Lack of relationship between quinidine pharmacokinetics and the sparteine oxidation polymorphism

Quinidine is a very potent inhibitor of CYP2D6, but the role of the enzyme in the biotransformation of quinidine has only been investigated in a single in vitro study and in two small in vivo experiments, with contradictory results. The present investigation was designed to present definite evaluation of whether quinidine is metabolised by CYP2D6. Eight poor metabolizers (PM) and 8 extensive metabolizers (EM) of sparteine each took one oral dose of 200 mg quinidine. In the EM, the total clearance, the clearance via 3-hydroxylation and the clearance via N-oxidation, were 33, 3.7 and 0.23 l·h^–1, respectively. In the PM, the corresponding values were 29, 3.1 and 0.18 l·h^–1, respectively. There were no statistically significant differences between EM and PM in any of these pharmacokinetic parameters. It is concluded that CYP2D6 is not an important enzyme for the oxidation of quinidine.     

Quinidine inhibits the 2-hydroxylation of imipramine and desipramine but not the demethylation of imipramine

Summary On separate occasions 6 extensive metabolizers of sparteine took a single oral dose of 100 mg imipramine and desipramine before and during the intake of quinidine sulphate 200 mg/day.During quinidine the total oral clearance of imipramine on average was reduced by 35%, and that of desipramine by 85%. The clearance of imipramine via demethylation was not significantly reduced during quinidine administration, whereas its clearance by other pathways, largely 2-hydroxylation, was reduced by more than 50%. 2-OH-Imipramine and 2-OH-desipramine were detected in plasma before (maximum concentrations 30–100 nmol · l^–1) but not during quinidine.It appears that quinidine is a potent inhibitor of the sparteine/debrisoquine oxygenase, P450dbl, which is responsible for the 2-hydroxylation of imipramine and desipramine, but not of the P450 isozyme responsible for the demethylation of imipramine.     

Enzymatic basis of the debrisoquine/sparteine-type genetic polymorphism of drug oxidation. Characterization of bufuralol 1'-hydroxylation in liver microsomes of in vivo phenotyped carriers of the genetic deficiency

The genetically controlled polymorphic oxidation of debrisoquine and sparteine is caused by the absence or functional deficiency of a cytochrome P-450 isozyme. In order to elucidate the mechanisms underlying the differences in cytochrome P-450 function we have studied the 1'-hydroxylation of the prototype drug bufuralol in human liver microsomes of individuals phenotyped in vivo as extensive metabolizers (EM, N = 10), poor metabolizers (PM, N = 5) and in subjects with an intermediate rate of metabolism (IM, N = 4). PM- as compared to EM-microsomes were characterized by a decreased Vmax for (+)-bufuralol 1'-hydroxylation (7.51 +/- 2.03 nmol X mg-1 X hr-1 vs 11.95 +/- 4.80 nmol X mg-1 X hr-1) but not for (-)-bufuralol 1'-hydroxylation (4.72 +/- 0.87 nmol X mg-1 X hr-1 vs 5.55 +/- 1.49 nmol X mg-1 X hr-1). The apparent Km for (+)-bufuralol 1'-hydroxylation was increased in PM microsomes (118 +/- 84.9 microM vs 17.9 +/- 6.30 microM). Inhibition of bufuralol 1'-hydroxylation by quinidine was biphasic in EM microsomes, providing further support for the involvement of at least two cytochrome P-450 isozymes. Quinidine acted as a competitive inhibitor of only the high affinity/stereoselectivity component of the reaction. Our data suggest that the debrisoquine/sparteine type of oxidation polymorphism is caused by an almost complete loss of a minor cytochrome P-450 isozyme which has a high affinity and stereoselectivity for (+)-bufuralol and a high sensitivity to inhibition by quinidine.     

Sparteine oxidation polymorphism in Denmark

Sparteine oxidation was polymorphic among 301 healthy Danish volunteers. Hence 22 subjects or 7.3% were phenotyped as poor metabolizers (PM) whereas 279 subjects were classified as extensive metabolizers (EM). The metabolic ratio (MR) between sparteine and 2- and 5-dehydrosparteine (% of dose) in 12 hrs urine ranged from 0.11-12.6 in EM and from 30-394 in PM. Urinary excretion of 2- and 5-dehydrosparteine also discriminated between PM and EM. Age, sex, and smoking habits did not influence the MR. This study confirms that sparteine is a useful probe drug in pharmacogenetic investigations.     

Lack of relationship between debrisoquine oxidation phenotype and the pharmacokinetics of quinine.

The relationship between debrisoquine oxidation phenotype and the pharmacokinetics of quinine after a single dose (600 mg) of quinine sulphate was studied in eight extensive metabolizers (EM) and five poor metabolizers (PM). The mean elimination half-life of quinine in the PMs (10.2 +/- 1.6 (s.d.)h) was similar to that in the EMs (10.9 +/- 1.7 h). The oral clearance of quinine in the PM subjects was 0.092 +/- 0.021 l h-1 kg-1 and was not significantly different (P greater than 0.05) from that observed in the EM subjects (0.073 +/- 0.019 l h-1 kg-1). This suggests that even though quinine is extensively metabolized by oxidative biotransformation, this is carried out largely by P450 isoenzymes different from P450IID6 which oxidizes debrisoquine.     

Sparteine oxidation polymorphism in Greenlanders living in Denmark.

Sparteine oxidation appeared to be polymorphic in 185 healthy Greenlanders living in Denmark. Six subjects (3.2%) were phenotyped as poor metabolizers (PM) and 179 subjects as extensive metabolizers (EM). The metabolic ratio (MR) between sparteine and 2- + 5-dehydrosparteine in a 12 h urine sample ranged from 0.06-3.12 in EM and from 30-480 in PM. The excretion of dehydrosparteines accounted for less than 2.2% of the dose in PM and ranged from 5.6%-63% in EM. The urinary recovery (% of dose) of sparteine, 2-dehydrosparteine and total sparteine + dehydrosparteines was lower in Greenlander EM than in Danish EM (Brøsen et al., 1985). Incomplete urine collection in a substantial proportion of the Greenlanders could explain these discrepancies.     

Genetically-determined interaction between propafenone and low dose quinidine: role of active metabolites in modulating net drug effect.

1. Quinidine is a potent inhibitor of the genetically-determined debrisoquine 4-hydroxylation. Oxidation reactions of several other drugs, including the 5-hydroxylation of the new antiarrhythmic drug propafenone, depend on the isozyme responsible for debrisoquine 4-hydroxylation. 2. The effect of quinidine on the debrisoquine phenotype-dependent 5-hydroxylation and the pharmacological activity of propafenone was studied in seven 'extensive' metabolizers and two 'poor' metabolizers of the drug receiving propafenone for the treatment of ventricular arrhythmias. 3. In patients with the extensive metabolizer phenotype, quinidine increased mean steady-state plasma propafenone concentrations more than two fold, from 408 +/- 351 (mean +/- s.d.) to 1096 +/- 644 ng ml-1 (P less than 0.001), decreased 5-hydroxypropafenone concentrations from 242 +/- 196 to 125 +/- 97 ng ml-1 (P less than 0.02) and reduced propafenone oral clearance by 58 +/- 23%. 4. Despite these changes in plasma concentrations, electrocardiographic intervals and arrhythmia frequency were unaltered by quinidine coadministration, indicating that 5-hydroxypropafenone contributes to the pharmacologic effects of propafenone therapy in extensive metabolizers. 5. In contrasts, plasma concentrations of propafenone and 5-hydroxypropafenone remained unchanged in the two patients with the poor metabolizer phenotype. 6. Biotransformation of substrates for the debrisoquine pathway can be markedly perturbed by even low doses of quinidine; interindividual variability in drug interactions may have a genetic component.     

Rifampicin treatment greatly increases the apparent oral clearance of quinidine

We investigated the effect of cytochrome P450 induction by rifampicin on the in vivo oxidative metabolism of quinidine. The pharmacokinetics of a 200 mg oral single dose quinidine were studied before and after one week of daily treatment with 600 mg rifampicin in six healthy young male volunteers. Biomarker reactions of cytochrome P450 isozyme activities in the form of caffeine, sparteine, mephenytoin, tolbutamide and cortisol metabolism were applied. The median total apparent oral clearance and partial clearance by 3-hydroxylation of quinidine increased 9 times. The partial clearance by N-oxidation increased 6 times. The Cmax and the elimination half life were reduced 3 times. No statistically significant changes were found for quinidine tmax and renal clearance. The cortisol metabolic ratio increased 5 times, while no statistically significant effects were seen for other CYP marker reactions. The results indicate that the inductive effect of rifampicin is likely to be of clinical relevance particularly when used concomitantly with drugs metabolized by CYP3A4.     

Oxidation of quinidine by human liver cytochrome P-450

The anti-arrhythmic quinidine has been reported to be a competitive inhibitor of the catalytic activities of human liver P-450DB, including sparteine delta 2-oxidation and bufuralol 1'-hydroxylation, and we confirmed the observation that submicromolar concentrations are strongly inhibitory. Human liver microsomes oxidize quinidine to the 3-hydroxy (Km 4 microM) and N-oxide (Km 33 microM) products, consonant with in vivo observations. Both bufuralol and sparteine inhibited microsomal quinidine 3-hydroxylation. Liver microsomes prepared from DA strain rats showed a relative deficiency in quinidine 3-hydroxylase activity in females compared to males. These observations might suggest that quinidine oxidation is catalyzed by the same P-450 forms that oxidize debrisoquine, bufuralol, and sparteine; i.e., rat P-450UT-H and P-450DB. However, neither of these two purified enzymes catalyzed quinidine 3-hydroxylation, and anti-P-450UT-H, which strongly inhibits human liver microsomal bufuralol 1'-hydroxylation, did not substantially inhibit quinidine 3-hydroxylation or N-oxygenation. P-450MP, the human S-mephenytoin 4-hydroxylase, also does not appear to oxidize quinidine but P-450NF, the human nifedipine oxidase, does. Anti-P-450NF inhibited greater than 95% of the 3-hydroxylation and greater than 85% of the N-oxygenation of quinidine in several microsomal samples. Quinidine inhibited microsomal nifedipine oxidation and, in a series of human liver samples, rates of nifedipine oxidation were correlated with rates of quinidine oxidation. Thus, quinidine oxidation appears to be catalyzed primarily by P-450NF and not by P-450DB. Quinidine binds 2 orders of magnitude more tightly to P-450DB, which does not oxidize it, than to P-450NF, the major enzyme involved in its oxidation. The substrate specificity of human P-450NF is discussed further in terms of its regioselective oxidations of complex molecules including quinidine, aldrin, benzphetamine, cortisol, testosterone and androstenedione, estradiol, and several 2,6-dimethyl-1,4-dihydropyridines.     

2-Hydroxylation of ethinyloestradiol in relation to the oxidation of sparteine and antipyrine.

The metabolism of [3H]ethinyloestradiol (EE2) was investigated in six male subjects who had been phenotyped with respect to sparteine metabolism (three metabolizers and three non-metabolizers). Urinary metabolite profiles of EE2 were virtually identical. Following enzyme hydrolysis of sulphate and glucuronide conjugates the major urinary metabolite was 2-methoxyEE2. The ratio EE2:2-methoxyEE2 was taken as a measure of EE2 2-hydroxylation (metabolizers, 2.4 +/- 0.3; non-metabolizers, 2.5 +/- 0.4). Primaquine (45 mg), previously shown to inhibit antipyrine metabolism, had no effect on EE2 2-hydroxylation. Supporting studies in rats showed that acute administration of primaquine (50 mg/kg) and 1-methylimidazole (50 mg/kg) inhibited antipyrine but not EE2 metabolism. It is concluded that the cytochrome P-450 enzyme responsible for 2-hydroxylation of EE2 is distinct from the enzymes involved in the oxidation of sparteine and antipyrine.     

Fluoxetine inhibits the metabolism of tolterodine-pharmacokinetic implications and proposed clinical relevance

AIMS: To investigate the change in disposition of tolterodine during coadministration of the potent cytochrome P450 2D6 (CYP2D6) inhibitor fluoxetine. METHODS: Thirteen patients received tolterodine l-tartrate 2 mg twice daily for 2.5 days, followed by fluoxetine 20 mg once daily for 3 weeks and then concomitant administration for an additional 2.5 days. They were characterized as extensive metabolizers (EM1 with one functional CYP2D6 gene, EM2 with two functional genes) or poor metabolizers (PM). RESULTS: Nine patients, three EM2 and four EM1 and two PM, completed the trial. Following tolterodine administration, the area under the serum concentration-time curve (AUC) of tolterodine was 4.4-times and 30-times higher among EM1 and PM, respectively, compared with EM2. The AUC of the 5-hydroxymethyl metabolite (5-HM) was not quantifiable in PM. Fluoxetine significantly decreased (P<0.002) the oral clearance of tolterodine by 93% in EM2 and by 80% in EM1. The AUC of 5-HM increased in EM2 and decreased in EM1. However, the exposure to the active moiety (unbound tolterodine +5-HM) was not significantly increased in the two phenotypes. The subdivision of the EM group showed a 2.1-fold increase in active moiety in EM2 but the exposure was still similar to EM1 compared with before the interaction. CONCLUSIONS: The study suggests a difference in the pharmacokinetics of tolterodine and its 5-hydroxymethyl metabolite depending on the number of functional CYP2D6 genes. Fluoxetine significantly inhibited the hydroxylation of tolterodine. Despite the effect on the pharmacokinetics of tolterodine in extensive metabolizers, the clinical effect is expected to be within normal variation.     

The influence of enzyme induction on polymorphic sparteine oxidation.

The effects of antipyrine (1200 mg day-1), phenobarbitone (100 mg day-1) and rifampicin (600 mg and 1200 mg day-1, respectively) administration for 7 days on sparteine metabolism and 6 beta-hydroxycortisol excretion were studied in panels of extensive (EM) and poor metaboliser (PM) subjects. Drug metabolism was induced in both EM and PM subjects by antipyrine and rifampicin pretreatment as indicated by increased excretion of 6 beta-hydroxycortisol. A 30% increase in metabolic clearance of sparteine was observed in EM subjects following rifampicin administration whereas in PM subjects no effect on the overall elimination of the drug was seen. The data indicate that the regulation of cytochrome P-450 isozyme involved in polymorphic debrisoquine/sparteine metabolism is predominantly under genetic control and that enzyme induction exerts only a marginal effect.     

Mephenytoin and sparteine oxidation: genetic polymorphisms in Denmark.

The oxidation of mephenytoin was polymorphic in 358 healthy Danish volunteers. The ratio between the chromatographic peak areas of (S)- and (R)-mephenytoin (S/R) in 12 h urine was less than or equal to 0.48 in 349 extensive metabolizers (EM) and greater than or equal to 1 in 9 (2.5%) poor metabolizers (PM). Concomitant intake of mephenytoin and sparteine and subsequent assay by gas chromatography had no influence on the test results (mephenytoin S/R ratio or sparteine metabolic ratio). Among ten parents and seven siblings to six unrelated PM of mephenytoin only one (1/17 = 5.9%) was a PM. The pedigrees were compatible with an autosomal recessive mode of inheritance.     

Rifampicin Treatment Greatly Increases the Apparent Oral Clearance of Qninidine

Abstract We investigated the effect of cytochrome P450 induction by rifampicin on the in vivo oxidative metabolism of quinidine. The pharmacokinetics of a 200 mg oral single dose quinidine were studied before and after one week of daily treatment with 600 mg rifampicin in six healthy young male volunteers. Biomarker reactions of cytochrome P450 isozyme activities in the form of caffeine, sparteine, mephenytoin, tolbutamide and cortisol metabolism were applied. The median total apparent oral clearance and partial clearance by 3-hydroxylation of quinidine increased 9 times. The partial clearance by N-oxidation increased 6 times. The C_max and the elimination half life were reduced 3 times. No statistically significant changes were found for quinidine t_max and renal clearance. The cortisol metabolic ratio increased 5 times, while no statistically significant effects were seen for other CYP marker reactions. The results indicate that the inductive effect of rifampicin is likely to be of clinical relevance particulary when used concomitantly with drugs metabolized by CYP3A4.     

CYP2B6, CYP2D6, and CYP3A4 catalyze the primary oxidative metabolism of perhexiline enantiomers by human liver microsomes.

The cytochrome P450 (P450)-mediated 4-monohydroxylations of the individual enantiomers of the racemic antianginal agent perhexiline (PHX) were investigated in human liver microsomes (HLMs) to identify stereoselective differences in metabolism and to determine the contribution of the polymorphic enzyme CYP2D6 and other P450s to the intrinsic clearance of each enantiomer. The cis-, trans1-, and trans2-4-monohydroxylation rates of (+)- and (-)-PHX by human liver microsomes from three extensive metabolizers (EMs), two intermediate metabolizers (IMs), and two poor metabolizers (PMs) of CYP2D6 were measured with a high-performance liquid chromatography assay. P450 isoform-specific inhibitors, monoclonal antibodies directed against P450 isoforms, and recombinantly expressed human P450 enzymes were used to define the P450 isoform profile of PHX 4-monohydroxylations. The total in vitro intrinsic clearance values (mean +/- S.D.) of (+)- and (-)-PHX were 1376 +/- 330 and 2475 +/- 321, 230 +/- 225 and 482 +/- 437, and 63.4 +/- 1.6 and 54.6 +/- 1.2 microl/min/mg for the EM, IM, and PM HLMs, respectively. CYP2D6 catalyzes the formation of cis-OH-(+)-PHX and trans1-OH-(+)-PHX from (+)-PHX and cis-OH-(-)-PHX from (-)-PHX with high affinity. CYP2B6 and CYP3A4 each catalyze the trans1- and trans2-4-monohydroxylation of both (+)- and (-)-PHX with low affinity. Both enantiomers of PHX are subject to significant polymorphic metabolism by CYP2D6, although this enzyme exhibits distinct stereoselectivity with respect to the conformation of metabolites and the rate at which they are formed. CYP2B6 and CYP3A4 are minor contributors to the intrinsic P450-mediated hepatic clearance of both enantiomers of PHX, except in CYP2D6 PMs.     

The contribution of genetically determined oxidation status to inter-individual variation in phenacetin disposition

The oxidative O-de-ethylation and aromatic 2-hydroxylation of phenacetin have been investigated in panels of extensive (EM, n = 13) and poor (PM, n = 10) metabolizers of debrisoquine. The EM group excreted in the urine significantly more paracetamol (EM: 40.8 +/- 14.9% dose/0-8 h; PM: 29.2 +/- 8.7% dose/0-8 h, 2P less than 0.05) and significantly less 2-hydroxylated metabolites (EM: 4.7 +/- 2.3% dose/0-8 h; PM: 9.7 +/- 3.5% dose/0-8 h, 2P less than 0.005) than the PM group. Apparent first-order rate constants, calculated from pooled phenotype data, for overall elimination of phenacetin (k) and formation of paracetamol (kml) were higher in the EM group (EM: k = 0.191 +/- 0.151 h-1; kml = 0.091 +/- 0.025 h-1; PM: k = 0.098 +/- 0.035 h-1, 2P less than 0.05, kml = 0.052 +/- 0.019 h-1, 2P less than 0.05) than the PM group. The apparent first-order rate constant for 2-hydroxylation displayed no significant inter-phenotype differences. Correlation analysis demonstrated that genetically determined oxidation status accounted for approximately 50% of the inter-individual variability in phenacetin disposition encountered in this study.     

Stereoselective inhibition of nortriptyline hydroxylation in man by quinidine.

1. Four volunteers phenotyped as extensive metabolizers of sparteine took 25 mg nortriptyline hydrochloride and collected urine for 72-80 h. Total free and conjugated 10-hydroxynortriptyline (10-OH-NT) accounted for 54-58% of the dose and it was reduced to 25-40% when 50 mg quinidine sulphate was ingested on the first and second day. 2. Of the four isomers of 10-OH-NT, (-)-E-10-OH-NT was selectively decreased in quantity by quinidine coadministration, while the (+)-isomer and (-)- and (+)-Z-10-OH-NT were found in unchanged or slightly increased quantities. The contribution of (-)-E-10-OH-NT to total E-10-OH-NT and the E-/Z-ratio in total 10-OH-NT were significantly reduced. 3. The quantity of the phenol, 2-hydroxynortriptyline in urine was decreased by quinidine; the relative amounts of metabolites with a primary amino group were not affected. 4. Liver microsomes from a donor in which cytochrome P450IID6 was shown to be present by in vitro phenotyping metabolized NT to E-10-OH-NT containing 86% of the (-)-isomer. Quinidine reduced the hydroxylation rate in (-)-E-10-position much more than that in (+)-E-10-position. 5. Since quinidine selectively impairs the function of cytochrome P450IID6, it is concluded that this isoform catalyses NT hydroxylation predominantly in (-)-E-10- and in 2-position.     

The genetic polymorphism of sparteine metabolism

The formation of the two major metabolites of the antiarrhythmic and oxytocic drug sparteine (2- and 5-dehydrosparteine) exhibits a genetic polymorphism. Two phenotypes, extensive (EM) and poor metabolizers (PM) are observed in the population. The frequency of the PM phenotype in various populations (Caucasian and Japanese) ranges from 2.3 to 9%. The metabolism of sparteine is determined by two allelic genes at a single gene locus. PM subjects are homozygous for an autosomal recessive gene. The metabolism of sparteine is predominantly under genetic control as treatment with drugs such as antipyrine and rifampicin known to induce oxidative drug metabolism elicited only marginal changes in sparteine metabolism. The formation of 2-dehydrosparteine in human liver microsomes from EM and PM subjects showed a more than 40-fold difference in Km between EM and PM subjects. However, Vmax-values were almost identical in both groups. These data indicate that the basis of the differences in oxidative capacity between EM and PM subjects is more likely to be due to a variant isozyme with defective catalytic properties than to a decreased amount of the isozyme.     

Stereoselective inhibition of nortriptyline hydroxylation in man by quinidine

1. Four volunteers phenotyped as extensive metabolizers of sparteine took 25?mg nortriptyline hydrochloride and collected urine for 72–80?h. Total free and conjugated 10-hydroxynortriptyline (10-OH-NT) accounted for 54–58% of the dose and it was reduced to 25–40% when 50?mg quinidine sulphate was ingested on the first and second day.

2. Of the four isomers of 10-OH-NT, (-)-E-10-OH-NT was selectively decreased in quantity by quinidine coadministration, while the (+)-isomer and (-)- and (+)-Z-10-OH-NT were found in unchanged or slightly increased quantities. The contribution of (-)-E-10-OH-NT to total E-10-OH-NT and the E-/Z-ratio in total 10-OH-NT were significantly reduced.

3. The quantity of the phenol, 2-hydroxynortriptyline in urine was decreased by quinidine; the relative amounts of metabolites with a primary amino group were not affected.

4. Liver microsomes from a donor in which cytochrome P450IID6 was shown to be present by in vitro phenotyping metabolized NT to E-10-OH-NT containing 86% of the (-)-isomer. Quinidine reduced the hydroxylation rate in (-)-E-10-position much more than that in (+)-E-10-position.

5. Since quinidine selectively impairs the function of cytochrome P450IID6, it is concluded that this isoform catalyses NT hydroxylation predominantly in (-)-E-10-and in 2-position     

Antipyrine metabolism in relation to polymorphic oxidations of sparteine and debrisoquine.

Thirty-five healthy subjects who had been classified as extensive or poor metabolizers of both sparteine and debrisoquine were given a single oral dose of antipyrine. Saliva concentration of antipyrine and urinary excretion of its three major oxidation metabolites were measured. All the parameters of antipyrine metabolism which were estimated had similar distributions in both the 28 EM and 7 PM genetic phenotypes defined by the metabolism of sparteine and debrisoquine. The clearance of antipyrine by the formation of 4-hydroxy-antipyrine and 3-hydroxy-antipyrine respectively were closely correlated (r = 0.83, P less than 0.001) and both were significantly higher in smokers than in non-smokers. Demethylation of antipyrine also seemed to be influenced by smoking, but not to a statistically significant extent. These findings confirm the influence of the environmental factor of smoking in antipyrine oxidative biotransformations.