-450 Involved in Debrisoquine 4-Hydroxylation and Phenacetin -Deethylation, Two Prototypes for Genetic Polymorphism in Oxidative Drug Metabolism (Distlerath, L. M., Reilly, P. E. B., Martin, M. V., Davis, G. G., Wilkinson, G. R., and Guengerich, F. P. (1985) 260, 9057–9067) Human Liver Microsomal Cytochrome P-450 Mephenytoin 4-Hydroxylase, a Prototype of Genetic Polymorphism in Oxidative Drug Metabolism. Purification and Characterization of Two Similar Forms Involved in the Reaction (Shimada, T., Misono, K. S., and Guengerich, F. P. (1986) 261, 909–921) Characterization of Rat and Human Liver Microsomal Cytochrome P-450 Forms Involved in Nifedipine Oxidation, a Prototype for Genetic Polymorphism in Oxidative Drug Metabolism (Guengerich, F. P., Martin, M. V., Beaune, P. H., Kremers, P., Wolff, T., and Waxman, D. J. (1986) 261, 5051–5060)
Topics: Cytochrome P-450 Enzyme System; Drug Industry; History, 20th Century; History, 21st Century; Humans; Isoenzymes; Microsomes, Liver; Pharmaceutical Preparations; United States
PubMed: 22563099
DOI: 10.1074/jbc.O112.000003 -
BMC Medical Genetics Mar 2012There are no known causes for progressive supranuclear palsy (PSP). The microtubule associated protein tau (MAPT) H1 haplotype is the major genetic factor associated...
BACKGROUND
There are no known causes for progressive supranuclear palsy (PSP). The microtubule associated protein tau (MAPT) H1 haplotype is the major genetic factor associated with risk of PSP, with both oxidative stress and mitochondrial dysfunction also implicated. We investigated whether specific single nucleotide polymorphisms (SNPs) in genes encoding enzymes of xenobiotic detoxification, mitochondrial functioning, or oxidative stress response, including debrisoquine 4-hydroxylase, paraoxonase 1 and 2, N-acetyltransferase 1 and 2 (NAT2), superoxide dismutase 1 and 2, and PTEN-induced putative kinase are associated with PSP.
METHODS
DNA from 553 autopsy-confirmed Caucasian PSP cases (266 females, 279 males; age at onset 68 ± 8 years; age at death 75 ± 8) from the Society for PSP Brain Bank and 425 clinical control samples (197 females, 226 males; age at draw 72 ± 11 years) from healthy volunteers were genotyped using Taqman PCR and the SequenomiPLEX Gold assay.
RESULTS
The proportion of NAT2 rapid acetylators compared to intermediate and slow acetylators was larger in cases than in controls (OR = 1.82, p < 0.05). There were no allelic or genotypic associations with PSP for any other SNPs tested with the exception of MAPT (p < 0.001).
CONCLUSIONS
Our results show that NAT2 rapid acetylator phenotype is associated with PSP, suggesting that NAT2 may be responsible for activation of a xenobiotic whose metabolite is neurotoxic. Although our results need to be further confirmed in an independent sample, NAT2 acetylation status should be considered in future genetic and epidemiological studies of PSP.
Topics: Aged; Aged, 80 and over; Arylamine N-Acetyltransferase; Aryldialkylphosphatase; Case-Control Studies; Cytochrome P-450 CYP2D6; Female; Genetic Predisposition to Disease; Humans; Inactivation, Metabolic; Isoenzymes; Male; Middle Aged; Mitochondria; Oxidative Stress; Polymorphism, Single Nucleotide; Protein Kinases; Superoxide Dismutase; Superoxide Dismutase-1; Supranuclear Palsy, Progressive; tau Proteins
PubMed: 22424094
DOI: 10.1186/1471-2350-13-16 -
European Journal of Clinical... Sep 2012The influence of the cytochrome P450 enzyme CYP2D6 in the metabolism of the novel dopaminergic stabilizer pridopidine was investigated in healthy Swedish Caucasians. (Clinical Trial)
Clinical Trial
PURPOSE
The influence of the cytochrome P450 enzyme CYP2D6 in the metabolism of the novel dopaminergic stabilizer pridopidine was investigated in healthy Swedish Caucasians.
METHODS
Six extensive metabolizers (EM) and six poor metabolizers (PM) of debrisoquine were given a single oral dose of pridopidine (EM, 50 mg; PM, 25 mg).
RESULTS
The mean total plasma clearance of pridopidine was 541 and 138 mL/min in EM and PM, respectively (p = 0.003), and was slightly higher in PM than the mean renal plasma clearance (105 mL/min; p = 0.11). The mean plasma area under the time-concentration curve between time zero and 32 h (AUC(0-32 h)) of the N-depropyl metabolite ACR30 was higher in EM than in PM (1,377 vs. 61 nmol h/mL, respectively; p < 0.001). The urinary excretion of pridopidine + ACR30 was high in both EM (85 %) and PM (78 %). The dose-adjusted peak concentration (C(max)) was not statistically different in EM and PM; consequently, the oral absorption of pridopidine was close to complete.
CONCLUSIONS
Following a single dose of pridopidine, the drug is N-depropylated by CYP2D6 in EM, while in PM the most important elimination pathway is renal glomerular filtration. Results of studies examining the effects of multiple repeat dosing suggest that the CYP2D6 enzyme is at least partly inactivated by pridopidine.
Topics: Administration, Oral; Adult; Area Under Curve; Biotransformation; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP2D6 Inhibitors; Dealkylation; Dopamine Antagonists; Enzyme Inhibitors; Glomerular Filtration Rate; Half-Life; Humans; Intestinal Absorption; Kidney; Metabolic Clearance Rate; Middle Aged; Phenotype; Piperidines; Sweden; White People; Young Adult
PubMed: 22399238
DOI: 10.1007/s00228-012-1248-z