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PubMed1992

Enantioselective amitriptyline metabolism in patients phenotyped for two cytochrome P450 isozymes.

In 26 hospitalized patients with depression, a combined pharmacogenetic test with dextromethorphan, a substrate of cytochrome P450IID6, and mephenytoin, the S-form of which is hydroxylated by a P450IIC isozyme, was carried out before amitriptyline therapy. Metabolites were determined in 24-hour urine samples collected on treatment day 8, and the contributions of individual compounds, including the four isomers of 10-hydroxyamitriptyline and 10-hydroxynortriptyline to total excretion were calculated. Formation of (-)-E-10-hydroxyamitriptyline and (-)-E-10-hydroxynortriptyline apparently depends on the activity of cytochrome P450IID6 because negative correlations existed between the log metabolic ratio of dextromethorphan and the relative quantities of these enantiomers. In contrast, correlations were positive for nortriptyline, (+)-E-10-hydroxynortriptyline, (-)-Z-10-hydroxynortriptyline, and (+)-Z-10-hydroxynortriptyline. The mephenytoin hydroxylase seems to participate in side-chain demethylation to the secondary and primary amines, because the log metabolic ratio of mephenytoin correlated negatively with the relative quantity of E-10-hydroxydidesmethylamitriptyline and positively with that of amitriptyline and its N-glucuronide.

Adult↗

PubMed2003

Role of conserved Asp293 of cytochrome P450 2C9 in substrate recognition and catalytic activity.

Human cytochrome P450 2C9 (CYP2C9) is important in the metabolism of non-steroidal anti-inflammatory compounds such as diclofenac, the antidiabetic agent tolbutamide and other clinically important drugs, many of which are weakly acidic. Multiple sequence alignment of CYPs identified CYP2C9 Asp(293) as corresponding to Asp(301) of CYP2D6, which has been suggested to play a role in the binding of basic substrates to the latter enzyme. Replacement of Asp(293) with Ala (D293A) decreased activity by more than 90%, and led to an approx. 3- to 10-fold increase in K (m) values for the three test substrates tolbutamide, dextromethorphan and diclofenac. Conservative replacement of the carboxyl side chain in a Glu (D293E) mutant produced no significant changes in K (m) values and slight increases in k (cat) values. Changes in regiospecificity were observed for both the Ala and Glu substitutions; low levels of both dextromethorphan O- and N-demethylation were observed in the D293A mutant, whereas increased preference for O-demethylation was observed for the D293E mutant. Expression of constructs coding for Asn (D293N) and Gln (D293Q) substitutions failed to form a P450 correctly. Our analysis suggests a structural role for the carboxyl side chain of Asp(293) in CYP2C9 substrate binding and catalysis. The conservation of an Asp residue in other CYP families in a position equivalent to Asp(293) indicates a common mechanism for maintaining the active-site architecture.

Amino Acid Sequence↗

PubMed1999

CYP2D6 polymorphism in a Gabonese population: contribution of the CYP2D62 and CYP2D617 alleles to the high prevalence of the intermediate metabolic phenotype.

AIMS: To determine the molecular basis of the intermediate extensive metaboliser (EM) CYP2D6 phenotype in healthy Gabonese subjects. METHODS: The CYP2D6 phenotype of 154 healthy Gabonese subjects was assessed by giving the subject a single dose of 30 mg dextromethorphan, and collecting their urine for the next 8 h. The CYP2D6 genotype was determined for 50 individuals of the EM phenotypic group by Southern blotting and various PCR-based procedures aimed at identifying different CYP2D6 alleles. RESULTS: We found that in the studied Gabonese population, as compared with a French population, there is significantly higher frequency of intermediate EM phenotype having lower frequency of CYP2D6 PM alleles. To clarify this discrepancy phenotype-genotype relationship was studied. We found that the CYP2D6*17 and CYP2D6*2 alleles, prevalent in this black population, are characterised by their low capacity for dextromethorphan demethylation. Our data also show that the CYP2D6*1 allele is associated with the highest in vivo activity followed by the CYP2D6*2 allele and then the CYP2D6*17 allele. CONCLUSIONS: The higher frequencies of the CYP2D6*2 and CYP2D6*17 alleles than the CYP2D6*1 allele account for the high frequency of the intermediate EM phenotype in this black population. The polymorphism of the CYP2D6 enzyme activity in African populations could have important implications for use of drugs that are substrates for CYP2D6 and have a narrow therapeutic window.

Alleles↗

PubMed2000

In vivo and in vitro induction of human cytochrome P4503A4 by dexamethasone.

PURPOSE: The aims of these experiments were to determine the effect of a therapeutic regimen of dexamethasone on cytochrome P4503A4 (CYP3A4) activity in healthy volunteers; and the concentration-effect relationship between dexamethasone and CYP3A4 activity in primary human hepatocyte cultures. METHODS: The effect of dexamethasone (8 mg administered by mouth two times a day for 5 days) on CYP3A4 activity in 12 healthy volunteers was assessed with the erythromycin breath test and urinary ratio of dextromethorphan to 3-methoxymorphinan. Concentration-effect of dexamethasone on CYP3A4-dependent testosterone 6-beta-hydroxylation was determined in human hepatocytes treated with 2 to 250 micromol/L dexamethasone. RESULTS: The percent of erythromycin metabolized per hour increased from 2.20% +/- 0.60% (mean +/- SD) at baseline to 2.67% +/- 0.55% on day 5 of dexamethasone (mean increase in hepatic CYP3A4 activity 25.7% +/- 24.6%; P = .004). The mean urinary ratio of dextromethorphan to 3-methoxymorphinan was 28 (4.8 to 109) and 7 (1 to 23) at baseline and on day 5 of dexamethasone (mean decrease = 49%; P = .06). Substantial intersubject variability was observed in the extent of CYP3A4 induction. The extent of CYP3A4 induction was inversely correlated with baseline erythromycin breath test (r2 = 0.58). In hepatocytes, dexamethasone 2 to 250 micromol/L resulted in an average 1.7-fold to 6.9-fold increase in CYP3A4 activity, respectively. The extent of CYP3A4 induction with dexamethasone in hepatocyte preparations was inversely correlated with baseline activity (r2 = 0.59). CONCLUSIONS: These data demonstrate that dexamethasone at doses used clinically increased CYP3A4 activity with extensive intersubject variability and that the extent of CYP3A4 induction was, in part, predicted by the baseline activity of CYP3A4 in both healthy volunteers and human hepatocyte cultures.

Administration, Oral↗

PubMed2002

Unique CYP2D6 activity distribution and genotype-phenotype discordance in black Americans.

BACKGROUND: Although CYP2D6 has been studied extensively in different population groups, relatively little is known for black Americans. METHODS: CYP2D6 activity was assessed with dextromethorphan in 283 black American subjects and correlated with their genotype (2D6*2 to *12, 2D6*14, 2D6*15, 2D6*17, 2D6*18, and 2D6*29 and gene duplications). Volunteers provided information about ethnicity and concurrent medication, and they participated in either phenotyping (n = 225), genotyping (n = 251), or both (n = 193). RESULTS: The median urinary dextromethorphan/dextrorphan metabolic ratio (MR) indicated significantly lower CYP2D6 activity in the black American group (0.016) than in a white control population (0.0044; P =.0001) studied previously. The reduced function allele 2D6*17 was more common (frequency [f] = 0.395) among intermediate metabolizers (0.03 < or = MR < or= 0.3) than extensive metabolizers (MR < or = 0.03; f = 0.148; P =.0001). Consistent with reduced function toward dextromethorphan of COS cell-expressed 2D6.29 protein, 2D6*29 also was more frequent in intermediate metabolizers (f = 0.114) than in extensive metabolizers (f = 0.057; P = NS). Frequencies for 2D6*17 and 2D6*29 were f = 0.213 and 0.072, respectively. Of the 193 genotyped and phenotyped subjects, 14 were determined to be poor metabolizers, with dextromethorphan/dextrorphan ratios >0.3 (7.25%), but only 2 subjects (1.04%) carried 2 nonfunctional alleles (2D6*3/*4x2 and 2D6*4/*4). A new allelic variant, 2D6*40, was subsequently found in 2 discordant subjects (2D6*4/*40 and 2D6*6/*40), implying that the 18-base pair (bp) insertion found in 2D6*40 renders it nonfunctional. The frequency of 2D6*40 was 0.006. For genotypes that contain 2D6*2, median MR values were consistently higher in black Americans than in white subjects, indicating that other unidentified factors also contribute to lower CYP2D6 activity in black Americans. CONCLUSIONS: The lower CYP2D6 activity observed in a black American population is in part attributable to the presence of variant alleles that occur at a higher frequency in this population than in white subjects. Additional studies are required to ascertain the pharmacokinetic and pharmacodynamic consequences of these pharmacogenetic data in black Americans.

Adolescent↗

PubMed2002

Residues glutamate 216 and aspartate 301 are key determinants of substrate specificity and product regioselectivity in cytochrome P450 2D6.

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K(m) values increased 10-100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K(m) 108 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, k(cat) 1 min(-1)), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.

Alkylation↗

PubMed2000

Characterization of transgenic mouse strains using six human hepatic cytochrome P450 probe substrates.

1. Transgenic mice were evaluated with six human cytochrome P450 (CYP) selective probe substrates, as little is known about their metabolism in the mouse. Mouse strains characterized include C57BL/SJL, FVB/N, mdr 1a/1b (-/-), ob/ob and ACCA. 2. Human CYP probe substrates used for characterization of mouse CYP activities included bufuralol, testosterone, dextromethorphan, phenacetin, diclofenac and S-mephenytoin. Activities were compared with those obtained in human liver microsomes and in human recombinant enzyme preparations. All transgenic mouse strains showed similar apparent K(m) with bufuralol, testosterone and dextromethorphan which compared favourably with those observed in human liver microsomes. 3. K(m) for phenacetin O-deethylase and S-mephenytoin 4'-hydroxylation were more variable across strains and in some cases demonstrated biphasic kinetics. Phenacetin O-deethylase activity was low in all mouse strains except FVB/N and mdr 1a/1b (-/-). Diclofenac 4-hydroxylation did not occur to any significant extent in the five strains of mouse evaluated here. 4. The findings suggest the validity of using five of the probes for transgenic mouse hepatic CYP characterization and gross comparison with data generated with human CYP.

Animals↗

PubMed2003

mRNA and protein expression of dog liver cytochromes P450 in relation to the metabolism of human CYP2C substrates.

1. Interpretation of novel drug exposure and toxicology data from the dog is tempered by our limited molecular and functional knowledge of dog cytochromes P450 (CYPs). The aim was to study the mRNA and protein expression of hepatic dog CYPs in relation to the metabolism of substrates of human CYP, particularly those of the CYP2C subfamily. 2. The rate of 7-hydroxylation of S-warfarin (CYP2C9 in humans) by dog liver microsomes (mean +/- SD from 12 (six male and six female) dogs = 10.8 +/- 1.9 fmol mg(-1) protein min(-1)) was 1.5-2 orders of magnitude lower than that in humans. 3. The rate of 4'-hydroxylation of S-mephenytoin, catalysed in humans by CYP2C19, was also low in dog liver (4.6 +/-1.5 pmol mg(-1) protein min(-1)) compared with human liver. In contrast, the rate of 4'-hydroxylation of the R-enantiomer of mephenytoin by dog liver was much higher. The kinetics of this reaction (range of K(m) or K(0.5) 15-22 micro M, V(max) 35-59 pmol mg(-1) protein min(-1), n = 4 livers) were consistent with the involvement of a single enzyme. 4. In contrast to our findings for S-mephenytoin, dog liver microsomes 5'-hydroxylated omeprazole (also catalysed by CYP2C19 in humans) at considerably higher rates (range of K(m) 42-64 micro M, V(max) 22-46 pmol mg(-1) protein min(-1), n = 4 livers). 5. For all the substrates except omeprazole, a sex difference in their metabolism was observed in the dog (dextromethorphan N-demethylation: female range = 0.7-0.9, male = 0.4-0.8 nmol mg(-1) protein min(-1) (p < 0.02); S-warfarin 7-hydroxylation: female = 9-15.5, male = 8-12 fmol mg(-1) protein min(-1) (p < 0.02); R-mephenytoin 4'-hydroxylation: female = 16-35, male = 11.5-19 pmol mg(-1) protein min(-1) (p < 0.01); omeprazole 5'-hydroxylation: female = 15-20, male 13-22 pmol mg(-1) protein min(-1) (p < 0.2)). 6. All dog livers expressed mRNA and CYP3A12, CYP2B11, CYP2C21 proteins, with no sex differences being found. Expression of CYP2C41 mRNA was undetectable in the livers of six of 11 dogs. 7. Correlation analysis suggested that CYP2B11 catalyses the N-demethylation of dextromethorphan (mediated in humans by CYP3A) and the 4'-hydroxylation of mephenytoin (mediated in humans by CYP2C19) in the dog, and that this enzyme and CYP3A12 contribute to S-warfarin 7-hydroxylation (mediated in humans by CYP2C9). 8. In conclusion, we have identified a distinct pattern of hepatic expression of the CYP2C41 gene in the Alderley Park beagle dog. Furthermore, marked differences in the metabolism of human CYP2C substrates were observed in this dog strain compared with humans with respect to rate of reaction, stereoselectivity and CYP enzyme selectivity.

Algorithms↗

PubMed2004

Metabolism of human cytochrome P450 marker substrates in mouse: a strain and gender comparison.

The aim was to characterize mouse gender and strain differences in the metabolism of commonly used human cytochrome (CYP) P450 probe substrates. Thirteen human CYP probe substrates (phenacetin, coumarin, 7-ethoxy-4-trifluoromethyl coumarin, amiodarone, paclitaxel, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, chlorzoxazone, p-nitrophenol, testosterone and lauric acid) were used in activity measurements. The metabolism of the probe substrates was compared in liver microsomes from male and female NMRI, CBA, C57bl/6, 129/SvJ and CD1 strains. The expression of proteins identified on Western blots with commonly available antibodies selective for specific human and rat CYP enzymes were compared in the different mouse strains. Males had higher metabolism than corresponding females for phenacetin O-deethylation (human marker for CYP1A2 activity), and a high correlation was found between phenacetin activity and immunoreactivity in Western blots produced with rat CYP1A2 antibodies. Protein detected by antibodies cross-reacting with human CYP2B6 and rat CYP2B1/2 antibodies was female specific except for the 129/SvJ strain, where it was absent in both genders. Females generally had a higher metabolism of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation (human markers for CYP2D activity). Bufuralol 1'-hydroxylation correlated with a female-dominant mouse CYP, which was detected with antibodies against rat CYP2D4. p-Nitrophenol 2-hydroxylation correlated better than chlorzoxazone 6-hydroxylation with the protein detected with antibodies against rat CYP2E1, indicating that p-nitrophenol is a more specific substrate for mouse CYP2E1.

Amiodarone↗

PubMed2002

Venous malformations associated with central pain: report of a case.

IMPLICATIONS: The authors describe an unusual case of central pain (CP) that resulted from giant venous hemangiomas. The patient was treated with a variety of medications, including the N-methyl-D-aspartate antagonist dextromethorphan. We report the first known association between venous malformations and CP and briefly describe why the use of dextromethorphan in this disorder requires further evaluation.

Analgesics, Opioid↗

PubMed2002

Ketamine, an N-methyl-D-aspartate receptor antagonist, inhibits the spinal neuronal responses to distension of the rat urinary bladder.

BACKGROUND: The effect of ketamine as a treatment of visceral pain is not known. The current study investigated the effect of ketamine on spinal dorsal horn neurons excited by urinary bladder distension (UBD). The effect of other clinically available N-methyl-D-aspartate receptor antagonists on these responses was also studied. METHODS: Extracellular recordings of neurons located in the L6-S2 spinal dorsal horn of cervical spinal cord-transected, decerebrate female rats were obtained. Cutaneous receptive fields of neuronal units excited by UBD were characterized for responses to segmental noxious and nonnoxious stimuli. Nonsegmental noxious stimuli were also applied, and neurons were classified as type I (inhibited) and type II (noninhibited) by the stimulus. The effect of intravenous ketamine (1, 3, and 10 mg/kg), dextromethorphan (5 mg/kg), and memantine (16 mg/kg) on neuronal responses of these units was measured. RESULTS: Spontaneous and evoked neuronal activity to UBD was reduced in a dose-dependent fashion by ketamine. Responses to nonnoxious cutaneous stimuli were also significantly reduced after treatment. Dextromethorphan inhibited neuronal activity evoked by UBD in type I neurons. A similar selective effect of treatment on type I versus type II neurons was observed after intravenous ketamine and memantine. CONCLUSIONS: Intravenous ketamine produces dose-dependent inhibition of the spinal cord neuronal responses evoked by UBD. All three N-methyl-D-aspartate receptor antagonists showed selective effects on spinal cord neurons subject to counterirritation. This neurophysiologic evidence supports a spinally mediated analgesic effect of ketamine in this model of urinary bladder nociception, an effect likely caused by N-methyl-D-aspartate receptor antagonism.

Animals↗

PubMed1998

The extent and determinants of changes in CYP2D6 and CYP1A2 activities with therapeutic doses of sertraline.

The extent of changes in CYP2D6 and CYP1A2 activities with higher therapeutic dosages (>50 mg/day) of sertraline is not well established in vivo. This study assessed the extent and determinants of changes in CYP2D6 and CYP1A2 isozyme activities after treatment with clinically relevant doses of sertraline. Patients and healthy volunteers aged 19 to 85 years (N = 21) were treated with sertraline for 5 to 55 days. The dosage of sertraline ranged from 25 to 150 mg/day (93.5+/-26.4 mg/day; mean +/- SD). All subjects had an extensive metabolizer phenotype for CYP2D6 and received a single oral dose of dextromethorphan (30 mg) and caffeine (100 mg) before and after sertraline treatment. The log O-demethylation ratio (ODMR) of dextromethorphan and the caffeine metabolic ratio (CMR) in overnight urine were used as in vivo indices of the CYP2D6 and CYP1A2 isozyme activities, respectively. Concurrent medications and lifestyle habits (e.g., smoking and diet) were monitored during the study. Baseline log ODMR (-2.33+/-0.45) but not CMR (5.1+/-1.9) (mean +/- SD) significantly changed after sertraline treatment (-2.19+/-0.62; 4.5+/-1.6, respectively) (p: ODMR = 0.04, CMR = 0.10). There was no significant effect of age, dose, duration of treatment, gender, sertraline and/or desmethylsertraline plasma concentration, subject type (patient or volunteer), and weight on the extent of changes in log ODMR or CMR (p > 0.05). In conclusion, sertraline treatment at a mean daily dosage of 94.0 mg did not significantly change CYP1A2 activity and resulted in a modest inhibition of CYP2D6 activity.

1-Naphthylamine↗

PubMed1998

Determinants of interindividual variability and extent of CYP2D6 and CYP1A2 inhibition by paroxetine and fluvoxamine in vivo.

Major depression may require antidepressant treatment for several years. This necessitates consideration of the long-term effects of antidepressants on multiple clinical endpoints. The antidepressants paroxetine and fluvoxamine are potent in vitro inhibitors of CYP2D6 and CYP1A2 isozymes, respectively. CYP2D6 and CYP1A2 are important for the clearance of 30 or more frequently used medications. Moreover, CYP1A2 also contributes to metabolism of 17beta-estradiol and metabolic activation of environmental procarcinogens (e.g., arylamines in cigarette smoke). The aim of this study was to assess the determinants of interindividual variability and extent of CYP2D6 and CYP1A2 inhibition during paroxetine and fluvoxamine treatment. Healthy volunteers and patients received caffeine (100 mg) and dextromethorphan (30 mg) at baseline and at steady state of paroxetine (10-20 mg/day, 5-74 days, N = 13) or fluvoxamine (50-100 mg/day, 5-43 days, N = 8). The caffeine metabolic ratio (CMR) and the log O-demethylation ratio (ODMR) of dextromethorphan in overnight urine were used as in vivo indices of the CYP1A2 and CYP2D6 isozyme activities, respectively. All subjects had an extensive metabolizer phenotype for CYP2D6. After fluvoxamine treatment, baseline CMR 5.1 +/- 1.4 (mean +/- SD) decreased to 2.7 +/- 1.1 (p < 0.01). Paroxetine did not have a significant effect on CMR (p > 0.05). In seven of eight subjects in the fluvoxamine group, posttreatment CMR was comparable with the minimum CMR value (2.0) attainable in nonsmoking healthy volunteers. After paroxetine treatment, log ODMR changed from a baseline value of -2.28 +/- 0.37 to -1.13 +/- 0.44, indicating significant inhibition of CYP2D6 (p < 0.001). Subjects' CYP2D6 phenotype did not change after paroxetine treatment. Fluvoxamine had no significant effect on log ODMR (p > 0.05). The extent of inhibition of CYP2D6 and CYP1A2 by paroxetine and fluvoxamine, respectively, displayed a positive correlation with baseline enzyme activity (p < 0.05). In addition, a negative association was found between the plasma paroxetine concentration and the CYP2D6 activity after paroxetine treatment (r = -0.47, p < 0.05). These data indicate that paroxetine and fluvoxamine treatment with minimum clinically effective doses significantly inhibit CYP2D6 and CYP1A2, respectively. The extent of inhibition of CYP2D6 by paroxetine and of CYP1A2 by fluvoxamine is dependent in part on the baseline enzyme activity. The interindividual variability in CYP2D6 inhibition by paroxetine can also be explained by variability in plasma paroxetine concentration. Most patients treated with fluvoxamine (50-100 mg/day) will reach population minimums for CYP1A2 activity. These results have potential implications for interindividual variability in the risk for drug-drug interactions mediated by CYP2D6 and CYP1A2 as well as for the disposition of 17beta-estradiol and environmental procarcinogens.

Adult↗

PubMed1990

Potent inhibition of cytochrome P450IID6 (debrisoquin 4-hydroxylase) by flecainide in vitro and in vivo.

Flecainide acetate, a class Ic antiarrhythmic agent, is eliminated to a larger extent by renal excretion and to a minor extent by the liver. In patients with impaired renal function or with elevated urinary pH, however, its elimination is dominated by hepatic metabolism. Recent evidence suggests that the in vivo metabolism of flecainide is controlled by the genetic polymorphism of the debrisoquin/sparteine type; i.e., it is a substrate of cytochrome P450IID6. We investigated the inhibitory effect of flecainide on bufuralol 1'-hydroxylation in human liver microsomes in vitro and on the metabolic dextromethorphan urinary ratio in eight healthy male volunteers. Both bufuralol and dextromethorphan are well-known substrates of cytochrome P450IID6. Microsomal bufuralol 1-hydroxylation was competitively inhibited by flecainide with an apparent Ki of 0.954 mumol/L. Moreover, a statistically significant increase in the urinary metabolic ratio (MR) of dextromethorphan/dextrorphan after 1 week of administration of oral flecainide was observed (p = 0.013) in all subjects. One individual increased the urinary MR to a value consistent with the poor metabolizer phenotype. We conclude that flecainide is a potent inhibitor of cytochrome P450IID6 in vitro and in vivo and that careful drug monitoring is required with respect to renal function, debrisoquine phenotype, and concomitant drug administration.

Adult↗

PubMed1995

Stability of debrisoquine (CYP2D6) phenotype in liver transplant patients.

Liver metabolism may be modified after liver transplantation according to the phenotype of the donor and may be influenced by posttransplantation complications. The CYP2D6 phenotype was assessed in 13 patients (group I) before and after liver transplantation using debrisoquine. CYP2D6 activity was also assessed in vitro on microsomes from the liver of the recipients and the donors, using dextromethorphan. Twelve patients were extensive metabolizers both before and after transplantation. One apparently poor metabolizer was transplanted with the liver of another poor metabolizer. The intrinsic clearance of dextromethorphan (CL(int)) measured on recipient liver microsomes was significantly lower than that on donor liver microsomes (p < 0.05). In extensive metabolizers, the debrisoquine metabolic ratio was correlated with CL(int) before (r = 0.78, p < 0.05) and after (r = 0.89, p < 0.0005) transplantation. Debrisoquine phenotype was measured repeatedly in nine additional patients (group II) up to 3 years after liver transplantation. Their phenotype was stable during the follow-up observation, although the variations observed may be clinically relevant. Therefore, no change in CYP2D6 phenotype (extensive/poor metabolizer) was observed because of the liver transplantation, and the debrisoquine log metabolic ratio was largely unaffected by the liver complications observed during the posttransplantation follow-up observation.

Cytochrome P-450 CYP2D6↗

PubMed1999

No effect of the new antidepressant reboxetine on CYP2D6 activity in healthy volunteers.

The effect of the new antidepressant reboxetine on the activity of the cytochrome P450 (CYP) 2D6 isoenzyme was investigated in 10 healthy volunteers using dextromethorphan as a model CYP2D6 substrate. Each volunteer received a single 30 mg oral dose of dextromethorphan on three different occasions separated by an interval of at least 4 weeks: a) in a control session; b) after 1 week of treatment with reboxetine, 8 mg/day; and c) after 1 week of treatment with paroxetine (an inhibitor of CYP2D6 activity) 20 mg/day. Urine was collected over the next 8 hours for the determination of the dextromethorphan/dextrorphan metabolic ratio. All subjects were classified as extensive metabolizers (EM) with a dextromethorphan/dextrorphan ratio < 0.3. There were no notable changes in the urinary dextromethorphan/dextrorphan ratio in the reboxetine phase as compared to the control session. By contrast, there was a statistically significant increase in the metabolic ratio in the paroxetine phase (p < 0.001), with 4 subjects switching to poor metabolizer (PM) phenotype. These results suggest that reboxetine is unlikely to cause clinically significant interactions with substrates of CYP2D6.

Adult↗

PubMed2000

On the assessment of drug metabolism by assays of codeine and its main metabolites.

Codeine and its main metabolites appear to have advantages for assessing drug metabolic phenotypes. The authors have further developed a high-performance liquid chromatography (HPLC) method for the quantification of codeine and six of its metabolites in urine. Quantification was performed by electrochemical detection for morphine, normorphine, morphine-6-glucuronide, and the internal standard 4-O-methyldopamine; and by ultraviolet detection for codeine, norcodeine, and morphine-3-glucuronide. The method had a detection limit of 2 nmol/L(-1) for morphine and normorphine, 4 nmol/L(-1) for morphine-6-glucuronide, 3 nmol/L for the internal standard, 20 nmol/L(-1) for morphine-3-glucuronide, and 60 nmol/L(-1) for codeine and norcodeine. The coefficients of variations were <9% for intraday and <10% for interday analyses. The recovery of codeine and its metabolites ranged from 55% (for morphine-3-glucuronide) to 90% (for codeine, norcodeine, morphine, and morphine-6-glucuronide). Eleven healthy volunteers were phenotyped for CYP2D6 using codeine as well as debrisoquine and dextromethorphan. Ten subjects were extensive metabolizers (EM) and one a poor metabolizer (PM) of codeine, debrisoquine, and dextromethorphan. Significant correlations between the metabolic ratios (MRs) of the different probe drugs were obtained (r2 > 0.95, p < 0.001). This HPLC method is simple, sensitive, accurate, and reproducible for assessing the CYP2D6 phenotype.

Adult↗

PubMed1998

Metabolism of dexfenfluramine in human liver microsomes and by recombinant enzymes: role of CYP2D6 and 1A2.

Dexfenfluramine has been widely used as an appetite suppressant in the treatment of obesity. It was recently shown that the apparent non-renal clearance of dexfenfluramine was significantly lower in poor metabolizers than in extensive metabolisers of debrisoquine which suggested the involvement of the polymorphically expressed enzyme, CYP2D6, in dexfenfluramine metabolism. In this study, human liver microsomes and yeast-expressed recombinant enzymes were used to examine dexfenfluramine metabolism in vitro. In human liver microsomes, the major product of dexfenfluramine was nordexfenfluramine with lesser amounts of a novel metabolite, N-hydroxynordexfenfluramine, and ketone and alcohol derivatives being formed. Eadie-Hofstee plots (v against v/[s]) of nordexfenfluramine formation between 1 and 1000 microM substrate concentration were biphasic in three of four liver microsome samples examined, with mean Km values of 3 and 569 microM for the high and low affinity enzymes, respectively. At a substrate concentration (0.5 microM) around the known therapeutic plasma concentration, there was negligible inhibition of microsomal dexfenfluramine N-dealkylation by sulphaphenazole and ketoconazole, but between 33 and 100% inhibition by quinidine, and 0-58% inhibition by 7,8-naphthoflavone in seven liver samples. In human liver microsomes, there was also a significant correlation (rs= 0.79, n = 10, P < 0.01) between dextromethorphan O-demethylation and dexfenfluramine (at 1 microM) N-dealkylation activities. Dexfenfluramine was a specific inhibitor (IC50 46 microM) of CYP2D6-mediated dextromethorphan O-demethylation in human liver microsomes but did not appreciably inhibit six other cytochrome P450 isoform-selective activities for CYP1A2, 2A6, 2C9, 2C19, 2E1 and 3A activities in human liver microsomes. Yeast-expressed recombinant human CYP2D6 metabolized dexfenfluramine with high affinity (Km 1.6 microM, Vmax 0.18 nmol min(-1) nmol P450(-1)) to nordexfenfluramine which was the sole product observed. Recombinant CYP1A2 was a lower affinity enzyme (Km 301 microM, Vmax 1.12 nmol min(-1) nmol P450(-1)) and produced nordexfenfluramine with small amounts of N-hydroxynordexfenfluramine. This is the first detailed study to examine the in-vitro metabolism of dexfenfluramine in human liver microsomes and by recombinant human P450s. We were able to identify CYP2D6 (high affinity) and CYP1A2 (low affinity) as the major enzymes catalysing the N-dealkylation of dexfenfluramine in human liver microsomes.

Cytochrome P-450 CYP1A2↗