Biomodification of acenocoumarol by bifidobacteria.

The increased interest of consumers in probiotic foods requires a deeper knowledge on the possible interactions with drugs, because their pharmacological properties could be modified. In this context, these studies are relevant for drugs such as acenocoumarol, whose dosage must be controlled due to, among other factors, food-drug interactions. Acenocoumarol is an oral anticoagulant with a narrow therapeutic range. The aim of the present research is to evaluate, in vitro, the effect of bifidobacteria on acenocoumarol. The drug was incubated with Bifidobacterium bifidum CIDCA 5310 or Bifidobacterium adolescentis CIDCA 5317 in MRS broth at 37°C for 24 h in anaerobic conditions. The effect of incubation with sterilized spent culture supernatants (SSCS) was also evaluated. Analysis by RP-HPLC showed that both bifidobacterial strains reduced the area of the acenocoumarol peak and two new peaks were evidenced. In addition, a decrease in the intensity of the bands at 1650, 1390 and 1110/cm was observed in the FTIR spectroscopic determinations. Moreover, a new band appeared at 1720/cm. No effect on the drug was observed when incubation was performed with SSCS. The present study showed a significant change in the concentration of the anticoagulant after incubation with bifidobacteria and results are compatible with biomodification of the drug due to enzymatic activity of bifidobacteria.

Development of potential candidate reference materials for drugs in bottom sediment, cod and herring tissues.

Regular use of a reference material and participation in a proficiency testing program can improve the reliability of analytical data. This paper presents the preparation of candidate reference materials for the drugs metoprolol, propranolol, carbamazepine, naproxen, and acenocoumarol in freshwater bottom sediment and cod and herring tissues. These reference materials are not available commercially. Drugs (between 7 ng/g and 32 ng/g) were added to the samples, and the spiked samples were freeze-dried, pulverized, sieved, homogenized, bottled, and sterilized by γ-irradiation to prepare the candidate materials. Procedures for extraction and liquid chromatography coupled with tandem mass spectrometry were developed to determine the drugs of interest in the studied material. Each target drug was quantified using two analytical procedures, and the results obtained from these two procedures were in good agreement with each other. Stability and homogeneity assessments were performed, and the relative uncertainties due to instability (for an expiration date of 12 months) and inhomogeneity were 10-25% and 4.0-6.8%, respectively. These procedures will be useful in the future production of reference materials.

Genotype-guided vs clinical dosing of warfarin and its analogues: meta-analysis of randomized clinical trials.

IMPORTANCE: Significant variations in dose requirements of warfarin and its analogues (acenocoumarol and phenprocoumon) make selecting the appropriate dose for an individual patient difficult. Genetic factors account for approximately one-third of the variation in dose requirement. The clinical usefulness of genotype-guided dosing of warfarin has been previously assessed in randomized clinical trials that were limited by lack of power and inconsistent results. OBJECTIVE: To compare genotype-guided initial dosing of warfarin and its analogues with clinical dosing protocols. DATA SOURCES AND STUDY SELECTION: MEDLINE (inception to December 31, 2013), EMBASE (inception to December 31, 2013), and the Cochrane Library Central Register of Controlled Trials (inception to December 31, 2013) were searched for randomized clinical trials comparing genotype-guided warfarin dosing vs clinical dosing for adults with indications for anticoagulation. DATA EXTRACTION AND SYNTHESIS: Two investigators extracted data independently on trial design, baseline characteristics, and outcomes. High-quality studies were considered those that described an appropriate method of randomization, allocation concealment, blinding, and completeness of follow-up. MAIN OUTCOMES AND MEASURES: The outcomes analyzed included the percentage of time that the international normalized ratio (INR) was within the therapeutic range, the percentage of patients with an INR greater than 4, and the incidence of major bleeding and thromboembolic events. Summary standardized differences in means (or Mantel-Haenszel risk ratios) were obtained using a random-effects model. RESULTS: In 9 trials, 2812 patients were randomized to receive warfarin, acenocoumarol, or phenprocoumon according to a genotype-guided algorithm or a clinical dosing algorithm. Follow-up ranged from 4 weeks to 6 months (median, 12 weeks). The standardized difference in means of the percentage of time that the INR was within the therapeutic range was 0.14 (95% CI, -0.10 to 0.39) in the genotype-guided dosing cohort (P = .25). The risk ratio for an INR greater than 4 was 0.92 (95% CI, 0.82 to 1.05) for genotype-guided dosing vs clinical dosing. The risk ratios for major bleeding and thromboembolic events were 0.60 (95% CI, 0.29 to 1.22) and 0.97 (95% CI, 0.46 to 2.05), respectively, for genotype-guided vs clinical dosing. CONCLUSIONS AND RELEVANCE: In this meta-analysis of randomized clinical trials, a genotype-guided dosing strategy did not result in a greater percentage of time that the INR was within the therapeutic range, fewer patients with an INR greater than 4, or a reduction in major bleeding or thromboembolic events compared with clinical dosing algorithms.

Genetic contribution of CYP2C9, CYP2C19, and APOE variants in acenocoumarol response.

Oral anticoagulants of the coumarin type have an inconveniently narrow therapeutic window, making their use difficult. In Mexico, genetic variables that participate in the heterogeneity of the therapeutic response remain poorly investigated. With the focus on warfarin, extensive pharmacogenomic studies have been performed, including those on the CYP450 family and APOE. The objective of this study was to determine the contribution of CYP2C9, CYP2C19, and APOE polymorphisms to the variations in response to the doses of acenocoumarol, which is the main anticoagulant prescribed to the Mexican population. The polymerase chain reaction-restriction fragment length polymorphism method was applied to identify 2 and 3 of CYP2C9, 2 of CYP2C19, and APOE variants. The genetic distribution of every polymorphism tested showed high variability when compared with other populations worldwide. Our results showed statistical differences only in the CYP2C19 gene between the 1 1 and 1 2 groups, with effective acenocoumarol doses of 2.56 ± 1.34 mg/day vs 1.35 ± 0.84 mg/day (P = 0.005), respectively. Multiple regression analysis, including patient age and both the CYP2C9 and CYP2C19 genes, showed that these variables explained more than 20% of the dose variations. This is the first report in Mexico searching for the relationship between CYP450 and APOE polymorphisms and the dose requirements of acenocoumarol. Our results suggest that, in the Mexican population, CYP2C19 is more involved in acenocoumarol metabolism than CYP2C9 and APOE. Besides considering the age factor, pharmacogenetic testing for CYP2C19 2 before initiating acenocoumarol treatment could lead to a safer anticoagulation therapy in Mexican patients.

Implication of novel CYP2C9*57 (p.Asn204His) variant in coumarin hypersensitivity.

INTRODUCTION: Polymorphisms in CYP2C9 can vary the rate of metabolic clearance of oral anticoagulants, risking toxicity in patients. The present study focused on exploring the genetic etiology of idiopathic hyper sensitivity to coumarin anticoagulants in a patient who presented with multiple bleeding episodes and supra-elevated International Normalized Ratios. MATERIALS AND METHODS: Bidirectional gene sequencing of CYP2C9 and VKORC1 was carried out. Using allele-specific polymerase chain reaction, the identified novel variant was genotyped in 309 patients on anticoagulation therapy. The pharmacoproteomic significance of the novel genetic variant was elucidated by structural demonstration of binding of coumarin molecules within the mutant CYP2C9 204His protein model and in silico bioinformatic evolutionary analyses. Three-dimensional structure model of the mutant protein was constructed on the basis of the published X-ray crystal structure of human CYP2C9 protein (Protein Data Bank, 1R9O). RESULTS: The patient was identified to have a novel heterozygous missense mutation in exon 4 of CYP2C9 gene (g.9172A > C; p.Asn204His; CYP2C9*57). The variant was absent in the 309 genotyped patients. In silico bioinformatic analyses indicated the variant to have a deleterious effect on the protein. Analysis of 3D structure model of the mutant protein revealed that the substituted His204 led to restricted binding of the coumarin drug within the binding site of CYP2C9 enzyme, thereby inhibiting its metabolic clearance and thus explaining the enhanced pharmacologic effect and bleeding in the patient. CONCLUSIONS: The study elucidates the structurally deleterious role of the novel CYP2C9*57 missense mutation in coumarin toxicity.

Selective binding of coumarin enantiomers to human alpha1-acid glycoprotein genetic variants.

Coumarin-type anticoagulants, warfarin, phenprocoumon and acenocoumarol, were tested for their stereoselective binding to the human orosomucoid (ORM; AGP) genetic variants ORM 1 and ORM 2. Direct binding studies with racemic ligands were carried out by the ultrafiltration method; the concentrations of free enantiomers were determined by capillary electrophoresis. The binding of pure enantiomers was investigated with quinaldine red fluorescence displacement measurements. Our results demonstrated that all investigated compounds bind stronger to ORM 1 variant than to ORM 2. ORM 1 and human native AGP preferred the binding of (S)-enantiomers of warfarin and acenocoumarol, while no enantioselectivity was observed in phenprocoumon binding. Acenocoumarol possessed the highest enantioselectivity in AGP binding due to the weak binding of its (R)-enantiomer. Furthermore, a new homology model of AGP was built and the models of ORM 1 and ORM 2 suggested that difference in binding to AGP genetic variants is caused by steric factors.

Allelic variants of cytochrome P450 2C9 modify the interaction between nonsteroidal anti-inflammatory drugs and coumarin anticoagulants.

INTRODUCTION: Cytochrome P450 (CYP) plays a key role in the metabolism of coumarin anticoagulants and nonsteroidal anti-inflammatory drugs (NSAIDs). Because CYP2C9 is a genetically polymorphic enzyme, genetic variability could play an important role in the potential interaction between NSAIDs and coumarins. We investigated whether NSAIDs were associated with overanticoagulation during therapy with coumarins and evaluated the effect of the CYP2C9 polymorphisms on this potential interaction. METHODS: We conducted a population-based cohort study among patients of an anticoagulation clinic who were treated with acenocoumarol or phenprocoumon between April 1, 1991, and May 31, 2003, and whose CYP2C9 status was known. Patients were followed up until an international normalized ratio (INR) of 6.0 or greater was reached or until the end of treatment, death, or the end of the study. Proportional hazards regression analysis was used to estimate the risk of an INR of 6.0 or greater in relation to concomitant use of a coumarin anticoagulant and NSAIDs after adjustment for several potentially confounding factors. To study effect modification by CYP2C9 genotype, stratified analyses were performed for wild-type patients and patients with a variant genotype. RESULTS: Of the 973 patients in the cohort, 415 had an INR of 6.0 or greater. Several NSAIDs increased the risk of overanticoagulation. The risk of overanticoagulation was 2.98 (95% confidence interval, 1.09-7.02) in coumarin-treated patients taking NSAIDs with a CYP2C9*2 allele and 10.8 (95% confidence interval, 2.57-34.6) in those with a CYP2C9*3 allele. CONCLUSIONS: Several NSAIDs were associated with overanticoagulation. For NSAIDs that are known CYP2C9 substrates, this risk was modified by allelic variants of CYP2C9. More frequent INR monitoring of patients taking NSAIDs is warranted.

The risk of bleeding complications in patients with cytochrome P450 CYP2C9*2 or CYP2C9*3 alleles on acenocoumarol or phenprocoumon.

The principal enzyme involved in coumarin metabolism is CYP2C9. Allelic variants of CYP2C9, CYP2C9*2 and CYP2C9*3, code for enzymes with reduced activity. Despite increasing evidence that patients with these genetic variants require lower maintenance doses of anticoagulant therapy, there is lack of agreement among studies on the risk of bleeding and CYP2C9 polymorphisms. It was, therefore, our objective to study the effect of the CYP2C9 polymorphisms on bleeding complications during initiation and maintenance phases of coumarin anticoagulant therapy. The design of the study was a population-based cohort in a sample of the Rotterdam Study, a study in 7,983 subjects. All patients who started treatment with acenocoumarol or phenprocoumon in the study period from January 1, 1991 through December 31, 1998 and for whom INR data were available were included. Patients were followed until a bleeding complication, the end of their treatment, death or end of the study period. Proportional hazards regression analysis was used to estimate the risk of a bleeding complication in relation to CYP2C9 genotype after adjustment for several potentially confounding factors such as age, gender, target INR level, INR, time between INR measurements, and aspirin use. The effect of variant genotype on bleeding risk was separately examined during the initiation phase of 90 days after starting therapy with coumarins. The 996 patients with analysable data had a mean follow-up time of 481 days (1.3 years); 311 (31.2%) had at least 1 variant CYP2C9 allele and 685 (68.8%) had the wild type genotype. For patients with the wild type genotype, the rate of minor bleeding, major bleeding and fatal bleeding was 15.9, 3.4 and 0.2 per 100 treatment-years, respectively. For patients with a variant genotype, the rate of minor, major and fatal bleeding was 14.6, 5.4 and 0.5 per 100 treatment-years. Patients with a variant genotype on acenocoumarol had a significantly increased risk for a major bleeding event (HR 1.83, 95% CI: 1.01-3.32). During the initiation phase of therapy we found no effect of variant genotype on bleeding risk. In this study among outpatients of an anticoagulation clinic using acenocoumarol or phenprocoumon, having a variant allele of CYP2C9 was associated with an increased risk of major bleeding events in patients on acenocoumarol, but not in patients on phenprocoumon. Although one might consider the assessment of the CYP2C9 genotype of a patient for dose adjustment before starting treatment with acenocoumarol, a prospective randomised trial should demonstrate whether this reduces the increased risk of major bleeding events.

Oral anticoagulant drug interactions with statins: case report of fluvastatin and review of the literature.

A 67-year-old man receiving a stable maintenance dosage of warfarin experienced an increased international normalized ratio (INR) without bleeding when his atorvastatin therapy was switched to fluvastatin. His warfarin dosage was reduced and his INR stabilized. The fluvastatin was switched back to atorvastatin, and the warfarin dosage was increased to maintain the patient's goal INR. The literature supports a drug interaction between warfarin and fluvastatin due to the strong affinity of fluvastatin for the cytochrome P450 enzyme 2D6. This interaction has not been seen with atorvastatin. Lovastatin also reportedly has caused increases in INR when coadministered with warfarin. It is unclear whether simvastatin interacts with warfarin, but it may increase INRs slightly or increase serum simvastatin levels. One case report describes an interaction between simvastatin and the anticoagulant acenocoumarol, which resulted in an elevated INR. Pravastatin does not appear to interact with warfarin but has caused an increased INR when combined with the anticoagulant fluindione. Thus, until more definitive data are available, clinicians should monitor the INR closely after starting statin therapy in any patient receiving anticoagulation therapy.

Pharmacokinetic study of the digoxin-acenocoumarol interaction in rabbits.

A study was carried out to evaluate the potential pharmacokinetic interaction between digoxin and acenocoumarol. The binding of digoxin to rabbit cardiac tissue homogenates was assessed in vitro, using the equilibrium dialysis technique. An increase in the first-order constant (p<0.05) and a reduction in the partition coefficient in the equilibrium situation (p<0.001) of digoxin were observed when the cardiac homogenates were previously treated with acenocoumarol. In the in vivo study, the kinetics of digoxin administered in single and multiple dosage regimens were compared in control rabbits and in rabbits treated simultaneously with acenocoumarol. Kinetic analysis of the results was performed using Non-linear Mixed Effects Modeling (NONMEM). In the presence of acenocoumarol, the population distribution volume (Vd) of digoxin was increased by 40-60%, no differences being found as regards the elimination clearance. Also, joint administration of both drugs led to a reduction in digoxin concentrations in the heart (p<0.01) at the end of the dosage regimen. Both sets of results point to the hypothesis of a hitherto unreported possible pharmacokinetic interaction between the two drugs affecting the distribution process. This interaction could lead to lower plasma digoxin levels, in view of the increased Vd, and a possible reduction in the therapeutic effect, owing to the decrease in affinity and in concentration in heart tissue.

Characterization of site I on human serum albumin: concept about the structure of a drug binding site.

Human serum albumin (HSA) possesses at least three sites or areas for high-affinity binding of drugs. Of these sites, site I was investigated by series of ultrafiltration and equilibrium dialysis experiments. Three ligands, acenocoumarol, dansyl-L-asparagine (DNSA) and n-butyl p-aminobenzoate (n-butyl p-ABE) were employed as marker ligands. Each ligand binds to a single high-affinity site on HSA, and binding studies with different pairs of the ligands revealed independent high-affinity binding. Preliminary displacement studies performed with the typical site I binding drugs warfarin, phenylbutazone and iodipamide showed different displacement patterns of the three marker ligands. These studies were followed by stringent competition experiments involving all possible combinations of the three test ligands themselves and of these and the three marker ligands. On the basis of the results obtained it seems that the acenocoumarol and DNSA binding regions correspond to the warfarin and azapropazone binding regions, respectively, of site I reported by others (Fehske, Schläfer, Wollert and Müller (1982) Mol. Pharmacol. 21, 387-393). The new binding region, represented by n-butyl p-ABE, is probably located adjacent to the acenocoumarol binding region but apart from that of DNSA. We have elaborated a model for binding site I in which we propose novel nomenclatures, region Ia, Ib, and Ic for the acenocoumarol, DNSA and n-butyl p-ABE binding regions, respectively. Furthermore, the relation between these regions and the high-affinity binding sites for other drugs have been discussed.

Human liver microsomal metabolism of the enantiomers of warfarin and acenocoumarol: P450 isozyme diversity determines the differences in their pharmacokinetics.

1. To explain the large differences in (the stereoselectivity of) the clearances of the enantiomers of warfarin and acenocoumarol (4'-nitrowarfarin) their human liver microsomal metabolism has been studied and enzyme kinetic parameters determined. The effects of cimetidine, propafenone, sulphaphenazole, and omeprazole on their metabolism has been investigated. 2. The 4-hydroxycoumarins follow similar metabolic routes and are mainly hydroxylated at the 6- and 7-position (accounting for 63 to 99% of the metabolic clearances). 3. Due to the lower Km values of R- and S-acenocoumarol and higher Vmax values of S-acenocoumarol, the overall metabolic clearances of R/S acenocoumarol exceed those of R/S warfarin 6 and 66 times respectively. 4. The metabolism of both compounds is stereoselective for the S-enantiomers, which is 10 times more pronounced in the case of acenocoumarol. 5. Except for the 7-hydroxylation of the R-enantiomers (r = 0.90; P < 0.025), the 6- and 7-hydroxylation rates of R/S warfarin do not correlate with those of R/S acenocoumarol. 6. Sulphaphenazole competitively inhibits the 7- and in some samples partly (up to 50%) the 6-hydroxylation of S-warfarin as well as the 7-hydroxylation of R- and S-acenocoumarol and the 6-hydroxylation of S-acenocoumarol (Kis ranging from 0.5-1.3 microM). 7. Omeprazole partly (40-80%) inhibits the 6- and 7-hydroxylation of R-warfarin (Ki = 99 and 117 microM) and of R- (Ki = 219 and 7.2 microM) and S-acenocoumarol (Ki = 6.1 and 7.7 microM) but not S-warfarin in a competitive manner. 8. Differences in the partial (up to 40%) inhibition of the metabolism of the enantiomers of the 4-hydroxycoumarins were also observed for the relatively weak inhibitors, propafenone and cimetidine.9. The results suggest that the coumarin ring hydroxylations of both compounds are catalysed by different combinations of P450 isozymes. The 7-hydroxylation of R/S acenocoumarol and the 6-hydroxylation of S-acenocoumarol are at least partly conducted by (a) P450 isozyme(s) of the 2C subfamily different from P450 2C9 (the main S-warfarin 7- and 6-hydroxylase).

Comparison of the rat liver microsomal metabolism of the enantiomers of warfarin and 4'-nitrowarfarin (acenocoumarol).

1. Rat liver microsomal metabolism of the enantiomers of warfarin and acenocoumarol (4'-nitrowarfarin) has been studied. The enantiomers of both compounds were hydroxylated mainly at the 6- and 7-positions. Acenocoumarol enantiomers were much better substrates for cytochromes P-450 than the corresponding warfarin enantiomers; Km values for the 6- and 7-hydroxylations were 2 to 19 times lower for R- and S-acenocoumarol than for warfarin. 2. Formation of the 6-, 7-, and 8-hydroxy-metabolites of warfarin was stereoselective for the R-enantiomer (the R/S ratio for total intrinsic clearance was about 3). 4'-Hydroxylation was not stereoselective. In contrast, formation of acenocoumarol metabolites was stereoselective for the S-enantiomer (the S/R ratio for total intrinsic clearance was about 3). 3. From the effects of phenobarbitone and methylcholanthrene induction, and inhibition by cimetidine, on in vitro metabolism of the enantiomers of both compounds, it was concluded that the differences between warfarin and acenocoumarol can be explained partly by the involvement of different enzymes.

The in vitro ketone reduction of warfarin and analogues. Substrate stereoselectivity, product stereoselectivity and species differences.

The in vitro metabolic ketone reduction of warfarin and its 4'-analogues acenocoumarol (4'-nitrowarfarin) and 4'-chlorowarfarin has been investigated using microsomal and cytosolic fractions of several species. Both subcellular fractions showed ketone reductase activity. The cytosolic fractions, in most species, exhibited strong substrate stereoselectivity as well as product stereoselectivity, i.e. the R(+)enantiomer was preferred as a substrate to be reduced mainly to the RS alcohol. Phenobarbital and methylcholanthrene induced cytosolic ketone reductase activity in the rat 5- to 11-fold and 3- to 7.4-fold, respectively. The microsomal fractions also showed substrate and product stereoselectivity. Contrary to the cytosolic fractions a general pattern for substrate and product stereoselectivity could not be seen. Stereoselectivity seemed species dependent (e.g. sheep vs bovine and pig). Rat liver microsomes showed practically no ketone reductase activity. Induction by phenobarbital or methylcholanthrene resulted in only a slight rise, if any, in rat microsomal ketone reductase activity. Both cytosolic and microsomal ketone reductases proved to be NADPH dependent. Substitution of the 4'-hydrogen of warfarin resulted in a change in reduction rates and in some cases, even in a change in substrate or product stereoselectivity. The data indicate that microsomal ketone reductases are different from cytosolic ketone reductases. Prelog's rule for product stereoselectivity of metabolic ketone reduction, when applied to the ketone reduction of the warfarin analogues did not agree with all data presented here.

Phenylbutazone-hydroxycoumarol interactions. Effects on steady state disposition, hepatocellular distribution, and biliary excretion of (S)-acenocoumarol in rats.

The effect of phenylbutazone on the disposition of (S)-acenocoumarol in the rat was studied at steady state conditions of distribution and elimination. (S)-Acenocoumarol was administered by constant rate infusions (1 microgram/min). The biliary excretion of 6- and 7-hydroxylated acenocoumarol was followed and the intrahepatic distribution was investigated. Phenylbutazone (50 mg/kg) increased the plasma unbound fraction about 4-fold. (S)-Acenocoumarol plasma clearance was enhanced (2.8 +/- 0.15 vs. 1.54 +/- 0.14 ml/min) but the unbound plasma clearance was reduced by 50% (67 +/- 9 vs. 140 +/- 27 ml/min). Phenylbutazone caused an intrahepatic redistribution of (S)-acenocoumarol, i.e. the drug shifted from the cytosol to the 10,000g pellet. The cytosolic unbound concentration, however, was increased. The (S)-acenocoumarol content in the microsomal fraction was not affected. The biliary excretion rate of total metabolite (free plus conjugated) comprised 50% of the (S)-acenocoumarol infusion rate in controls and was slightly stimulated (+20%) by phenylbutazone. The biliary excretion of free metabolites, however, was greatly increased (62 +/- 7 vs. 22 +/- 6 ng/min for 6-hydroxy-acenocoumarol; 337 +/- 38 vs. 141 +/- 32 ng/min for 7-hydroxy-acenocoumarol). This effect is probably due to stimulation of a hepatic biliary transport system; the rate constant for transport of 7-hydroxy-acenocoumarol was enhanced 5-fold (0.107 +/- 0.03 vs. 0.021 +/- 0.007 min-1).

The biliary excretion of acenocoumarol in the rat: stereochemical aspects.

Within 24 h, 50% of a single dose of the acenocoumarol enantiomers was recovered in bile and 20% in urine of Wistar rats. The elimination products were mainly (greater than 90%) the 6- and 7-hydroxyacenocoumarol as conjugates in the bile but free in the urine. Only R-acenocoumarol, free and conjugated, was excreted in bile. There were no gross differences between the enantiomers in metabolic pattern or in the amount of metabolites formed. A significant difference was observed for the biliary excretion of the 7-hydroxy metabolite; the ratio of free and conjugated 7-hydroxyacenocoumarol was three times higher for the S- than for the R-isomer. An unknown third metabolite was recovered in bile in higher amounts with the S- than with the R-acenocoumarol. Only traces of this metabolite were recovered from urine. The data show an extensive biliary excretion of acenocoumarol and demonstrate stereoselective mechanisms in the excretion processes.

Apparent dose dependency of the hepatic (S)-acenocoumarol clearance in the rat: a study using open liver biopsies.

Saturable hepatic uptake processes may account for the apparent dose-dependent clearance of 4-hydroxycoumarins. We investigated the dose dependent clearance and dose dependent liver distribution of the (S)-enantiomer of acenocoumarol (4-hydroxy-3-[1-4-nitrophenyl)-3-oxobutyl]coumarin) in the rat applying the in situ liver biopsy technique. The drug was administered by constant-rate infusion. At infusion rates below 0.6 microgram/min, blood clearance appeared to be dose dependent, i.e., clearance of (S)-acenocoumarol declined gradually from 6.5 mL/min at a 0.15 microgram/min infusion rate to 3.9 mL/min at a 0.6 micrograms/min infusion rate. From 0.6 microgram/min up to the highest input tested, i.e., 15 micrograms/min, clearance was almost constant, 3.5 mL/min. At low infusion rates the steady-state liver concentration of (S)-acenocoumarol rose steeply in a convex way with infusion. Scatchard analysis of steady-state liver concentrations in relation to steady-state blood concentrations revealed a hepatic binding site for (S)-acenocoumarol, exhibiting a capacity of 1.4 microgram/g of liver tissue.