Genome-wide association study reveals a complex genetic architecture underpinning-induced CYP3A4 enzyme activity.

Atypical cytochrome P450 3A4 (CYP3A4) enzyme activity-induced and inhibited-is thought to be the driver of numerous poor or adverse therapeutic responses to up to 50 % of all commonly prescribed drugs. We carried out a genome-wide association study to identify common genetic variants associated with variation in induced CYP3A4 activity. A total of 310 twins were included in this study. Each participant had already completed a 14 days course of St John's Wort to induce CYP3A4, which was quantified through the metabolic ratio of exogenous 3-hydroxyquinine to quinine. We failed to detect any genome-wide significant associations (P < 1 × 10(-8)) with variation in induced CYP3A4 activity although several genomic regions were highlighted which may play minor roles. We report the first GWAS of variation in induced CYP3A4 activity and our preliminary results indicate a complex genetic architecture underpinning induced CYP3A4 enzyme activity.

Genetic epidemiology of induced CYP3A4 activity.

AIM: The cytochrome P450 3A4 (CYP3A4) enzyme is implicated in the metabolism of more than 50% of all prescribed medications and its activity - including induced or inhibited activity - is deemed to be a crucial determinant of interindividual variability in drug disposition, poor therapeutic efficacy, and adverse response to medication. METHODS: We used the classical twin model in conjunction with an induction experiment to uncover the relative contribution of genetic and environmental factors to interindividual variation in induced CYP3A4 activity. A total of 367 healthy twins participated in the study. Each volunteer was administered a potent inducer of CYP3A4 (St John's Wort) for 14 days and the activity of CYP3A4 was quantified through the metabolism of the exogenously administered probe drug quinine sulfate. RESULTS: Baseline and induced CYP3A4 activity were highly variable with a seven-fold and 11-fold difference among our population, respectively. Alcohol consumption, BMI, and smoking were significantly associated with induced CYP3A4 activity, collectively explaining 20% of the variation (P<1×10(-4)). The narrow-sense heritability of induced CYP3A4 activity was estimated at 66%, whereas the remainder of the variation was attributed to unique environmental factors. CONCLUSION: To our knowledge, this is the first genetic epidemiological study of induced CYP3A4 activity. Our results motivate further research to identify common and rarer genetic variants that underpin the heritable component of variation in induced CYP3A4 activity.

Rapid quantification of quinine and its major metabolite (3S)-3-hydroxyquinine in diluted urine by UPLC-MS/MS.

A rapid UPLC-MS/MS quantitative assay for the quantification of quinine and (3S)-3-hydroxyquinine requiring minimal sample pre-treatment - dilute-and-shoot type approach - has been developed. The assay was run at 0.6mL/min using gradient elution with (pH 10; 10mM) ammonium bicarbonate and methanol with a total cycle time of 2.5min on a 50mm×2.1mm ID, 1.7µm Acquity BEH column. Peak shapes were highly symmetrical allowing for accurate peak integration. Calibration curves for both analytes were constructed from 1.00 to 20.00ng/mL, yielding R(2) values >0.995. Intra- and inter-batch assay precision and accuracy were evaluated using 6 injections of QC solutions on 3 separate days (n=18) and were found to be within ±10% and 90-110% respectively. The method was shown to be suitable for quantitatively determining the ratio of quinine to (3S)-3-hydroxyquinine for a cohort of samples from an epidemiological study.

Resonance light scattering study on the interaction between quinidine sulfate and congo red and its analytical application.

The interaction between quinidine sulfate (QDS) and congo red (CR) was studied using resonance light scattering (RLS) technique, ultraviolet-visual spectrophotometry and fluorimetry. In weak acidic medium, QDS reacts with CR to form a supermolecular complex which results in the enhanced RLS intensity. Some important interacting parameters, such as the solution acidity and CR concentration, salt effect and addition order of the reagents, were investigated and optimized. Under the optimum conditions, it was found that the enhanced RLS intensity was in proportion to the concentration of QDS in the range 0.2-8.4 microg mL(-1). The corresponding detection limit was 12.0 ng mL(-1). The results showed that this new method enabled simple, sensitive and rapid determination of QDS and was used for the determination of QDS in urine and simulated huamn serum samples.

Metabolism and elimination of quinine in healthy volunteers.

OBJECTIVES: The aims were to investigate: (1) The renal elimination of quinine and its metabolites 3-hydoxyquinine, 2'-quininone, (10R) and (10S)-11-dihydroxydihydroquinine and (2) the relative importance of CYP3A4, CYP1A2 and CYP2C19 for the formation of 2'-quininone, (10R) and (10S)-11-dihydroxydihydroquinine in vivo. METHODS: In a randomised three-way crossover study, nine healthy Swedish subjects received a single oral dose of quinine hydrochloride (500 mg), on three different occasions: (A) alone, (B) concomitantly with ketoconazole (100 mg twice daily for 3 days) and (C) concomitantly with fluvoxamine (25 mg twice daily for 2 days). Blood and urine samples were collected before quinine intake and up to 96 h thereafter. All samples were analysed by means of high-performance liquid chromatography. RESULTS: Co-administration with ketoconazole significantly increased the area under the plasma concentration versus time curve (AUC) of 2'-quininone, (10S)-11-dihydroxydihydroquinine, and (10R)-11-dihydroxydihydroquinine, the geometric mean ratios (90% CI) of the AUC were 1.9 (1.8, 2.0), 1.3 (1.1, 1.7) and 1.6 (1.4, 1.8), respectively. Co-administration with fluvoxamine had no significant effect on the mean AUC of any of the metabolites. A mean of 56% of the administered oral quinine dose was recovered in urine after hydrolysis with beta-glucuronidase relative to the 40% recovered before hydrolysis. CONCLUSION: Quinine is eliminated in urine mainly as unchanged drug and as 3-hydroxyquinine. The major metabolite of quinine is 3-hydroxyquinine formed by CYP3A4. There is no evidence for the involvement of CYP3A4, 1A2 or 2C19 in the formation of 2'-quininone, (10S)-11-dihydroxydihydroquinine and (10R)-11-dihydroxydihydroquinine in vivo. Glucuronidation is an important pathway for the renal elimination of quinine, mainly as direct conjugation of the drug.

Quinine 3-hydroxylation as a biomarker reaction for the activity of CYP3A4 in man.

OBJECTIVE: To investigate the usefulness of the 3-hydroxylation of quinine as a biomarker reaction for the activity of CYP3A4 in man and to study the interindividual variation in the metabolic ratio (MR), i.e. quinine/3-hydroxyquinine. METHODS: Data from a previous study (A) was used for determination of the MR of quinine in plasma and urine at different time points. In study B, 24 healthy Swedish subjects received 250 mg quinine hydrochloride first alone and later together with four other CYP probe drugs [losartan (CYP2C9), omeprazole (CYP2C19), debrisoquine (CYP2D6) and caffeine (CYP1A2)] administered on the same day. Plasma and urine samples were collected before quinine intake and 16 h thereafter and analysed for quinine and 3-hydroxyquinine using high-performance liquid chromatography. Plasma and/or urine were collected for the other probes at different time points. MRs of all the probes were determined and correlations to quinine MR were studied. RESULTS: In study A, the MR in plasma was stable over 96 h. The ratio increased from 5.8 to 12.2 (P=0.006) during co-administration with ketoconazole, whereas no significant difference (P=0.76) was observed during co-administration with fluvoxamine (from 5.8 to 6.0). In study B, there was no significant difference (P=0.36) between the mean MRs when quinine was given alone (4.7) or together with the four other drugs (4.5). There was a significant correlation between the MR of quinine and omeprazole sulphone formation (r=0.52, P<0.01), but not to the MRs of the other probes. There was a fivefold interindividual variability in the MR. CONCLUSIONS: The MR of quinine in plasma or urine may serve as a stable measure of the activity of CYP3A4 in man. These results together with in vitro data show that quinine is also a specific CYP3A4 probe.

Simultaneous determination of quinine and four metabolites in plasma and urine by high-performance liquid chromatography.

The determination of quinine, (3S)-3-hydroxyquinine, 2'-quininone and (10R)- and (10S)-10,11-dihydroxydihydroquinine in plasma and urine samples is described. This is the first time the R and S configurations have been correctly assigned to the two metabolites of 10,11-dihydroxyquinine. One hundred microliter-plasma samples were protein precipitated with 200 microl cold methanol. Urine samples were 10-100 x diluted and then directly injected into the HPLC. A reversed-phase liquid chromatography system with fluorescence detection and a Zorbax Eclipse XDB phenyl column and gradient elution was used. The within and between assay coefficients of variation of the method for quinine and its metabolites in plasma and urine was less than 13%. The lower limit of quantitation was in the range of 0.024-0.081 microM.

Capillary electrophoretic separation, immunochemical recognition and analysis of the diastereomers quinine and quinidine and two quinidine metabolites in body fluids.

The capillary electrophoretic separation and immunochemical recognition of the two naturally fluorescing, cationic diastereomers quinine (QN) and quinidine (QD), their hydroderivatives and two major QD metabolites (3-hydroxyquinidine and quinidine-N-oxide) was investigated. Plain aqueous phosphate buffers and an alkaline buffer containing dodecyl sulfate micelles are shown to be incapable of resolving the two diastereomers. However, incorporation of an additional chemical equilibrium (with beta-cyclodextrin) in the case of capillary zone electrophoresis (CZE) and the presence of a small amount of an organic solvent as buffer modifier (2-propanol) in dodecyl sulfate based micellar electrokinetic capillary chromatography (MECC), were found to provide separation media which lead to complete resolution of QN, QD and the other compounds of interest. Furthermore, for MECC- and CZE-based immunoassay formats, a commercially available antibody against QD was found to be a perfect discriminator between QD and QN. It was determined to recognize QD and the two QD metabolites (cross reactivity of 20--30%) but not QN. MECC and CZE with laser induced fluorescence (LIF) detection are shown to be suitable to determine QD and metabolites in urine and plasma (quinidine-N-oxide only) collected after single dose intake of 50 mg QD sulfate and of QN in urine, saliva and serum samples that were collected after self-administration of 0.5 l of quinine water (25 mg of QN). With direct injection of a body fluid, MECC with LIF was found to provide 10 ng/ml detection limits for QD and QN. This ppb sensitivity is comparable to that obtained in HPLC assays that are based upon drug extraction. Furthermore, MECC and CZE assays with UV detection are shown to provide the ppm sensitivity required for therapeutic drug monitoring and clinical toxicology of QD and QN.

Quinidine as a probe for CYP3A4 activity: intrasubject variability and lack of correlation with probe-based assays for CYP1A2, CYP2C9, CYP2C19, and CYP2D6.

BACKGROUND: In vitro studies have shown that the formation of 3-hydroxyquinidine from quinidine is catalyzed almost exclusively by CYP3A4. In vivo this result has been supported in various interaction studies, and the use of this reaction as an in vivo biomarker reaction of CYP3A4 activity has been suggested. We studied the possible correlation of the formation clearance of 3-hydroxyquinidine with probe-based assays for CYP1A2, CYP2C9, CYP2C19, and CYP2D6. Descriptive analyses of the outcome of various biomarker reactions were performed. METHODS: Forty-two healthy, young male volunteers participated in an open study consisting of two identical test periods separated by a 12- to 14-week washout period. In each period biomarker reactions of CYP1A2 (caffeine), CYP2C9 (tolbutamide), CYP2C19 (mephenytoin), CYP2D6 (sparteine), CYP3A4 (urinary excretion of 6beta-hydroxycortisol), as well as the pharmacokinetics of quinidine after a 200-mg single oral dose of quinidine sulfate were studied. RESULTS: The median formation clearance of 3-hydroxyquinidine were 2.40 and 2.33 L/h in the two test periods. As measured by the formation clearance of 3-hydroxyquinidine, the intraindividual coefficient of variation for CYP3A4 activity was 18%, whereas the interindividual activity varied fourfold. The formation clearance of 3-hydroxyquinidine did not correlate with the outcome of indexes for activities of CYP1A2, CYP2C9, CYP2C19, or CYP2D6 or the urinary excretion of 6beta-hydroxycortisol. The formation clearance of 3-hydroxyquinidine correlated well to point values of 3-hydroxyquinidine to quinidine ratios in plasma and urine. CONCLUSION: The formation clearance of 3-hydroxyquinidine after a single oral dose of 200 mg quinidine sulfate may represent a useful index of CYP3A4 activity in vivo.

Grapefruit juice has no effect on quinine pharmacokinetics.

OBJECTIVE: As quinine is mainly metabolised by human liver CYP3A4 and grapefruit juice inhibits CYP3A4, the effect of grapefruit juice on the pharmacokinetics of quinine following a single oral dose of 600 mg quinine sulphate was investigated. METHODS: The study was carried out in ten healthy volunteers using a randomised cross-over design. Subjects were studied on three occasions, with a washout period of 2 weeks. During each period, subjects received a pretreatment of 200 ml orange juice (control), full-strength grapefruit juice or half-strength grapefruit juice twice daily for 5 days. On day 6, the subjects were given a single oral dose of 600 mg quinine sulphate with 200 ml of one of the juices. Plasma and urine samples for measurement of quinine and its major metabolite, 3-hydroxyquinine, were collected over a 48-h period and analysed by means of a high-performance liquid chromatography method. RESULTS: The intake of grapefruit juice did not significantly alter the oral pharmacokinetics of quinine. There were no significant differences among the three treatment periods with regard to pharmacokinetic parameters of quinine, including the peak plasma drug concentration (Cmax), the time to reach Cmax (tmax), the terminal elimination half-life (t1/2), the area under the concentration-time curve and the apparent oral clearance. The pharmacokinetics of the 3-hydroxyquinine metabolite were slightly changed when volunteers received grapefruit juice. The mean Cmax of the metabolite (0.25+/-0.09 mg l(-1), mean +/- SD) while subjects received full-strength grapefruit juice was significantly less than during the control period (0.31+/-0.06 mg l(-1), P < 0.05) and during the intake of half-strength grapefruit juice (0.31+/-0.07 mg l(-1), P < 0.05). CONCLUSION: These results suggest that there is no significant interaction between the parent compound quinine and grapefruit juice, so it is not necessary to advise patients against ingesting grapefruit juice at the same time that they take quinine. Since quinine is a low clearance drug with a relatively high oral bioavailability, and is primarily metabolised by human liver CYP3A4, the lack of effect of grapefruit juice on quinine pharmacokinetics supports the view that the site of CYP inhibition by grapefruit juice is mainly in the gut.

High-performance liquid chromatographic method for the determination of the major quinine metabolite, 3-hydroxyquinine, in plasma and urine.

The determination of 3-hydroxyquinine in urine and plasma samples is described. Extraction was performed using a mixture of toluene-butanol (75:25, v/v), followed by back-extraction into the mobile phase, which consisted of 0.1 M phosphate buffer, acetonitrile, tetrahydrofuran and triethylamine. A reversed-phase liquid chromatography system with fluorescence detection and a CT-sil C18 column were used. The within-assay coefficient of variation of the method was 2% at the higher concentration values in plasma, 2.95 microM, 4% at 227 nM and 9% at the lower limit of quantitation, 4.5 nM. In urine, the coefficient of variation was 11% at the lower concentration, 227 nM and was 3% at 56.8 microM. The between-assay coefficient of variation was 4% at the low concentration (5.1 nM) in plasma, 2% at 276.8 nM and 3% at 1.97 microM. In urine, the between assay coefficient of variation was 4% at 204.6 nM, 3% at 5.12 microM and 2% at 56.8 microM.

Modification of a tunable UV-visible capillary electrophoresis detector for simultaneous absorbance and fluorescence detection: profiling of body fluids for drugs and endogenous compounds.

Using fused-silica optical fibres for fluorescence light collection and bandpass filters for selection of emission wavelengths, a capillary electrophoresis detection cell of a conventional, tunable UV-Vis absorbance detector was adapted for simultaneous fluorescence (at selected emission wavelength) and absorbance (at selected excitation wavelength) detection. Detector performance is demonstrated with the monitoring of underivatized fluorescent compounds in body fluids by micellar electrokinetic capillary chromatography with direct sample injection. Compared with UV absorption detection, fluorescence detection is shown to provide increased selectivity and for selected compounds also up to tenfold higher sensitivity. Examples studied include screening for urinary indole derivatives (tryptophan, 5-hydroxytryptophan, tyrosine, 3-indoxyl sulfate and 5-hydroxyindole-3-acetic acid) and catecholamine metabolites (homovanillic acid and vanillylmandelic acid) and the monitoring of naproxen in serum, quinidine in serum and urine and of salicylate and its metabolites in serum and urine.

Determination of quinidine, dihydroquinidine, (3S)-3-hydroxyquinidine and quinidine N-oxide in plasma and urine by high-performance liquid chromatography.

A specific and sensitive method for the quantitation of quinidine, (3S)-3-hydroxyquinidine, quinidine N-oxide, and dihydroquinidine in plasma and urine has been developed. The method is based on a single-step, liquid-liquid extraction procedure, followed by isocratic reversed-phase high-performance liquid chromatography, with fluorescence detection. After extraction from 250 microliters plasma and 100 microliters urine, the limit of determination is 10 nM and 25 nM, respectively. For the use as standards, commercially available quinidine was purified from dihydroquinidine; quinidine N-oxide was synthesized.

Column liquid chromatographic analysis of quinine in human plasma, saliva and urine.

A new simple, selective and reproducible high-performance liquid chromatographic method for the determination of quinine in plasma, saliva and urine is described. The ion-pair method was carried out on a reversed-phase C18 column, using perchlorate ion as the counter ion and ultraviolet detection at 254 nm. Quinine was well resolved from its major metabolite, 3-hydroxyquinine, and the internal standard, primaquine. The limit of detection was 10 ng/ml and the recovery was greater than 90% from the three biological fluids.

Quinidine inhibition of debrisoquine S(+)-4- and 7-hydroxylations in Chinese of different CYP2D6 genotypes.

Pronounced differences in the CYP2D6 gene between Chinese and Caucasians have previously been described. There was a low frequency of detrimental mutations in the Chinese CYP2D6 gene causing the poor metabolizer (PM) phenotype. In contrast to Caucasians where the Xba I 44 kb allele is almost always associated with the PM phenotype, Chinese with the 44/44 kb RFLP pattern are extensive metabolizers (EM). In order to evaluate whether the debrisoquine hydroxylation seen in subjects with this haplotype is catalysed by a functionally similar enzyme to CYP2D6 or is catalysed by another type of P450 isozyme, product selectivity of the 4-hydroxylation was studied in 27 Chinese. The inhibition of CYP2D6 by quinidine was also investigated. In the 26 Chinese EM the S(+)-4-hydroxy enantiomer was found to be the major urinary metabolite of debrisoquine with an enantiomeric excess of 96.8-100%, which is similar to that in Caucasians. A correlation between the amount of S(+)-4-hydroxy and the minor 7-hydroxy metabolites excreted in urine (r = 0.72; p < 0.001) was seen. The amount of these two metabolites excreted was less in Chinese EM of debrisoquine with the 44/44 kb RFLP pattern, than in those with the wild type 29/29 kb pattern (p < 0.01). The stereoselectivity was very high in both groups. All Chinese homozygous for the 44 kb fragment (n = 5) were transformed to apparent PM after a single 100 mg dose of quinidine similarly to five Caucasian EM. Both the S(+)-4- and 7-hydroxylations of debrisoquine were inhibited by quinidine in both populations. This study shows that the cytochrome P450 catalysing the 4- and 7-hydroxylations of debrisoquine in Chinese EM has the same properties (product stereoselectivity and inhibition by quinidine) as the CYP2D6 in Caucasian EM.

Characterization of the principal metabolite of quinine in human urine by 1H-n.m.r. spectroscopy.

1. The major metabolite of quinine in human urine, which is also the sole metabolite in human plasma and saliva, has been identified and characterized by chemical ionization mass spectrometry and 1H-n.m.r. spectrometry. 2. The mass spectrum showed that an oxygen atom is incorporated in the quinuclidine nucleus, and the exact position of the oxidation was established from the n.m.r. spectrum to be at the C-3 position.

Quinidine inhibits in vivo metabolism of amphetamine in rats: impact upon correlation between GC/MS and immunoassay findings in rat urine.

Amphetamine is metabolized by cytochrome P-450 (P450) to p-hydroxyamphetamine and phenylacetone in mammalian species. P450 metabolism is affected by genetic polymorphisms and by xenobiotic interactions in an isozyme-specific fashion. Little is known concerning the isozyme selectivity of amphetamine metabolism. Quinidine selectively inhibits the debrisoquine-specific isozyme (P450db) which displays genetic polymorphism in humans and rats. We now report the effect of quinidine on the metabolism of amphetamine to p-hydroxyamphetamine in vivo. At 0 h male Lewis rats received (po): no treatment (I), 80 mg quinidine/kg in 50% ethanol (II), or 50% ethanol (III), followed at 2 h by 15 mg d-amphetamine sulfate/kg (po). Urine specimens were collected and pooled at 0, 24, and 48 h. Amphetamine and p-hydroxyamphetamine concentrations were determined using a new GC/MS method for simultaneous quantitation. The ethanol vehicle-control (III) had no significant effect on amphetamine metabolism. Quinidine pretreatment (II) resulted in a significant decrease in the excretion of p-hydroxyamphetamine at 24 and 48 h to 7.2 and 24.1% of the vehicle-control levels, respectively, accompanied by a significant increase in amphetamine excretion between 24 and 48 h to 542% of the control. These data show that quinidine inhibits in vivo metabolism of amphetamine in rats and suggest that amphetamine metabolism may, in part, be mediated by an isozyme of P450 which displays genetic polymorphism. The inhibition of amphetamine metabolism results in an increased ratio of parent drug to metabolite concentration (metabolic ratio) in the urine, which mimics the effect of genetic polymorphisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Quinidine oxidative metabolism. Identification and biosynthesis of quinidine 10,11-dihydrodiol stereoisomers.

The isocratic reversed phase high performance liquid chromatographic method proposed for quinidine metabolic studies facilitates particularly the separation of 10(R) and (S) isomers of quinidine 10,11-dihydrodiols. The finding of each of these forms following a new synthetic pathway allows us to identify and quantify them in biological fluids. These two isomers have especially been observed in rat bile and hepatocyte secretions. The metabolic inducing effect of phenobarbital on the oxidative metabolism of quinidine is verified in rat isolated hepatocytes. Simultaneous secretion of the two dihydrodiols is also verified in human urine by a gas chromatography/mass spectrometry procedure.

Quinidine kinetics after a single oral dose in relation to the sparteine oxidation polymorphism in man.

The kinetics at a single oral dose (400 mg) of quinidine were studied in four extensive metabolizers (EM) and four poor metabolizers (PM) of sparteine. The clearance of quinidine by 3-hydroxylation was significantly lower in PM than in EM, but the difference was small (25-30%). This finding suggests that 3-hydroxylation, in part, is catalyzed by the same isoenzyme of cytochrome P450, P450db1 which oxidizes sparteine. Otherwise, no significant phenotypic differences in total or metabolic clearance were found and it is concluded that the metabolism of quinidine is largely carried out by P450 isoenzymes different from P450db1. A biexponential decline in the log plasma quinidine concentration vs time curves was observed in all subjects, and the mean elimination half-life was 11-12 h. This is about twice as long as generally reported in the literature.

Effect of cimetidine on quinidine bioavailability.

Elevations in quinidine steady-state serum concentrations have been reported in patients who received cimetidine concurrently. Studies in normal volunteers have shown that areas under the serum concentration-time curve of orally administered quinidine are higher when quinidine is given during chronic cimetidine therapy as compared to under control conditions. The mechanism for this interaction is generally ascribed to decreased hepatic clearance as a consequence of enzyme inhibition. In this study, we show that cimetidine also decreases the bioavailable fraction of quinidine.