Determination of a quantitative relationship between hepatic CYP3A5*1/*3 and CYP3A4 expression for use in the prediction of metabolic clearance in virtual populations.

The creation of virtual populations allows the estimation of pharmacokinetic parameters, such as metabolic clearance in extreme individuals rather than the 'average human'. Prediction of variability in metabolic clearance within genetically diverse populations relies on understanding the covariation in the expression of enzymes. A number of statistically significant positive correlations have been observed in the hepatic expression of cytochrome P450 drug metabolising enzymes. However, these rarely provided a quantitative description of the relationships which is required in creating virtual populations. Collation of data from 40 human liver microsomal samples in the current study indicated a significant positive relationship between hepatic microsomal CYP3A5*1/*3 and CYP3A4 content. Having developed a model describing the relationship between hepatic CYP3A4 and CYP3A5*1/*3, the Simcyp Population-based Simulator(®) was used to investigate the consequences of either incorporating or ignoring the relationship between the two enzymes on estimates of drug clearance. Simulations indicated that for a compound with greater metabolism by CYP3A5 than CYP3A4, such as tacrolimus, incorporation of the correlation between CYP3A4 and CYP3A5 does have an impact on the prediction of oral clearance. Failure to consider the relationship between CYP3A4 and CYP3A5 when creating the virtual population led to a 32% lower estimate of oral clearance in individuals possessing both the CYP3A5*1/*3 genotype and high basal concentrations of CYP3A4. Potential clinical implications may include an inadequate dose estimation during clinical study design, the consequences of which may include organ rejection in transplant recipients using immunosuppressants such as tacrolimus or toxicity due to elevated concentrations of circulating metabolites.

Characterisation of zuclopenthixol metabolism by in vitro and therapeutic drug monitoring studies.

OBJECTIVE: Zuclopenthixol pharmacokinetics is incompletely characterised. We investigated potential interactions mediated through cytochrome P450 enzymes. METHOD: In vitro, we examined the impact of CYP2D6 and CYP3A4 inhibitors on zuclopenthixol metabolism in microsomes from six human livers. Subsequently, we compared dose-corrected serum zuclopenthixol concentrations in 923 samples from a therapeutic drug monitoring database from patients prescribed oral (n = 490) or injected (n = 423) zuclopenthixol alone or with fluoxetine, paroxetine, levomepromazine or carbamazepine. RESULTS: In vitro fluoxetine, paroxetine, ketoconazole and quinidine all significantly inhibited zuclopenthixol metabolism. Ketoconazole and quinidine together abolished zuclopenthixol disappearance. Clinically, dose-corrected oral zuclopenthixol serum concentrations increased significantly, after adjustment, by 93%, 78% and 46% during co-treatment with fluoxetine, paroxetine and levomepromazine and decreased 67% with carbamazepine. Carbamazepine caused dose-dependent reductions in the oral zuclopenthixol concentration-dose ratio (P < 0.001), fluoxetine (P < 0.001) and paroxetine (P = 0.011) dose-dependent increases and levomepromazine an increase related to its serum concentration (P < 0.001). Results for injected zuclopenthixol were similar but not all reached statistical significance. CONCLUSION: The In vitro study suggests zuclopenthixol is metabolised primarily by CYP2D6 and CYP3A4. The clinical study supports this, demonstrating the impact of co-prescribed inhibitors or inducers. Guidelines should incorporate these interactions noting the potential for zuclopenthixol-related toxicity or treatment failure.

Determination by liquid chromatography-mass spectrometry of clomiphene isomers in the plasma of patients undergoing treatment for the induction of ovulation.

A rapid, sensitive and selective LC-MS method is described for the simultaneous determination of zuclomiphene and enclomiphene in plasma from patients undergoing treatment with clomiphene citrate for the induction of ovulation. Samples spiked with N-didesmethyltamoxifen, the internal standard, were extracted into methyl tertiary butyl ether. The compounds were separated on a Luna C(18) analytical column, and a mobile phase of methanol-water (70:30 v/v) containing 0.05% trifluoroacetic acid at a flow rate of 1ml/min. The limits of determination were 35pg/ml and 7pg/ml for zu- and enclomiphene, respectively. Within-day coefficients of variation ranged from 2.1% to 7.2%.

A six-year evaluation of methadone prescribing practices at a substance misuse treatment centre in the UK.

OBJECTIVES: To document changes in prescribing practice at a specialized substance misuse service in the UK occurring since the introduction of the 1999 UK National Guidelines on the management of drug misuse, and to explore a possible link between the length of time spent in methadone maintenance therapy (MMT) and the dosage prescribed. METHODS: A retrospective analysis of a computerized prescription database between 1996 and 2002 obtained from Sheffield Care Trust Substance Misuse Service was performed. The relationship between various measures of dosage and the length of time spent in MMT was investigated. RESULTS: In accordance with the 1999 UK National Guidelines, the proportion of injectable methadone prescribed decreased from 22% to 16%. This was offset by an increase in the prescribing of methadone elixir from 74% to 79%. The 'maximum dose' of methadone prescribed correlated significantly with patient retention, explaining 14% of the variation in time spent in MMT. CONCLUSIONS: Our findings indicate that publication of the UK National Guidelines had a measurable effect on prescribing practice at the Service. We found that a higher methadone dose is associated with increased patient retention in MMT. However, as only a maximum of 14% of the variation in the length of stay is related to methadone dose, the importance of other aspects of treatment such as counselling and rehabilitation programmes, should be considered for the successful treatment of opioid abusers.

A critical evaluation of the experimental design of studies of mechanism based enzyme inhibition, with implications for in vitro-in vivo extrapolation.

The published literature on mechanism based inhibition (MBI) of CYPs was evaluated with respect to experimental design, methodology and data analysis. Significant variation was apparent in the dilution factor, ratio of preincubation to incubation times and probe substrate concentrations used, and there were some anomalies in the estimation of associated kinetic parameters (k(inact), K(I), r). The impact of the application of inaccurate values of k(inact) and K(I) when extrapolating to the extent of inhibition in vivo is likely to be greatest for those compounds of intermediate inhibitory potency, but this also depends on the fraction of the net clearance of substrate subject to MBI and the pre-systemic and systemic exposure to the inhibitor. For potent inhibitors, the experimental procedure is unlikely to have a material influence on the maximum inhibition. Nevertheless, the bias in the values of the kinetic parameters may influence the time for recovery of enzyme activity following re-synthesis of the enzyme. Careful attention to the design of in vitro experiments to obtain accurate kinetic parameters is necessary for a reliable prediction of different aspects of the in vivo consequences of MBI. The review calls for experimental studies to quantify the impact of study design in studies of MBI, with a view to better harmonisation of protocols.

Design, synthesis and evaluation of furanocoumarin monomers as inhibitors of CYP3A4.

A number of furanocoumarins isolated from grapefruit juice have been found to inhibit CYP3A4 activity in vitro. In this study, we have designed and synthesised a range of analogues based on bergamottin to investigate the relationship between chemical structure and inhibition of CYP3A4 activity. Studies were performed using human liver microsomes and human intestinal S9 fraction, with testosterone as the marker substrate. With the exception of the coumarin and phenolic furanocoumarin derivatives, which were inactive, the alkyloxy-furanocoumarin analogues were found to inhibit CYP3A4 activity in a dose dependent manner, with observed IC50 values ranging from 0.13 +/- 0.03 to 49.3 +/- 1.9 microM. The unsaturated furan derivatives were found to exhibit time-dependent inhibition, showing a 2-, 4- and 14-fold increase in potency for 6',7'-epoxybergamottin, 6',7'-dihydroxybergamottin and bergamottin, respectively after a preincubation period of ten minutes. Reduction of the furan moiety resulted in an 11-fold decrease in inhibitory potency, suggesting that this functional group is key to the interaction between these compounds and CYP3A4.

Synthesis of 8-geranyloxypsoralen analogues and their evaluation as inhibitors of CYP3A4.

Furanocoumarins have been shown to inhibit CYP3A4 in vitro with varying degrees of potency. In this study, we report the effects of a series of novel furanocoumarins based on the naturally occurring derivative 8-geranylepoxypsoralen which has been shown to be a more potent inhibitor of CYP3A4 than its 5-position-substituted counterpart bergamottin. Compounds were designed, synthesised and tested for their ability to inhibit CYP3A4 activity in human liver microsomes using testosterone as the marker substrate. Both the saturated and unsaturated phenolic furanocoumarin derivatives were found to be inactive. However, the 8-alkyloxy-furanocoumarin analogues were shown to inhibit CYP3A4 activity in a dose dependent manner, with IC(50) values ranging from 0.78+/-0.11 to 3.93+/-0.53 microM. The reduced furan derivative dihydro-8-geranyloxypsoralen showed a 4-fold decrease in inhibitory potency, suggesting that the furan moiety plays a role in the interaction between these compounds and CYP3A4.

Development of novel furanocoumarin dimers as potent and selective inhibitors of CYP3A4.

Grapefruit juice has been found to cause an increase in the oral bioavailability of many therapeutic agents. Such interactions are believed to result from the mechanism-based inhibition of CYP3A4 activity in the intestine. Furanocoumarin dimers present in the juice have been found to be extremely potent inhibitors of CYP3A4 activity. The aim of this work was to synthesize and test a series of dimers with a view to defining the relationship between structure and inhibitory activity and establish whether they might make suitable probes of CYP3A4 activity. Eleven furanocoumarin dimers were synthesized and evaluated as inhibitors of CYP3A4 using human liver microsomes, with testosterone as the marker substrate. Four of the most potent dimers were also investigated for their effects on CYP3A4 activity in the human intestine and on five additional hepatic cytochrome P450 isoforms. The dimers showed potent dose-dependent inhibition of CYP3A4 activity in both liver and intestine; IC50 values ranged from 0.021 +/- 0.002 to 0.146 +/- 0.041 microM (mean +/- S.D. n = 3). Of the four dimers evaluated further, all showed time-dependent inhibition of CYP3A4 activity. 88Prop showed moderate inhibition of both CYP2C19 and CYP1A2 with IC50 values of 4.42 +/- 0.01 and 1.98 +/- 0.34 microM, 88Octa was found to inhibit CYP2C19 (IC50 = 3.16 +/- 0.01 microM) and 58Prop to inhibit CYP1A2 (IC50 = 2.39 +/- 0.77 microM). Minimal inhibition of CYP2D6 and CYP2C9 was observed (IC50 > 10 microM). In conclusion, all the dimers tested were extremely potent inhibitors of CYP3A4 activity. In particular, dimer 55EE was highly selective toward the enzyme, suggesting that this compound is a suitable probe for determining the contribution of CYP3A4 to drug metabolism.

Potential for drug interactions involving cytochrome P450 in patients attending palliative day care centres: a multicentre audit.

AIM: To determine the potential for drug interactions involving cytochrome P450 (CYP) in patients receiving palliative day care. METHODS: Drugs used by patients attending four specialist palliative day care centres were reviewed to identify combinations that could result in a pharmacokinetic interaction via any of the five main human forms of CYP. RESULTS: Of 160 patients, 145 (91%) were prescribed at least one drug that was a substrate, inhibitor or inducer of one of the five main CYP isoforms. Twenty-four drug combinations, involving 34 patients, could have given rise to a clinically important interaction. CONCLUSIONS: Prescribers should be aware that in this group of patients, one in five are at risk of a clinically important CYP-mediated drug interaction.

Characterization of the human cytochrome P450 forms involved in metabolism of tamoxifen to its alpha-hydroxy and alpha,4-dihydroxy derivatives.

Tamoxifen is a known hepatocarcinogen in rats and is associated with an increased incidence of endometrial cancer in patients. One mechanism for these actions is via bioactivation, where reactive metabolites are generated that are capable of binding to DNA or protein. Several metabolites of tamoxifen have been identified that appear to predispose to adduct formation. These include alpha-hydroxytamoxifen, alpha,4-dihydroxytamoxifen, and alpha-hydroxy-N-desmethyltamoxifen. Previous studies have shown that cytochrome P450 (P450) enzymes play an important role in the biotransformation of tamoxifen. The aim of our work was to determine which P450 enzymes were capable of producing alpha-hydroxylated metabolites from tamoxifen. When tamoxifen (18 or 250 microM) was used as the substrate, P450 3A4, and to a lesser extent, P450 2D6, P450 2B6, P450 3A5, P450 2C9, and P450 2C19 all produced a metabolite with the same HPLC retention time as alpha-hydroxytamoxifen at either substrate concentration tested. This peak was well-separated from 4-hydroxy-N-desmethyltamoxifen, which eluted substantially later under the chromatographic conditions used. No alpha,4-dihydroxytamoxifen was detected in incubations with any of the forms with tamoxifen as substrate. However, when 4-hydroxytamoxifen (100 microM) was used as the substrate, P450 2B6, P450 3A4, P450 3A5, P450 1B1, P450 1A1, and P450 2D6 all produced detectable concentrations of alpha,4-dihydroxytamoxifen. These studies demonstrate that multiple human P450s, including forms found in the endometrium, may generate reactive metabolites in women undergoing tamoxifen therapy, which could subsequently play a role in the development of endometrial cancer.

Mechanism-based inactivation of CYP2D6 by methylenedioxymethamphetamine.

The potency of methylenedioxymethamphetamine (MDMA) as a mechanism-based inhibitor of CYP2D6 has been defined using microsomes prepared from yeast expressing the enzyme and from three human livers. The inhibitory effect was increased by preincubation through formation of a metabolic intermediate complex. Inactivation parameters (kinact and KI), defined with respect to the O-demethylation of dextromethorphan, were 0.29 +/- 0.03 (S.E.) min(-1) and 12.9 +/- 3.6 (S.E.) microM for yeast-expressed CYP2D6, and 0.26 +/- 0.02 min(-1) and 14.4 +/- 2.5 microM, 0.15 +/- 0.01 min(-1) and 8.8 +/- 2.6 microM, and 0.12 +/- 0.05 min(-1) and 45.3 +/- 32.1 microM for the liver microsomal preparations. The rate of inactivation of CYP2D6 by MDMA decreased when quinidine, a competitive inhibitor of CYP2D6, was added to the primary incubation mixture. However, inactivation was unaffected by the addition of glutathione. The results indicate that MDMA is a potent mechanism-based inhibitor of CYP2D6, with implications for understanding its in vivo disposition and drug interaction potential.

Potential for drug interactions involving cytochromes P450 2D6 and 3A4 on general adult psychiatric and functional elderly psychiatric wards.

AIMS: To assess the potential for interactions involving cytochromes P450 2D6 (CYP2D6) and 3A4 (CYP3A4) between drugs prescribed in a city in-patient psychiatric service. METHODS: Prescription information was obtained from all 236 patients in general adult wards and all 87 patients in functional elderly wards under a city psychiatric service. The frequencies with which combinations of drugs expected to interact via CYP2D6 or CYP3A4 were documented and compared between these two settings. RESULTS: All 2089 drug prescriptions, of which 1237 (59%) were administered, were analyzed. One hundred and seventy-two patients (73%) on adult wards and 59 (68%) on functional elderly wards were prescribed at least one drug metabolized by and/or inhibiting CYP2D6, the difference being nonsignificant (95% confidence interval on the difference -6.3%, 16.4%). Anticipated interactions from 62/82 CYP2D6-related combinations prescribed on adult wards (27/100 patients) and 19/30 prescribed to elderly patients (22/100 patients) were judged to be clinically important or potentially clinically important. The proportion of patients on functional elderly wards prescribed at least one drug interacting with CYP3A4 (87%) was significantly greater than that for patients on adult wards (57%, P < 0.001). The frequency of interactions involving CYP3A4 was significantly greater on functional elderly than adult wards (43/100 vs 22/100 patients, P < 0.025, 95% confidence interval on the difference 4, 38/100). CONCLUSIONS: Our findings confirm extensive polypharmacy on general adult psychiatric and functional elderly psychiatric wards. A substantial proportion of patients were receiving combinations of drugs that interact with CYP2D6 and/or CYP3A4, many of which are known to produce clinically important interactions. Doctors practising in old age psychiatry should be aware that patients on functional elderly wards are at increased risk of clinically important CYP3A4 interactions. Psychiatrists should consider the pharmacokinetic implications of drugs prescribed for use 'as needed', because of the potential for unpredictable interactions.

Biotransformation of chlorpyrifos and diazinon by human liver microsomes and recombinant human cytochrome P450s (CYP).

The cytochrome P450 (CYP)-mediated biotransformation of the organophosphorothioate insecticides chlorpyrifos and diazinon was investigated. Rates of desulphuration to the active oxon metabolite (chlorpyrifos-oxon and diazinon-oxon) and dearylation to non-toxic hydrolysis products were determined in human liver microsome preparations from five individual donors and in recombinant CYP enzymes. Chlorpyrifos and diazinon underwent desulphuration in human liver microsome with mean Km = 30 and 45 microM and V(max) = 353 and 766 pmol min(-1) mg(-1), respectively. Dearylation of these compounds by human liver microsome proceeded with Km = 12 and 28 microM and V(max) = 653 and 1186 pmol min(-1) mg(-1), respectively. The apparent intrinsic clearance (V(max)/Km) of dearylation was 4.5- and 2.5-fold greater than desulphuration for chlorpyrifos and diazinon, respectively. Recombinant human CYP2B6 possessed the highest desulphuration activity for chlorpyrifos, whereas CYP2C19 had the highest dearylation activity. In contrast, both desulphuration and dearylation of diazinon were catalysed at similar rates, in the rank order CYP2C19 > CYP1A2 > CYP2B6 > CYP3A4. Both organophosphorothioates were more readily detoxified (dearylation) than bioactivated (desulphuration) in all human liver microsome preparations. However, the role of individual CYP enzymes in these two biotransformation pathways varied according to the structure of the organophosphorothioate, which was reflected in different activation/detoxification ratios for chlorpyrifos and diazinon. Variability in activity of individual CYP enzymes may influence interindividual sensitivity to the toxic effects of chlorpyrifos and diazinon.

A discordance between cytochrome P450 2D6 genotype and phenotype in patients undergoing methadone maintenance treatment.

AIMS: To assess CYP2D6 activity and genotype in a group of patients undergoing methadone maintenance treatment (MMT). METHODS: Blood samples from 34 MMT patients were genotyped by a polymerase chain reaction-based method, and results were compared with CYP2D6 phenotype (n = 28), as measured by the molar metabolic ratio (MR) of dextromethorphan (DEX)/dextrorphan (DOR) in plasma. RESULTS: Whereas 9% of patients (3/34) were poor metabolizers (PM) by genotype, 57% (16/28) were PM by phenotype (P < 0.005). Eight patients, who were genotypically extensive metabolizers (EM), were assigned as PM by their phenotype. The number of CYP2D6*4 alleles and sex were significant determinants of CYP2D6 activity in MMT patients, whereas other covariates (methadone dose, age, weight) did not contribute to variation in CYP2D6 activity. CONCLUSIONS: There was a discordance between genotype and in vivo CYP2D6 activity in MMT patients. This finding is consistent with inhibition of CYP2D6 activity by methadone and may have implications for the safety and efficacy of other CYP2D6 substrates taken by MMT patients.

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.

Determination of midazolam and 1'-hydroxymidazolam by liquid chromatography-mass spectrometry in plasma of patients undergoing methadone maintenance treatment.

A rapid, sensitive and selective LC-MS method was developed for the simultaneous determination of midazolam (MDZ) and 1'-hydroxymidazolam (1'-OHMDZ) in plasma taken from 54 patients undergoing methadone maintenance therapy, most of whom were multidrug users. Samples spiked with prazepam, the internal standard, and were extracted into diethyl ether. Compounds were separated on a Phenomenex Luna C(18) column and a mobile phase of acetonitrile-ammonium acetate buffer (10 mM, pH 4.7) (52:48, v/v) at a flow-rate of 1 ml/min. The limit of detection was 0.65 and 0.68 (ng/ml) for MDZ and 1'-OHMDZ, respectively. Within-day relative standard deviations were less than 8%.

Influence of phenylalanine-481 substitutions on the catalytic activity of cytochrome P450 2D6.

Homology models of the active site of cytochrome P450 2D6 (CYP2D6) have identified phenylalanine 481 (Phe(481)) as a putative ligand-binding residue, its aromatic side chain being potentially capable of participating in pi-pi interactions with the benzene ring of ligands. We have tested this hypothesis by replacing Phe(481) with tyrosine (Phe(481)-->Tyr), a conservative substitution, and with leucine (Phe(481)-->Leu) or glycine (Phe(481)-->Gly), two non-aromatic residues, and have compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived protein. The Phe(481)-->Tyr substitution did not alter the kinetics [K(m) (microM) and V(max) (pmol/min per pmol) respectively] of oxidation of S-metoprolol (27; 4.60), debrisoquine (46; 2.46) or dextromethorphan (2; 8.43) relative to the respective wild-type values [S-metoprolol (26; 3.48), debrisoquine (51; 3.20) and dextromethorphan (2; 8.16)]. The binding capacities [K(s) (microM)] of a range of CYP2D6 ligands to the Phe(481)-->Tyr enzyme (S-metoprolol, 22.8; debrisoquine, 12.5; dextromethorphan, 2.3; quinidine, 0.13) were also similar to those for the wild-type enzyme (S-metoprolol, 10.9; debrisoquine, 8.9; dextromethorphan, 3.1; quinidine, 0.10). In contrast, the Phe(481)-->Leu and Phe(481)-->Gly substitutions increased significantly (3-16-fold) the K(m) values of oxidation of the three substrates [S-metoprolol (120-124 microM), debrisoquine (152-184 microM) and dextromethorphan (20-31 microM)]. Similarly, the K(s) values of the ligands to Phe(481)-->Leu and Phe(481)-->Gly mutants were also increased 3 to 10-fold (S-metoprolol, 33.2-41.9 microM; debrisoquine, 85-90 microM; dextromethorphan, 15.7-18.8 microM; quinidine 0.35-0.53 microM). However, contrary to a recent proposal that Phe(481) has the dominant role in the binding of substrates that undergo CYP2D6-mediated N-dealkylation routes of metabolism, the Phe(481)-->Gly substitution did not substantially decrease the capacity of the enzyme to N-deisopropylate metoprolol (wild-type, 1.12 pmol/min per pmol of P450; Phe(481)-->Gly, 0.71), whereas an Asp(301)-->Gly substitution decreased the N-dealkylation reaction by 95% of the wild-type rate. Overall, our results are consistent with the proposal that Phe(481) is a ligand-binding residue in the active site of CYP2D6 and that the residue interacts with ligands via a pi-pi interaction between its phenyl ring and the aromatic moiety of the ligand. However, the relative importance of Phe(481) in binding is ligand-dependent; furthermore, its importance is secondary to that of Asp(301). Finally, contrary to predictions of a recent homology model, Phe(481) does not seem to have a primary role in CYP2D6-mediated N-dealkylation.

Parkinson's disease and CYP1A2 activity.

UNLABELLED: AIMS MPTP, a neurotoxin which induces parkinsonism is partially metabolized by the enzyme CYP1A2. Smoking appears to protect against Parkinson's disease (PD) and cigarette smoke induces CYP1A2 activity. Thus, we investigated the hypothesis that idiopathic PD is associated with lower CYP1A2 activity using caffeine as a probe compound. METHODS CYP1A2 activity was assessed using saliva paraxanthine (PX) to caffeine (CA) ratios. Caffeine half-life was also estimated from salivary concentrations of caffeine at 2 and 5 h post dose. 117 treated and 40 untreated patients with PD and 105 healthy control subjects were studied. RESULTS PX/CA ratios were 0. 57, 0.93 and 0.77 in treated patients, untreated patients and healthy control subjects, respectively, with no significant differences between study groups (95% CI: treated patients vs controls -0.24, 0.57; untreated patients vs controls -0.75, 0.35). However, patients with PD (treated or untreated) had caffeine half-lives shorter than that in controls (treated patients: 262 min, untreated patients: 244 min, controls: 345 min; 95% CI: controls vs treated patients 23, 143 (P = 0.003); controls vs untreated patients 19, 184 (P = 0.011)). Amongst the patients with PD, caffeine half-life was also inversely related to the age of onset of disease (P = 0.012); gender and concomitant drugs did not influence this significantly. CONCLUSIONS: Based on PX/CA ratio, there was no evidence of decreased CYP1A2 activity in patients compared with control subjects. The observed decrease in the elimination half-life of caffeine in PD may be caused by increased CYP2E1 activity, an enzyme that also contributes to the metabolism of caffeine. The latter warrants further investigation.

The 1'-hydroxylation of Rac-bufuralol by rat brain microsomes.

The 1'-hydroxylation of rac-bufuralol, which is catalyzed by polymorphic CYP2D6 in humans, was studied in brain microsomes from male and female Wistar rats and from the female Dark Agouti rat, a model of the CYP2D6 poor metabolizer phenotype. The kinetics of the 1'-hydroxylation of bufuralol (1-1500 microM) by brain microsomes were biphasic. The activity of the high-affinity site of metabolism was consistent with Michaelis-Menten kinetics (apparent K(m1) = 0. 61-1.42 microM, V(max1) = 4.3-4.8 fmol/min/mg of protein), whereas the low-affinity activity was better described by a Hill function (K(50%(2)) = 253-258 microM, V(max2) = 817-843 fmol/min/mg of protein, n = 1.2-1.3). Values for kinetic constants were similar in all rat strains. Quinine was only a weak inhibitor of both the high- (apparent K(i) = 90 microM) and low-affinity (210 microM) sites of metabolism. In contrast, the kinetics of 1'-hydroxylation of bufuralol by rat liver microsomes were best described by a two-site Michaelis-Menten function. V(max) values were 3 to 5 orders of magnitude greater compared with those for brain microsomes (male and female Wistar), and liver microsomes from female Dark Agouti rats were significantly less active than those from Wistar rats. These data, together with the known potent inhibitory effect of quinine on bufuralol 1'-hydroxylation by rat liver microsomes, indicate tissue-specific differences in the enzymology of this reaction. The role of brain CYP2D enzymes remains to be clarified.