Erik Eliasson

Ligand-dependent maintenance of ethanol-inducible cytochrome P-450 in primary rat hepatocyte cell cultures

Administration of ethanol, dimethylsulphoxide, 2-propanol or imidazole to rats caused 2-7-fold increases in the level of hepatic ethanol-inducible cytochrome P-450 (P-450j), without any concomitant enhancement of corresponding mRNA. All the compounds were able to stabilize P-450j in hepatocyte cultures for at least three days, whereas P-450j mRNA rapidly disappeared from the cultures. A correlation was reached between the concentration of Me2SO, ethanol and 2-propanol necessary to maintain P-450j in the cell cultures and their binding affinities to the enzyme. It is suggested that the ligand-bound form of P-450j in the hepatocytes is protected from degradation.

Pharmacokinetics of losartan and its metabolite E-3174 in relation to the CYP2C9 genotype.

Losartan is metabolized by polymorphic CYP2C9 to E‐3174. Our aim was to evaluate the pharmacokinetics of losartan and E‐3174 in relation to the CYP2C9 genotype.

Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT The only existing study of CYP2C19*17-associated alterations in drug pharmacokinetics was retrospective and compared probe drug metabolic ratios. The CYP2C19*17 allele had been associated with a two- and fourfold decrease in omeprazole and S/R-mephenytoin metabolic ratios. WHAT THIS STUDY ADDS This study characterized the single-dose pharmacokinetics of omeprazole, along with the 5-hydroxy and sulphone metabolites, in CYP2C19*17/*17 and CYP2C19*1/*1 subjects. The observed differences in omeprazole AUC(infinity) suggest that the CYP2C19*17 allele is an important explanatory factor behind individual cases of therapeutic failure. AIMS To investigate the influence of the CYP2C19*17 allele on the pharmacokinetics of omeprazole, a commonly used CYP2C19 probe drug, in healthy volunteers. METHODS In a single-dose pharmacokinetic study, 17 healthy White volunteers genotyped as either CYP2C19*17/*17 or CYP2C19*1/*1 received an oral dose of 40 mg of omeprazole. Plasma was sampled for up to 10 h postdose, followed by quantification of omeprazole, 5-hydroxy omeprazole and omeprazole sulphone by high-performance liquid chromatography. RESULTS The mean omeprazole AUC(infinity) of 1973 h nmol l(-1) in CYP2C19*17/*17 subjects was 2.1-fold lower [95% confidence interval (CI) 1.1, 3.3] than in CYP2C19*1/*1 subjects (4151 h nmol l(-1), P = 0.04). A similar trend was observed for the sulphone metabolite with the CYP2C19*17/*17 group having a mean AUC(infinity) of 1083 h nmol l(-1), 3.1-fold lower (95% CI 1.2, 5.5) than the CYP2C19*1/*1 group (3343 h nmol l(-1), P = 0.03). A pronounced correlation (r(2) = 0.95, P < 0.0001) was seen in the intraindividual omeprazole AUC(infinity) and omeprazole sulphone AUC(infinity) values. CONCLUSIONS The pharmacokinetics of omeprazole and omeprazole sulphone differ significantly between homozygous CYP2C19*17 and CYP2C19*1 subjects. For clinically important drugs that are metabolized predominantly by CYP2C19, the CYP2C19*17 allele might be associated with subtherapeutic drug exposure.

The role of CYP2C9 genotype in the metabolism of diclofenac in vivo and in vitro

Abstract. Introduction: The polymorphic cytochrome P450 enzyme 2C9 (CYP2C9) catalyses the metabolism of many drugs including S-warfarin, acenocoumarol, phenytoin, tolbutamide, losartan and most of the non-steroidal anti-inflammatory drugs. Diclofenac is metabolised to 4′-hydroxy (OH), the major diclofenac metabolite, 3′-OH and 3′-OH-4′-methoxy metabolites by CYP2C9. The aim of the present study was to clarify the impact of the CYP2C9 polymorphism on the metabolism of diclofenac both in vivo and in vitro. Subjects, materials and methods: Twenty healthy volunteers with different CYP2C9 genotypes [i.e. CYP2C9*1/*1 (n=6), *1/*2 (n=3), *1/*3 (n=5), *2/*3 (n=4), *2/*2 (n=1), *3/*3 (n=1)] received a single 50-mg oral dose of diclofenac. Plasma pharmacokinetics [peak plasma concentration (Cmax), half-life (t1/2) and area under the plasma concentration–time curve (AUCtotal)] and urinary recovery of diclofenac and its metabolites were compared between the genotypes. Diclofenac 4′-hydroxylation was also analysed in vitro in 16 different samples of genotyped [i.e. CYP2C9*1/*1 (n=7), *1/*2 (n=2), *1/*3 (n=2), *2/*3 (n=2), *2/*2 (n=2), *3/*3 (n=1)] human liver microsomes. Results: Within each genotype group, a high variability was observed in kinetic parameters for diclofenac and 4′-OH-diclofenac (6- and 20-fold, respectively). No significant differences were found between the different genotypes either in vivo or in human liver microsomes. No correlation was found between the plasma AUC ratio of diclofenac/4′-OH-diclofenac and that of losartan/E-3174, previously determined in the same subjects. Conclusion: No relationship was found between the CYP2C9 genotype and the 4′-hydroxylation of diclofenac either in vivo or in vitro. This, together with the lack of correlation between losartan oxidation and diclofenac hydroxylation in vivo raises the question about the usefulness of diclofenac as a CYP2C9 probe.

Amyloid fibril formation by pulmonary surfactant protein C.

Lung surfactant protein C (SP‐C) is a lipopeptide that contains two fatty acyl (palmitoyl) chains bound via intrinsically labile thioester bonds. SP‐C can transform from a monomeric α‐helix into β‐sheet aggregates, reminiscent of structural changes that are supposed to occur in amyloid fibril formation. SP‐C is here shown to form amyloid upon incubation in solution. Furthermore, one patient with pulmonary alveolar proteinosis (PAP, a rare disease where lung surfactant proteins and lipids accumulate in the airspaces) and six healthy controls have been studied regarding presence and composition of amyloid fibrils in the cell‐free fraction of bronchoalveolar lavage (BAL) fluid. Abundant amyloid fibrils were found in BAL fluid from the patient with PAP and, in low amounts, in three of the six healthy controls. SDS‐insoluble fibrillar material associated with PAP mainly consists of SP‐C, in contrast to the fibrils found in controls. Fibrillated SP‐C has to a significant extent lost the palmitoyl groups, and removal of the palmitoyl groups in vitro increases the rate of fibril formation.

Hydroxylation of acetone by ethanol‐ and acetone‐inducible cytochrome P‐450 in liver microsomes and reconstituted membranes

Acetone oxidation in rat liver microsomes was induced 5‐ or 8‐fold by the treatment of the animals with ethanol or acetone, respectively. The apparent K m of the reaction was 0.9 mM, a value lower than the concentration reported for plasma acetone under starvation conditions. The major acetone metabolite was identified as acetol by GC‐MS. Acetone oxidation in microsomes was inhibited by typical P‐450 inhibitors as well as by compounds (e.g. imidazole) known to interact with the ethanol‐inducible P‐450 form. Antibodies against this P‐450 isozyme were inhibitory for the reaction in rabbit liver microsomes and this isozyme was the only one that showed acetone hydroxylation activity in reconstituted membranes. Imidazole inhibited the conversion of [14C]acetone into low‐M r compounds (e.g. glucose) in vivo. It is suggested that the ethanol‐and acetone‐inducible P‐450 make use of acetone as an endogenous substrate in the utilization of the compound for, e.g. glucose production under conditions of starvation and diabetic ketoacidosis.

Bioactivation of cyclophosphamide: the role of polymorphic CYP2C enzymes

Several-fold differences have been observed among patients in the biotransformation of cyclophosphamide. The aim of this study was to investigate the contribution of CYP2C9 and CYP2C19 and their polymorphisms to the variability of cyclophosphamide activation. The formation of 4-hydroxycyclophosphamide was studied in microsomes from a total of 32 different genotyped human livers, as well as in yeast microsomes expressing different genetic variants of CYP2C9 and CYP2C19. The kinetic data obtained in the yeast system revealed that the intrinsic clearance (Vmax/Km) of cyclophosphamide by CYP2C9.2 and CYP2C9.3 samples was approximately threefold lower than that by CYP2C9.1. However, in liver microsomes, there were no statistically significant differences in the intrinsic clearance of 4-hydroxycyclophosphamide formation between the group of seven CYP2C9*1/*1 livers and the remaining nine with one or two variant CYP2C9 alleles (P>0.7). We found a statistically significant correlation (rs=0.65, P=0.003) between 4-hydroxylation of cyclophosphamide and 5′-hydroxylation of R-omeprazole, a measure of CYP2C19 activity in human liver microsomes (n=19). No correlation was found between 4-hydroxylation of cyclophosphamide and the formation rate of hydroxycelecoxib, mainly catalysed by CYP2C9 (rs=0.17, P=0.55, n=32). In conclusion, based on the correlation with the formation of R-5′-hydroxyomeprazole, CYP2C19 may partly contribute to the bioactivation of cyclophosphamide in human liver microsomes, while the role of CYP2C9 appears minor.

Effect of ACE insertion/deletion and 12 other polymorphisms on clinical outcomes and response to treatment in the life study

Objective This pharmacogenetics substudy from the Losartan Intervention for Endpoint reduction in Hypertension study in patients with hypertension and left ventricular hypertrophy (LVH) treated with the angiotensin receptor blocker losartan versus the &bgr;-blocker atenolol for 4.8 years tested whether the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene and 12 other previously well-characterized polymorphisms of hypertension susceptibility genes affected blood pressure reduction, heart rate reduction, cardiovascular events, and/or response to treatment. These polymorphisms were chosen because they could affect blood pressure control or the pharmacological action of losartan or atenolol. Methods We genotyped 3503 patients, 1774 on losartan and 1729 on atenolol. Results ACE and the 12 other genotypes did not affect the reduction in systolic blood pressure, diastolic blood pressure, pulse pressure, mean arterial pressure, or heart rate, or treatment differences between losartan and atenolol on these endpoints, as assessed by general linear models. Also, ACE and the 12 other genotypes did not affect risk of the primary composite endpoint or its components stroke, myocardial infarction, and cardiovascular death, or treatment differences between losartan and atenolol on these endpoints, as assessed by Cox proportional hazards models including baseline Framingham risk score and LVH. Conclusion ACE insertion/deletion and 12 other polymorphisms of hypertension susceptibility genes did not affect blood pressure reduction, heart rate reduction, or cardiovascular events in patients with hypertension and LVH, or treatment differences between losartan and atenolol on these endpoints. These results suggest that the observed effects of losartan versus atenolol in the Losartan Intervention for Endpoint reduction in hypertension study do not depend on ACE and 12 other polymorphisms of hypertension susceptibility genes.

Stable expression of human cytochrome P450 2E1 in V79 Chinese hamster cells

A V79 Chinese hamster cell line was constructed for stable expression of human cytochrome P450 2E1 (CYP2E1) by integration of a SV40 Early promoter recombinant CYP2E1 cDNA into the chromosomal DNA. The cDNA encoded CYP2E1 was effectively expressed and enzymatically active, as shown by hydroxylation of chlorzoxazone and of p-nitrophenol, at rates of about 70 pmol x mg-1 total protein x min-1. CYP2E1 content and activity was increased upon cultivation in the presence of ethanol indicating a substrate mediated stabilization effect. A similar stabilizing effect was also observed for inhibitors of CYP2E1, e.g. imidazole, 4-methylpyrazole, and isoniazid. The feasibility of the newly established cell line V79MZh2E1 for toxicological studies was shown by CYP2E1-mediated activation of N-nitrosodimethylamine and p-nitrophenol and a dose-dependent cytotoxic and mutagenic effect.

Accumulation of celecoxib with a 7-fold higher drug exposure in individuals homozygous for CYP2C9*3

i c i e l c r c o ccumulation of celecoxib with a 7-fold higher rug exposure in individuals homozygous for YP2C9*3 o the Editor: In September 2004 the cyclooxygenase 2 inhibitor rofeoxib was withdrawn because of an increased risk of major ardiovascular events during long-term treatment. Thereafter he Adenoma Prevention with Celecoxib Study reported a imilar risk for celecoxib as well. Importantly, for both rugs, an apparent dose-risk relationship was evident, which d