Determination of 5-(p-hydroxyphenyl)-5-phenylhydantoin and studies relating to the disposition of phenytoin in man

A gas chromatographic on-column methylation technique was developed for the routine laboratory determination of 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), the principal urinary product of phenytoin )PHT) metabolism in man. 5-(p-Hydroxyphenyl)-5-(p-tolyl)hydantoin (HMPPH), a new internal standard, was synthesized and evaluated against 5-phenyl--5-(p-tolyl)hydantoin (MPPH), the compound normally used as the internal standard in p-HPPH assays. HMPPH withstood the challenges of intralaboratory quality control checks and tests of precision of p-HPPH values at times when MPPH provided erratic and unreliable values. Both an enzyme and acid treatment of urine were studied for the purpose of hydrolysis of p-HPPH-glucuronide, the form in which p-HPPH is excreted in urine. The use of both treatments in studies of three different patient urines pointed to the conclusion that acid-catalyzed decomposition of PHT dihydrodiol, a minor urinary metabolite of PHT in man, was unimportant from the analytical point of view, contributing little if anything to total urinary p-HPPH content. Some aspects of PHT disposition, as evidenced by studies of PHT plasma levels and p-HPPH urinary outputs in individual patients, are discussed.     

Stereoselective 4'-hydroxylation of phenytoin: relationship to (S)-mephenytoin polymorphism in Japanese

Aims The aim of this study was to clarify whether phenytoin (PHT) stereoselective hydroxylation cosegregates with (S)-mephenytoin phenotype.Methods A single dose of PHT (100 mg) was administered orally to six healthy Japanese subjects in whom the genotype and phenotype of CYP2C19 had been determined previously. The urinary excretion profiles of the metabolites of PHT, (R)- and (S)-p-HPPH [5-(4-hydroxyphenyl)-5-phenylhydantoin] up to 36 h postdose were compared between the two groups of poor metabolizers (PMs, n=3) and extensive metabolizers (EMs, n=3) with respect to CYP2C19. CYP2C9 genotype was also determined.Results All the alleles were found to be wild type (Arg₁₄₄Tyr₃₅₈Ile₃₅₉Gly₄₁₇ ) in each subject. The mean value for cumulative urinary excretion of unchanged PHT was not significantly different between the PMs and the EMs. However, recovery of (R)-p-HPPH at 36 h was 3.5-fold lower and that of (S)-p-HPPH 1.3-fold lower in PMs than in EMs. Although the mean urinary excretion values for both metabolites were significantly lower in the PMs than in the EMs, the difference between the two groups was larger for (R)-p-HPPH. A significant negative correlation was observed between the hydroxylation index of omeprazole (the ratio between the serum concentrations of omeprazole and hydroxyomeprazole in blood samples drawn 3 h after drug intake) and the log₁₀ 0–12 h urinary recovery of (R)-p-HPPH.Conclusions In humans, the 4′-hydroxylation of PHT is highly stereoselective towards formation of the (S)-enantiomer. Thus, (S)-hydroxylation by CYP2C9 might be the major determinant of the disposition of PHT. However, these results support the hypothesis that the stereoselective hydroxylation pathway of PHT to form (R)-p-HPPH cosegregates with the CYP2C19 metabolic polymorphism.     

Urinary excretion of phenytoin metabolites, 5-(4'-hydroxyphenyl)-5-phenylhydantoin and its O-glucuronide in humans and analysis of genetic polymorphisms of UDP-glucuronosyltransferases

The anticonvulsant agent phenytoin (5,5-diphenylhydantoin) is mainly excreted as 5-(4'-hydroxyphenyl)-5-phenylhydantoin (4'-HPPH) O-glucuronide in humans. Previously, we demonstrated that the glucuronidation of 4'-HPPH is catalyzed by multiple UDP-glucuronosyltransferases (UGTs) of UGT1A1, UGT1A4, UGT1A6, and UGT1A9. Since 4'-HPPH may be bioactivated to a reactive metabolite by peroxidase, the glucuronidation in considered to be a detoxification pathway. In the present study, we investigated the relationship between the extent of interindividual variability in the urinary excretion levels of 4'-HPPH and its O-glucuronide and genotyping of CYP2C9, CYP2C19, UGT1A1, UGT1A6, and UGT1A9. 4'-HPPH and its glucuronide in urine samples from 15 patients to whom phenytoin was administered were measured by liquid chromatography-tandem mass spectrometry. When the molar ratio of 4'-HPPH O-glucuronide/4'-HPPH was calculated as an index of glucuronidation, a large interindividual variability (11 fold) was observed in the 15 patients. Phenytoin is metabolized to 4'-HPPH by CYP2C9 and CYP2C19 in which there are genetic polymorphisms. Although 5 patients were genotyped as heterozygotes of mutated alleles of CYP2C9 or CYP2C19 genes, no relationship with the interindividual difference in the total excretion levels of 4'-HPPH and its O-glucuronide was observed. The UGT1A1*6, UGT1A1*28, UGT1A1*60 and UGT1A6*2 alleles were found in 1, 3, 6, and 8 patients, respectively. Although there was no relationship between the genetic polymorphisms of UGT1As and the interindividual difference in the 4'-HPPH glucuronidation, the large interindividual variability of 4'- HPPH glucuronidation may contribute to interindividual differences in toxic reactions to phenytoin.     

Effect of moderate haemodilution with Fluosol-DA or normal saline on low-dose phenytoin and (+/-)-5-(4-hydroxyphenyl)-5-phenylhydantoin kinetics

Phenytoin kinetics were determined in the rat following moderate (50%) blood exchange with either Fluosol-DA or normal saline. Rats received an intravenous phenytoin dose (10 mg kg-1) 0.5, 24, 48, or 72 h after exchange and were compared with non-exchanged controls. Phenytoin t 1/2 was not altered by exchange with either fluid. Its Cl and Vd were decreased and AUC increased 24, 48, and 72 h after saline exchange and 24 h after Fluosol-DA exchange. (+/-)-5-(4-Hydroxyphenyl)-5-phenylhydantoin (HPPH), a major metabolite of phenytoin, showed a decreased t1/2 and VHPPH 24, 48, and 72 h after exchange with either fluid; t1/2 was also reduced 0.5 h after Fluosol-DA exchange. The decreased Vd and VHPPH may result from changes in cardiac output secondary to haemodilution, or may represent a redistribution in the microcirculation. Fluosol-DA appears to enhance phenytoin and HPPH metabolism 48 and 72 h after exchange.     

Sensitive method for the determination of phenytoin in plasma, and phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in urine by high-performance liquid chromatography.

An isocratic high-performance liquid chromatographic method is described for the quantitative analysis of phenytoin (PHT) in plasma and both phenytoin and its main metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) in urine. Following ethyl acetate (plasma) and Extrelut-1 (urine) extraction, samples are analysed by means of a reversed-phase column (Nova-Pak C18), using a mobile phase consisting of methanol-water-tetrahydrofuran (40:60:4, v/v/v) with UV detection at 230 nm. The chromatography is complete in 10 min and the results show good precision (RSD 1.23-4.49%) and sensitivity for a linear range of 0.4-4.0 micrograms ml-1 for PHT in plasma, 0.1-1.0 microgram ml-1 for PHT in urine and 0.1-1.2 microgram ml-1 for p-HPPH in urine. The results indicate the method to be suitable for pharmacokinetic studies.     

In vivo effect of 5- and 8-methoxypsoralens and cimetidine on R,S-warfarin metabolism in rat

Several forms of cytochrome P-450 (CYP) metabolize R,S-warfarin in a regio- and enantioselective manner, therefore R,S-warfarin can be recognized as a metabolic probe for a number of CYP isoforms. We have applied a warfarin model in vivo in order to estimate the inhibitory properties of 5- and 8-methoxypsoralens on the activity of rat CYP isoforms. The area under the serum concentration versus time curve (AUC) values from time zero to 5 h for R- and S-warfarin and their metabolites were calculated. R,S-Warfarin kinetics measurements were made three times on each rat: a week before the 7-days inhibitor treatment, 3 h after the last dose of inhibitor and 3-7 days after the inhibitor was withdrawn. The inhibitory effect of cimetidine on CYP 2C11 and CYP 2C6 activities was confirmed in this approach and can be recognized as a positive control in validation of the in vivo experiment. Both 5- and 8-methoxypsoralen inhibited CYP 2C6 activity as the respective AUC for metabolite/warfarin enantiomer ratio decreased significantly. The activity of CYP 2C6 in 5- and 8-methoxypsoralen-treated rats increased over control values after the inhibitor was withdrawn. It was also observed that cimetidine additionally inhibits the absorption of R,S-warfarin and a decrease in the sum of AUC for R- and S-enantiomers became evident in spite of inhibition of the activity of both CYPs. 5-Methoxypsoralen modified the serum R-warfarin/S-warfarin ratio and a selective increase in AUC(S-warfarin) was observed, the most pronounced being after the inhibitor was withdrawn. This effect is not likely to be mediated by P-glycoprotein (P-gp) because quinidine--, a P-gp inhibitor at a dose of 15 mg kg(-1) body wt.--did not influence the AUC for either enantiomer.     

Effects of Guanxinning injection on rat cytochrome P450 isoforms activities in vivo and in vitro

Abstract

1. We aimed to investigate the regulatory effects of Guanxinning injection (GXNI) on activities of cytochrome P1A2 (CYP1A2), CYP2C11, CYP2D1 and CYP3A1/2 by probe drugs in rats in vivo and in vitro.

2. GXNI-treated and blank control groups were administered GXNI and physiological saline by caudal vein for 14 days consecutively, then they were given the probe drugs of caffeine (10?mg/kg), tolbutamide (10?mg/kg), metoprolol (20?mg/kg) and dapsone (10?mg/kg) by intraperitoneal injection. The blood samples were collected at different times for ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Changes of the pharmacokinetics parameters between the GXNI-treated and the blank control groups were used to evaluate the effects of GXNI on the four CYP450 isoforms in rats in vivo. After blood collection, the livers of rats were taken and made microsomes for in vitro tests. The relevant metabolites of phenacetin, tolbutamide, dextromethorphan and testosterone were analyzed quantitatively by high-performance liquid chromatography (HPLC) after microsome incubation. The statistical differences between the two groups were observed to detect the effects of GXNI on the four CYP450 isoforms in rats in vitro.

3. The in vivo and in vitro results demonstrated that GXNI could induce CYP1A2 activity in rats, but had no significant effects on CYP2C11, CYP2D1 and CYP3A1/2.     

Amodiaquine, its desethylated metabolite, or both, inhibit the metabolism of debrisoquine (CYP2D6) and losartan (CYP2C9) in vivo

Objective To study the extent of in vivo inhibition by the antimalarial drug amodiaquine, its active metabolite N-desethylamodiaquine, or both, of the metabolism of four probe drugs of the enzymes CYP2D6, CYP2C19, CYP2C9 and CYP1A2.Methods Twelve healthy Swedish volunteers received a cocktail of four probe drugs (debrisoquine, omeprazole, losartan and caffeine) to determine their baseline metabolic capacities. After a washout period, they received a 600 mg oral dose of amodiaquine hydrochloride; and 2–3 h later the cocktail was administered again. One week after the intake of amodiaquine, the subjects received the cocktail a third time. The levels of probe drugs and their metabolites as well as amodiaquine and its metabolite were determined by HPLC.Results Plasma levels of amodiaquine and N-desethylamodiaquine could be followed in all subjects for 6 h and 28 days, respectively. Among the 12 subjects, a 3-fold variation in amodiaquine AUC and a 2-fold variation in N-desethylamodiaquine AUC, were observed. The CYP2D6 and CYP2C9 activities of the subjects were measured by debrisoquine and losartan phenotyping tests, respectively. There were significant mean increases in debrisoquine metabolic ratio (MR) between baseline and the second cocktail [MR_{2 h}−MR_baseline 1.426 (95% confidence interval 1.159, 1.755), P=0.002; ANOVA, Fisher LSD test] and in mean losartan MR between baseline and the second cocktail [MR_{2 h}−MR_baseline 1.724 (95% confidence interval 1.076, 2.762), P=0.026; ANOVA, Fisher LSD test]. The effects on CYP2D6 and CYP2C9 activities subsided within a week after intake of amodiaquine as tested by the phenotyping cocktail. The changes in omeprazole MRs and caffeine MRs were not statistically significant between any of the study phases.Conclusion A single dose of amodiaquine decreased CYP2D6 and CYP2C9 activities significantly compared to baseline values. Amodiaquine has the potential to cause drug-drug interactions and should be further investigated in malarial patients treated with drug combinations containing amodiaquine.     

Effect of hepatic CYP inhibitors on the metabolism of sildenafil and formation of its metabolite,N‐desmethylsildenafil,in rats in vitro and in vivo

Objectives It has been reported that hepatic cytochrome P450 (CYP)2C9 and CYP3A4 are responsible for the metabolism of sildenafil and formation of its metabolite, N‐desmethylsildenafil, in humans. However, in‐vivo studies in rats have not been reported. Methods Sildenafil (20 mg/kg) was administered intravenously to rats pretreated with sulfaphenazole, cimetidine, quinine hydrochloride or troleandomycin, inhibitors of CYP2C6, CYP2C11, CYP2D subfamily and CYP3A1/2, respectively. In‐vitro studies using rat liver microsomes were also performed. Key findings The area under the plasma‐concentration time curve (AUC) was increased and clearance of sildenafil decreased in rats pretreated with cimetidine or troleandomycin. The AUC ratio for N‐desmethylsildenafil (0–4 h): sildenafil (0–∞) was significantly decreased only in rats pretreated with cimetidine. Similar results were obtained in the in‐vitro study using rat liver microsomes. Conclusions Sildenafil is metabolised via hepatic CYP2C11 and 3A1/2, and N‐desmethylsildenafil is mainly formed via hepatic CYP2C11 in rats. Thus, rats could be a good model for pharmacokinetic studies of sildenafil and N‐desmethylsildenafil in humans.     

Metabolism of citalopram enantiomers in CYP2C19/CYP2D6 phenotyped panels of healthy Swedes

AIMS: To investigate pharmacokinetics of the enantiomers of citalopram (CT) and its metabolites desmethylcitalopram (DCT) and didesmethylcitalopram (DDCT) in Swedish healthy volunteers in relation to CYP2C19 and CYP2D6 geno- and phenotypes. METHODS: Racemic CT was given for seven days to panels with different genotypes and the following mephenytoin (Me) and debrisoquine (De) hydroxylation phenotypes: EMDe/EMMe, PMDe/EMMe, EMDe/PMMe (n = 6 in all groups), and one PMDe/PMMe subject. Blood sampling was carried out during day 7, and all urine was collected for 12 h after the last dose of CT. RESULTS: The AUC of S-CT was significantly higher in the EMDe/PMMe panel compared to the EMDe/EMMe and PMDe/EMMe panels (P < 0.05), whereas the AUC of R-CT did not differ between the panels. Similar differences, although they did not reach statistical significance, were noted for S-DCT and R-DCT. The enantiomers of DDCT were not quantifiable in PMDe, and there was no difference in DDCT enantiomer concentrations between the other two panels. A PMDe/PMMe subject stopped taking CT after five days due to severe adverse effects. Based on two time points, this subject had a very long CT half-life of 95 h. The value of 1.0 for the S/R ratio of the CT trough in this subject was similar to the mean S/R CT trough ratio of the EMDe/PMMe panel, but higher than the S/R CT ratio of the EMDe/EMMe panel (0.56; 95% CI 0.49-0.63) and the PMDe/EMMe panel (0.44; 95% CI 0.31-0.57). Thus the latter two phenotypes eliminated S-CT more rapidly via CYP2C19. An adverse effect described as an 'alcohol hangover' feeling was reported by one subject from each of the three panels. These individuals had the highest concentrations of both CT enantiomers. CONCLUSIONS: The AUC of S-, but not R-(CT) was found to be significantly higher in PM of mephenytoin compared to EMs, PMs may need a lower dosage of CT.     

Effects of verapamil enantiomers and major metabolites on the cytotoxicity of vincristine and daunomycin in human lymphoma cell lines

Summary Verapamil, a calcium channel blocker, is used as the racemate. Recently, racemic verapamil has been shown to increase the cytotoxicity of vinca alkaloid and anthracycline derivatives in several resistant tumour cell lines. With respect to its cardiovascular activity S-verapamil is an order of magnitude more potent than R-verapamil. Since it was not known whether the effect on multidrug resistance was also enantioselective a comparison has been made of the potency of the R and S enantiomers and racemic verapamil in their ability to increase the cytotoxicity of vincristine and daunomycin in sensitive (MOLT 4B) and drug resistant human T-lymphoma cell lines (MOLT/VCR-5×9, MOLT/DAU-8 and VCR 1000, a highly resistant subline of CCRF-CEM). Two major metabolites, norverapamil and D617 were tested in the same system.(+)-R, (–)-S-, racemic verapamil, norverapamil and D617 alone had no effect on cell growth in sensitive or resistant cell lines in concentrations up to 20 M. In combination with vincristine, verapamil and norverapamil but not D617 produced a concentration dependent increase in the sensitivity of the resistant lines. Racemic verapamil, its individual enantiomers and norverapamil were equipotent. The concentration of the modifiers required to elicit 50% of the maximum effect (EC₅₀) was of the order of 0.5 M. No significant difference in the slopes of the concentration-effect curves were observed. The effect of verapamil and norverapamil was additive. In the sensitive MOLT 4B cell line both enantiomers and norverapamil increased sensitivity towards vincristine. However, the EC₅₀ values were at least an order of magnitude higher (2.5–8 M) than in the resistant cell lines. In contrast to the vincristine resistant cell lines, no effect on the potentiation of daunomycin cytotoxicity was observed in sensitive and daunomycin-resistant cells in the presence of a modifier. Since the activity of verapamil as a modifier of drug resistance is not enantioselective, the weaker calcium antagonist R-enantiomer appears to be better suited for clinical trials. Because of its lesser cardiovascular activity much higher doses could be given, and a higher plasma concentration would be achieved. As norverapamil, the major plasma metabolite formed during first pass elimination, is as potent as the parent drug as a modifier of drug resistance, the oral route of administration is preferable to i. v. administration in clinical trials employing R-verapamil as a modifier.Presented in part at the 30^th Spring Meeting of the Deutsche Gesellschaft für Pharmakologie und Toxikologie, Mainz, 1989, and at the IV. World Conference on Clinical Pharmacology and Therapeutics, Mannheim-Heidelberg, 1989     

Effects of phillyrin and forsythoside A on rat cytochrome P450 activities in vivo and in vitro

1. Phillyrin and forsythoside A are two important active ingredients in Forsythia suspensa. However, the effects of phillyrin and forsythoside A on the activities of cytochrome P450 (CYP450) remain unclear.

2. This study aimed to investigate the effects of phillyrin and forsythoside A on the activities of CYP1A2, CYP2C11, CYP2D1 and CYP3A1/2 by cocktail probe drugs in rats both in vivo and in vitro.

3. Many pharmacokinetic parameters of caffeine and metoprolol in phillyrin pretreatment group, caffeine and tolbutamide in forsythoside A pretreatment group were affected significantly. In rat liver microsomal incubation system, the concentrations of acetaminophen and dextrophan in the phillyrin pretreatment group are higher than blank control group by 207.69% and 125.00%, however, the concentrations of 4-hydroxytolbutamide and 6β-hydroxytestosterone were not significantly altered. The concentrations of acetaminophen and 4-hydroxytolbutamide in the forsythoside A pretreatment group are higher than blank control group by 223.07% and 154.16%, whereas the concentrations of dextrophan and 6β-hydroxytestosterone were not significantly altered.

4. These results indicated that Phillyrin had potential inductive effects on rat CYP1A2 and CYP2D1 activities, without affecting CYP2C11 and CYP3A1/2 activities. Moreover, forsythoside A had inductive effects on the activities of CYP1A2 and CYP2C11, without affecting CYP2D1 and CYP3A1/2 activities.     

Evidence for an arene oxide-NIH shift pathway in the metabolic conversion of phenytoin to 5-(4-hydroxyphenyl)-5-phenylhydantoin in the rat and in man

To determine whether the hydroxylation of 5,5-diphenylhydantoin (DPH) to 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) occurs by an arene oxide-NIH shift process, racemic 5-(4-deuteriophenyl)-5-phenylhydantoin (p-2H-DPH) was subjected to in vivo metabolic experiments in the rat and in man. After enzymatic hydrolysis of the urine, para-hydroxylated metabolites were separated by HPLC. Deuterium retention in the isolated metabolites determined by gas chromatography-mass spectrometry, was 68-72%. The results are interpreted as the predominance of an arene oxide-NIH shift pathway in those two metabolic systems. Induction of rats with phenobarbital or 3-methylcholanthrene showed no effect on the value of deuterium retention.     

Comparison of (S)-mephenytoin and proguanil oxidation in vitro: contribution of several CYP isoforms

AIMS: To compare the oxidative metabolism of (S)-mephenytoin and proguanil in vitro and to determine the involvement of various cytochrome P450 isoforms. METHODS: The kinetics of the formation of 4'-hydroxymephenytoin and cycloguanil in human liver microsomes from 10 liver samples were determined, and inhibition of formation was studied using specific chemical inhibitors and monoclonal antibodies directed towards specific CYP450 isoforms. Expressed CYP450 enzymes were used to characterize further CYP isoform contribution in vitro. Livers were genotyped for CYP2C19 using PCR amplification of genomic DNA followed by restriction endonuclease digestion. RESULTS: All livers were wildtype with respect to CYP2C19, except HLS#5 whose genotype was CYP2C19*1/CYP2C19*2. The Km, Vmax and CLint values for the formation of 4'-hydroxymephenytoin from (S)-mephenytoin and the formation of cycloguanil from proguanil ranged from 50.8 to 51.6 and 43-380 microm, 1.0-13.9 and 0.5-2.5 nmol mg-1 h-1, and 20.2-273.8 and 2.7-38.9 microl h-1 mg-1, respectively. There was a significant association between the Vmax values of cycloguanil and 4'-hydroxymephenytoin formation (rs=0.95, P=0.0004). Cycloguanil formation was inhibited significantly by omeprazole (CYP2C19/3A), troleandomycin (CYP3A), diethyldithiocarbamate (CYP2E1/3A), furafylline (CYP1A2), and (S)-mephenytoin. 4'-Hydroxymephenytoin formation was inhibited significantly by omeprazole, diethyldithiocarbamate, proguanil, furafylline, diazepam, troleandomycin, and sulphaphenazole (CYP2C9). Human CYP2E1 and CYP3A4 monoclonal antibodies did not inhibit the formation of cycloguanil or 4'-hydroxymephenytoin, and cycloguanil was formed by expressed CYP3A4 and CYP2C19 supersomes. However, only expressed CYP2C19 and CYP2C19 supersomes formed 4'-hydroxymephenytoin. CONCLUSIONS: The oxidative metabolism of (S)-mephenytoin and proguanil in vitro is catalysed by CYPs 2C19 and 1A2, with the significant association between Vmax values suggesting that the predominant enzymes involved in both reactions are similar. However the degree of selectively of both drugs for CYP isoforms needs further investigation, particularly the involvement of CYP3A4 in the metabolism of proguanil. We assert that proguanil may not be a suitable alternative to (S)-mephenytoin as a probe drug for the CYP2C19 genetic polymorphism.     

A clinical study to assess CYP1A2 and CYP3A4 induction by AZD7325, a selective GABA(A) receptor modulator - an in vitro and in vivo comparison

AIM(S)

To investigate the potential of AZD7325 to induce CYP1A2 and CYP3A4 enzyme activities.

METHODS

Induction of CYP1A2 and CYP3A4 by AZD7325 was first evaluated using cultured human hepatocytes. The effect of multiple doses of 10 mg AZD7325 on the pharmacokinetics of midazolam and caffeine was then examined in healthy subjects.

RESULTS

The highest CYP1A2 and CYP3A4 induction responses were observed in human hepatocytes treated with 1 or 10 µm of AZD7325, in the range of 17.9%–54.9% and 76.9%–85.7% of the positive control responses, respectively. The results triggered the further clinical evaluation of AZD7325 induction potential. AZD7325 reached a plasma C_max of 0.2 µm after 10 mg daily dosing to steady-state. AZD7325 decreased midazolam geometric mean AUC by 19% (0.81-fold, 90% CI 0.77, 0.87), but had no effect on midazolam C_max (90% CI 0.82, 0.97). The mean CL/F of midazolam increased from 62 l h⁻¹ (midazolam alone) to 76 l h⁻¹ when co-administered with AZD7325. The AUC and C_max of caffeine were not changed after co-administration of AZD7325, with geometric mean ratios (90% CI) of 1.17 (1.12, 1.23) and 0.99 (0.95, 1.03), respectively.

CONCLUSIONS

While AZD7325 appeared to be a potent CYP3A4 inducer and a moderate CYP1A2 inducer from in vitro studies, the expected efficacious dose of AZD7325 had no effect on CYP1A2 activity and only a weak inducing effect on CYP3A4 activity. This comparison of in vitro and in vivo results demonstrates the critical role that clinical exposure plays in evaluating the CYP induction risk of a drug candidate.     

The effect of the cytochrome P450 CYP2C8 polymorphism on the disposition of (R)-ibuprofen enantiomer in healthy subjects

AIMS: To study the effect of CYP2C8*3, the most common CYP2C8 variant allele on the dis-position of (R)-ibuprofen and the association of CYP2C8*3 with variant CYP2C9 alleles. METHODS: Three hundred and fifty-five randomly selected Spanish Caucasians were screened for the common CYP2C8 and CYP2C9 mutations. The pharmacokinetics of (R)-ibuprofen were studied in 25 individuals grouped into different CYP2C8 genotypes. RESULTS: The allele frequency of CYP2C8*3 (0.17) was found to be higher than that reported for other Caucasian populations (P = 0.0001). The frequencies of CYP2C9*2 and CYP2C9*3 were 0.19 (0.16-0.21) and 0.10 (0.08-0.12), respectively. An association between CYP2C8*3 and CYP2C9*2 alleles was observed, occurring together at a frequency 2.4-fold higher than expected for a random association of alleles (P = 0.0001). The presence of the CYP2C8*3 allele was found to influence the pharmacokinetics of (R)-ibuprofen in a gene-dose effect manner. Thus, after administration of 400 mg ibuprofen, the plasma half-life (95% confidence intervals) for individuals with genotypes CYP2C8*1/*1, CYP2C8*1/*3 and CYP2C8*3/*3, was 2.0 h (1.8-2.2), 4.2 h (1.9-6.5; P < 0.05) and 9.0 h (7.8-10.2; P < 0.002), respectively. A statistically significant trend with respect to the number of variant CYP2C8*3 alleles was also observed for the area under the concentration-time curve (P < 0.025), and drug clearance (P < 0.03). CONCLUSION: Polymorphism of the CYP2C8 gene was found to be common, with nearly 30% of the population studied carrying the variant CYP2C8*3 allele. The presence of the latter caused a significant effect on the disposition of (R)-ibuprofen. This suggests that a substantial proportion of Caucasian subjects may show alterations in the disposition of drugs that are CYP2C8 substrates.