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Acenocoumarol (Sintrom®) is an oral anticoagulant prescribed for the treatment of a variety of thromboembolic disorders such as atrial fibrillation and thrombosis or embolism. It inhibits fibrin production preventing clot formation. Acenocoumarol has a narrow therapeutic range, and its effects depend on several factors, such as body weight, age, metabolism, diet, certain medical conditions or the intake of additional drugs, among others. A higher dose may result in the risk of bleeding, while if it is too low, the risk of blood clot can increase. Complementary tools that allow the therapeutic drug monitoring (TDM) of acenocoumarol plasmatic levels from the starting of the treatment would be of paramount importance to personalize the treatment. Point-of-care (POC) devices can offer an added value in facilitating on-site monitoring (i.e. hospitals, primary care doctor or even by the patient itself) and can aid in dosage management. With this aim, we have developed a compact and simple nanoplasmonic sensing device based on gold nanodisks for the rapid monitoring of acenocoumarol, using highly specific polyclonal antibodies produced against this drug. A specific and reproducible label free indirect competitive assay has been developed and the viability of performing the evaluation directly in plasma diluted 1:1 has been demonstrated. A limit of detection (LOD) of only 0.77 ± 0.69 nM, an IC 50 of 48.2 ± 5.12 nM and a dynamic range between 3.38 ± 1.33 nM and 1154 ± 437 nM were achieved, which easily fit within the drug plasma levels of acenocoumarol, making this approach a highly attractive option for its decentralized monitoring in human plasma., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: Our aim was to determinate the acenocoumarol dose requirement in highly sensitive geriatric patients, based on a minimum of genotype (VKORC1 and CYP2C9) data., Methods: We used a Gaussian kernel density estimation test to identify patients highly sensitive to the drug and PHARMACHIP®-Cuma test (Progenika Biopharma, SA, Grifols, Spain) to determine the CYP2C9 and VKORC1 genotype., Results: All highly sensitive geriatric patients were taking ≤5.6 mg/week of acenocoumarol (AC), and 86% of these patients presented the following genotypes: CYP2C9*1/*3 or CYP2C9*1/*2 plus VKORC1 A/G, CYP2C9*3/*3, or VKORC1 A/A., Conclusion: VKORC1 A and CYP2C9*2 and/or *3 allelic variants extremely influence on AC dose requirement of highly sensitive geriatric patients. These patients display acenocoumarol dose requirement of ≤5.6 mg/week.

Chronic diseases are the major contributors to the global burden of disease and involve prodigious consumption of various drugs that usually affect platelet function. The autologous technology of plasma rich in growth factors (PRGF) provides a biological approach using autologous platelets as a reservoir and local delivery of proteins to promote tissue healing. The purpose of this study was to evaluate the effect of the consumption of acetylsalicylic acid, acenocoumarol and glucosamine sulfate on the preparation as well as on the biological properties of the PRGF technology. Clotting time and platelet activation of PRGF was evaluated. The latter was performed by flow cytometry. PRGF growth factor content and the release of various biomolecules by gingival fibroblasts were quantified by enzyme-linked immunosorbent assay. Cell proliferation was evaluated by means of a fluorescence-based method and cell migration was performed on culture inserts. None of the parameters evaluated was modified by the consumption of any of the three drugs tested; only the plasma of patients who had consumed acetylsalicylic acid and acenocoumarol expressed greater gingival fibroblast migration compared to plasma control. The intake of acetylsalicylic acid, acenocoumarol and glucosamine sulfate does not alter the preparation and biological properties of the autologous technology of PRGF.

Background and Objective: Vitamin K epoxide reductase complex, subunit 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) polymorphisms are taken into account when predicting a safe oral dose of coumarin anticoagulant therapy, but little is known about the effects of genetic predictors on the response to fluindione and acenocoumarol. The aims of this study were to characterize the relationship between fluindione and acenocoumarol concentrations and the international normalized ratio (INR) response, and to identify genetic predictors that are important for dose individualization., Methods: Fluindione concentrations, S- and R-acenocoumarol concentrations, the INR and genotype data from healthy subjects were used to develop a population pharmacokinetic-pharmacodynamic model in Monolix software. Twenty-four White healthy subjects were enrolled in the pharmacogenetic study. The study was an open-label, randomized, two-period cross-over study. The subjects received two doses of an oral anticoagulant: 20 mg of fluindione (period A) or 4 mg of acenocoumarol (period B). The pharmacokinetics and pharmacodynamics were studied from day 2 to day 3., Results: A two-compartment model with a first-order input model was selected as the base model for the two drugs. The pharmacodynamic response was best described by an indirect action model with S-acenocoumarol concentrations and fluindione concentrations as the only exposure predictors of the INR response. Three covariates (CYP2C9 genotype, VKORC1 genotype and body weight) were identified as important predictors for the pharmacokinetic-pharmacodynamic model of S-acenocoumarol, and four covariates (CYP2C9 genotype, VKORC1 genotype, CYP1A2 phenotype and body weight) were identified as predictors for the pharmacokinetic-pharmacodynamic model of fluindione. Because some previous studies have shown a dose-response relationship between smoking exposure and the CYP1A2 phenotype, it was also noted that smokers have greater CYP1A2 activity., Conclusion: During initiation of therapy, CYP2C9 and VKORC1 genetic polymorphisms are important predictors of fluindione and acenocoumarol pharmacokinetic-pharmacodynamic responses. Our result suggests that it is important to take the CYP1A2 phenotype into account to improve individualization of fluindione therapy, in addition to genetic factors.

A simple high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection has been developed and validated for simultaneous identification and quantification of three antivitamin K drugs (acenocoumarol, warfarin and phenprocoumon) in whole blood. The aim of this development was to propose an analytical technique adapted to the situations of forensic toxicology, i.e. intoxication with massive anticoagulant doses, when the usual coagulation tests could not be used. The blood sample, after spiked with prazepam as an internal standard (IS), was submitted to a liquid-liquid extraction (LLE) prior to HPLC analysis. A chromatographic separation was achieved on a C8 Symmetry column with a mobile phase consisting of an acetonitrile and phosphate buffer (pH 3.8) mixture in a gradient mode. Detection was carried out at a wavelength between 200 and 400 nm. This method has been validated with the concept of total error as decision criterion. Trueness ranged from 99.1% to 105.0% and precision was good with RSD between 1.3% and 6.7%. Consequently, this rapid and simple chromatographic technique is well adapted to focus intoxications with most important coumarinic drugs available on pharmaceutical market and is now routinely used in our laboratory for forensic "general unknown" screening.

Objective: Aliskiren is a direct renin inhibitor approved for the treatment of hypertension. This study investigated the effects of aliskiren on the pharmacokinetics and pharmacodynamics of a single dose of acenocoumarol in healthy volunteers., Methods: This two-sequence, two-period, randomized, double-blind crossover study recruited 18 healthy subjects (ages 18-45) to receive either aliskiren 300 mg or placebo once daily on days 1-10 of each treatment period and a single dose of acenocoumarol 10 mg on day 8. Treatment periods were separated by a 10-day washout. Blood samples were taken frequently for determination of steady-state plasma concentrations of aliskiren (LC-MS/MS) and of R(+)- and S(-)-acenocoumarol (HPLC-UV), prothrombin time (PT) and international normalized ratio (INR)., Results: Co-administration with aliskiren had no effect on exposure to R(+)-acenocoumarol. Geometric mean ratios (GMR; aliskiren:placebo co-administration) for R(+)-acenocoumarol AUC(0-t) and C(max) were 1.08 and 1.04, respectively, with 90% CI within the range 0.80-1.25. Co-administration of aliskiren resulted in a 19% increase in S(-)-acenocoumarol AUC(0-t) (GMR 1.19; 90% CI 0.92, 1.54) and a 9% increase in C(max) (GMR 1.09; 90% CI 0.88, 1.34). The anticoagulant effect of acenocoumarol was not affected by co-administration of aliskiren. Geometric mean ratios were 1.01 for all pharmacodynamic parameters (AUC(PT), PT(max), AUC(INR) and INR(max)), with 90% CI within the range 0.97-1.05., Conclusion: Aliskiren has no clinically relevant effect on the pharmacokinetics or pharmacodynamic effects of a single dose of acenocoumarol in healthy volunteers, hence no dosage adjustment of acenocoumarol is likely to be required during co-administration with aliskiren.

E7070 is a novel sulfonamide anticancer agent that arrests cancer cells at the G1/S boundary of the cell cycle. Three patients receiving chronic therapy with the oral anticoagulant acenocoumarol experienced bleeding and/or a prolonged prothrombin time after treatment with E7070 at a dose of 700 mg/m2 given as a 1-h infusion. In vitro studies have shown that E7070 has the potential to inhibit several cytochrome P450 (CYP)-enzymes, including CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. The major enzyme involved in the metabolism of acenocoumarol in man is CYP2C9. This study was performed to investigate the interaction between E7070 and acenocoumarol. Blood samples were obtained from two patients receiving daily oral maintenance treatment with acenocoumarol both prior to and following treatment with E7070. In addition, we incubated acenocoumarol enantiomers with pooled human microsomes with and without E7070 and measured the in vitro plasma protein binding of acenocoumarol after incubation with E7070. Pharmacokinetic parameters of acenocoumarol were calculated by noncompartmental analysis and revealed that in both patients the area under the concentration-time curve up to 24 h after the acenocoumarol administration was higher following E7070 (2.56 and 1.58 h*micromol/L) compared to the systemic exposure in the absence of E7070 (1.87 and 1.23 h*micromol/l). The formation of acenocoumarol metabolites was retarded by E7070 at already low concentrations (2.1 microM). The plasma protein binding of acenocoumarol was reduced at higher concentrations of E7070 (259 microM). These results indicate that E7070 may primarily interact with acenocoumarol by reducing its systemic clearance. Displacement of acenocoumarol's plasma protein binding by E7070 may also occur but to a minor extent. In the absence of careful monitoring this drug-drug interaction may result in hypoprothrombinemia and a hemorrhagic tendency.

We evaluated a new portable monitor (AvoSure PT PRO, Menarini Diagnostics, Firenze, Italy) developed to test the prothrombin time in capillary blood and plasma by comparing it with the standard laboratory determination. We studied 62 patients receiving acenocoumarol therapy. The international normalized ratio (INR) in capillary blood was analyzed by 2 methods: AvoSure PT PRO and Thrombotrack Nycomed Analyzer (Axis-Shield, Dundee, Scotland). Parallel studies were performed in plasma samples by a reference method using the Behring Coagulation Timer (Behring Diagnostics, Marburg, Germany). Plasma samples also were tested with the AvoSure PT PRO. Correlation was good for INR values for capillary blood and plasma samples by AvoSure PT PRO and our reference method (R2 = 0.8596) and for capillary blood samples tested by the AvoSure PT PRO and Thrombotrack Nycomed Analyzer (R2 = 0.8875). The correlation for INR in capillary blood and plasma samples by AvoSure PT PRO was 0.6939 (P < .0004). Capillary blood determinations are rapid and effective for monitoring oral anticoagulation therapy and have a high correlation to plasma determinations. AvoSure PT PRO is accurate for controlling INR in plasma and capillary blood samples, may be used in outpatient clinics, and has advantages over previous portable monitors.

Background and Objectives: Cytochrome P450 (CYP) 2C9 is one of the major CYP enzymes involved in the biotransformation of drugs, among others, the oral anticoagulant acenocoumarol. The enzyme has several polymorphisms, with the CYP2C9*2 and CYP2C9*3 variants most commonly present in white patients. Patients with the CYP2C9*3 variant are known to require a lower maintenance dose of racemic acenocoumarol. We investigated the impact of the polymorphisms CYP2C9*2 and CYP2C9*3 on the pharmacokinetics of R- and S-acenocoumarol., Methods and Results: In the first study 26 healthy volunteers with the genotype *1/*1 (n = 9), *1/*2 (n = 7), *1/*3 (n = 6), *2/*3 (n = 3), and *2/*2 (n = 1) were given 8 mg of racemic acenocoumarol as a single oral dose. Plasma R- and S-acenocoumarol concentrations were assayed at 4, 7, and 24 hours. Mean plasma S-acenocoumarol concentrations at 7 hours were higher in subjects with a variant allele; the differences were significant (P =.01) for the *1/*3 and *2/*3 genotypes. In the second study, the oral pharmacokinetics of acenocoumarol was investigated in 6 subjects (*1/*1 [n = 3] and *1/*3 [n = 3]). The mean oral clearance of S-acenocoumarol was 45% lower in the CYP2C9*1/*3 genotypes (10.9 +/- 3.0 L/h versus 19.8 +/- 3.1 L/h, P =.02). Plasma half-life was prolonged from 1.0 +/- 0.2 hours to 2.0 +/- 0.7 hours (P =.09). R-acenocoumarol pharmacokinetics did not differ between the genotypes. There was no difference in mean international normalized ratio at 24 hours, which was 1.2 in both groups. In vitro enzyme kinetics showed reduced (85%) intrinsic activity of the *3 enzyme to catalyze the hydroxylations of S-acenocoumarol. The lower activity resulted from higher Michaelis-Menten constant (2-fold) and lower maximum rate of metabolism by an enzyme-mediated reaction (by 70%). The activity of the *2 enzyme was 50% of the wild-type one., Conclusion: The results show S-acenocoumarol pharmacokinetics to be dependent on CYP2C9 polymorphism. In particular, the presence of the CYP2C9*3 allele impairs oral clearance of the coumarin.

Background: Coumarin-type anticoagulants are used for the long-term treatment and prevention of thromboembolic disorders. The identification of these drugs is crucial in patients with an increased prothrombin time of unknown origin. The aim of this study was to develop a sensitive and specific method for the simultaneous determination of phenprocoumon, acenocoumarol, and warfarin in human plasma by HPLC-electrospray ionization tandem mass spectrometry., Methods: After addition of the internal standard, p-chlorowarfarin, plasma samples were extracted using Oasis MCX solid-phase extraction cartridges. The compounds were separated on a Symmetry C18 column (Waters) with a mobile phase of acetonitrile-1 g/L formic acid (75:25 by volume) at a flow rate of 0.5 mL/min., Results: Extraction and separation of the three drugs and the internal standard were accomplished in 9 min. The overall extraction efficiency was >89% for all three compounds. The limits of detection were 1 microg/L for phenprocoumon and warfarin and 10 microg/L for acenocoumarol. Regression analysis of the calibration data revealed good correlation (r(2) >or=0.995) for all compounds. Within-run accuracies for quality-control samples were +/- 1% to 7% of the target concentration, with CVs <9%., Conclusions: The proposed method enables the unambiguous identification and quantification of phenprocoumon, warfarin, and acenocoumarol in both clinical and forensic specimens. This method combines a new, rapid solid-phase extraction procedure with an extremely fast chromatographic analysis, which is especially advantageous for clinical laboratories.

We describe a normal-phase HPLC method for the stereospecific determination of R- and S-acenocoumarol and R- and S-phenprocoumon with S-warfarin as internal standard. The compounds were separated using a Whelk-O1 chiral stationary phase, detected by UV at 310 nm and quantified in the internal standard mode. Linearity was verified for acenocoumarol in the range of 15-2000 microg/l and for phenprocoumon from 15 to 2200 microg/l, respectively. The detection limits were 5 microg/l for all compounds. The recovery was >84% for R- and S-acenocoumarol and >74% for R- and S-phenprocoumon. The imprecision (C.V.) (50-1800 microg/l) for R- and S-acenocoumarol was <4.7% within-day and <7.8% between-day. For R- and S-phenprocoumon the respective values were <5.6% and <5.9%. The accuracy for all compounds was 96.5-110%.

Reduced vitamins K are acting as cofactors of glutamic carboxylation of procoagulant factors (II, VII, IX and X). The reduction of these vitamins is inhibited by oral anticoagulants. The response to antivitamins K is individual and may change during the treatment of a patient. The determinations of plasmatic vitamin K and acenocoumarol may help to explain the mechanism of a resistance to the anticoagulant therapy. Quantifications of vitamin K1 are realized after liquid-liquid extraction with liquid chromatography and fluorescence detection after post-column reduction. For acenocoumarol, plasma concentration is measured, after liquid-liquid extraction, by liquid chromatograph, with ion trap mass spectrometer detector.

1. An open-label study was performed to assess the effect of piroxicam on the pharmacokinetics of acenocoumarol enantiomers. 2. Eight healthy male volunteers received an oral dose of 4 mg rac-acenocoumarol on days 1 and 8, plus 40 mg piroxicam orally 2 h before the anticoagulant on day 8. R- and S-acenocoumarol, piroxicam and their metabolites were measured in plasma over a 24 h interval. 3. The pharmacokinetics of R-acenocoumarol were markedly modified by piroxicam: Cmax+28.0% (s.d.23.8), P < 0.05; AUC(0, 24 h)+47.2% (21.5), P < 0.005; and t1/2 +38.0% (34.5), P < 0.01. A concomitant decrease of CL/F was observed: -30.8% (10.0), P < 0.0001. A similar, but statistically non-significant trend, was observed on the S-enantiomer: Cmax: +9.5% (s.d.36.6), AUC(0, 24 h): + 15.4% (23.4), t1/2: +19.9% (42.0), and CL/F: -9.8% (20.5). V/F remained unchanged for both enantiomers. 4. Piroxicam plasma AUC(0, 24 h) correlated closely with R- and S-acenocoumarol AUCs on day 1 (r = 0.901, P < 0.005 and r = 0.797, P < 0.05, respectively), as well as with the difference of AUC between days 1 and 8 for R-acenocoumarol (r = 0.903, P < 0.001) and S-acenocoumarol (r = 0.711, P < 0.05). 5. Piroxicam markedly reduced acenocoumarol enantiomer clearance, with a greater effect on the more active R-isomer. This interaction, which occurs in addition to the well documented pharmacodynamic one (effect on platelets), is expected to result in increased anticoagulant effect.

Purpose: To compare two initial doses of oral anticoagulant (acenocoumarin) studying the haemorrhagic and thromboembolic episodes occurred during the first month of treatment, the mean time and necessary controls until achievement of the desired level of anticoagulation., Patients and Methods: From january 1992 to december 1993; a comparative study of two groups of patients was performed: group 1, compiling 129 patients chosen at random and retrospectively, who begun oral anticoagulant treatment with 4 daily mg of acenocoumarin; and group 2, compiling 129 patients chosen prospectively, who begun with 2 mg daily. In both groups the mean time and the number of controls performed until achieving the desired level of anticoagulation were analyzed, as well as the haemorrhagic episodes occurred during the first month of treatment, their severity (classified into major and minor ones), the level of anticoagulation when they occurred and their possible causes. In the same way the thromboembolic processes occurred during that period in both groups were studied., Results: The mean time necessary to achieve the desired level of anticoagulation was 3.8 days in group 1 and 6.3 in group 2; the mean number of controls performed in group 1 was 1.2 and in group 2 it was 1.8. We have observed 19 haemorrhagic episodes, 15 in group 1 (4 minor and 11 major); and 4 in group 2 (2 minor and 2 major). We have found significant differences with respect to the mean time (p < 0.01), number of controls (p < 0.01) and incidence of hemorrhages (p = 0.017) between groups 1 and 2. One thromboembolic episode was registered in each group: in group 1 a deep venous thrombosis and in group 2 a stroke., Conclusion: The initial daily doses of acenocoumarin of 2 mg is as effective as the 4 mg one in the prevention of thromboembolic episodes, with a significant reduction in the number of haemorrhages observed during the first month of treatment. However this produces a prolongation in the necessary mean time and more number of controls performed until the achievement of the desired level of anticoagulation.

A method for the determination of acenocoumarol in human plasma by capillary gas chromatography-mass-selective detection is described. After addition of a structurally related analogue as the internal standard, the compounds are extracted from plasma at acidic pH into toluene, back-extracted with a basic solution and re-extracted from hydrochloric acid solution with toluene, which is then evaporated to dryness. The compounds are converted into their methyl derivatives, which are determined by gas chromatography using a mass-selective detector at m/z 324 for acenocoumarol and m/z 338 for the internal standard. The reproducibility and accuracy of the method were found to be suitable over the acenocoumarol concentrations range 2.2-74 nmol/l. The method could be considered as selective for acenocoumarol in the presence of its major metabolites in plasma.

Stereoselectivities for the binding of rac-acenocoumarol to human serum albumin (HSA), alpha 1-acid glycoprotein (AGP), and human plasma were determined by chiral HPLC analysis of the ultrafiltrates on a Chiral-AGP column. The results confirmed the previously detected inverse stereoselectivities; for HSA the ratio of the enantiomeric constants was KR/KS approximately 2, while for AGP it was KR/KS approximately 0.3. In plasma the contribution of HSA dominates, although in pathological states, elevated AGP levels may compensate for stereoselective distribution.

Pharmacokinetic interaction between ponsinomycin-nicoumalone was studied in six subjects who received an 8 mg oral dose of racemic nicoumalone alone and 4 days into an oral regimen of ponsinomycin 800 mg twice daily. The concentrations of R(+) and S(-)-nicoumalone in plasma were measured using a stereospecific h.p.l.c. assay. The disposition characteristics of nicoumalone enantiomers in the control phase of this study were similar to those reported previously with the exception of the data for one subject whose oral clearance for S(-)-nicoumalone was seven times lower than those in the other subjects. A statistically significant effect of ponsinomycin on the kinetics of R(+) and S(-)-nicoumalone was not demonstrated.

1. A stereospecific h.p.l.c. assay of nicoumalone in plasma has been developed. 2. The assay was applied to a study in which 20 mg racemic nicoumalone was given orally to three volunteers and blood samples taken for 168 h. 3. The mean pharmacokinetic parameters of the individual enantiomers were: clearance/bioavailability 1.28 1 h-1, R-enantiomer; 17.5 1 h-1, S-enantiomer: volume of distribution/bioavailability 12.5 1, R-enantiomer; 22.6 1, S-enantiomer: terminal half-life 6.8 h, R-enantiomer; 0.91 h, S-enantiomer. 4. The data are consistent with a substantial first-pass hepatic loss of S-nicoumalone.

A method for the quantitative estimation of acenocoumarol in plasma is described. Plasma containing acenocoumarol, to which a known amount of gamma-oxo derivative of phenylbutazone is added as an internal standard, is acidified and extracted with ethylene dichloride. The drug and the internal standard are then back-extracted into alkali, which, in turn, is acidified and reextracted with ethylene dichloride. The organic extract is evaporated and treated with an ethereal solution of diazomethane (100 microliter). The reacted mixture is evaporated, and the residue is dissolved in 25 microliter of carbon disulfide. Aliquots (2-3 microliter) are injected into a gas chromatograph equipped with a flame-ionization detector. The methyl derivatives of acenocoumarol and the internal standard give sharp, well-separated, symmetrical peaks. The method is of sufficient sensitivity to determine 0.25 microgram/ml of the drug in plasma with a relative standard deviation of 4%.

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.

A method based on solvent extraction, formation of a fluorinated derivative and quantitation by gas-liquid chromatography with electron-capture detection has been developed for the determination of acenocoumarin in plasma. The specificity and sensitivity of the procedure appear to be satisfactory for drug level measurements in human plasma. Its relative simplicity permits its use in routine analysis.

Marcumar [3-(1-phenylpropyl)-4-hydroxycoumarin] was determined by means of high-performance liquid chromatography from serum extracts after the oral application of its sodium salt solution. 4-Hydroxycoumarin was used as the internal standard and rats were used as the test animals. Furthermore mixtures containing anticoagulants and anti-epileptics were also analysed with the help of high-performance liquid chromatography.

The interactions of acidic and basic drugs with alpha 1-acid glycoprotein (alpha 1-AGP) were investigated using circular dichroism (CD) measurements. Extrinsic Cotton effects were generated by the binding of drugs to alpha 1-AGP. The CD data suggested the presence of a single binding site on the alpha 1-AGP molecule. The induced ellipticities of the acidic drug-alpha 1-AGP system decreased with increasing pH, while the ellipticities for the basic drugs increased with pH. The ellipticities for all drugs were reduced by the addition of fatty acids. Furthermore, the induced ellipticities decreased in the presence of cesium chloride for basic drugs bound to alpha 1-AGP. The extrinsic Cotton effects therefore appear to result from hydrophobic interaction with alpha 1-AGP for the acidic drugs and from hydrophobic and electrostatic interactions for the basic drugs.

The effect of pentobarbitone anaesthesia and the effect of hypothermia associated with the anaesthesia on the blood clearance of indocyanine green (ICG) and of S(-)-acenocoumarol (AC) was investigated in male Wistar rats using the constant rate infusion technique. During anaesthesia, body temperature was regulated by heat supply. At normothermia (rectal temperature of 37.5 degrees C), pentobarbitone anaesthesia decreased ICG clearance from 8.0 +/- 1.7 ml min-1 (mean +/- s.e.m., n = 3) in the conscious state to 4.3 +/- 0.6 ml min-1 and AC clearance from 4.9 +/- 0.4 ml min-1 to 3.4 +/- 0.3 ml min-1. Hypothermia further reduced the clearances of the compounds to 0.9 +/- 0.1 and 2.5 +/- 0.2 ml min-1 for ICG and AC, respectively. The effect of hypothermia was reversible. The results show that pharmacokinetic constants obtained in the anaesthetized animal may differ greatly from those in the conscious one. If pharmacokinetic experiments are performed in the anaesthetized animal, body temperature should be controlled and a slight hyperthermia is most favourable. Our pharmacokinetic data on ICG seriously question the use of this drug to estimate liver blood flow in the rat.

The kinetics and dynamics of single doses (5 mg p.o.) of the optical isomers of acenocoumarol (R-AC and S-AC) were followed in healthy subjects and the effect on them of cimetidine 800 mg/day was also investigated. The AC enantiomers differed greatly in their pharmacokinetics. The mean residence time (MRT) of R-AC was about 10 times longer than that of S-AC, 15 h vs 1.2 h. There was no difference in the volume of distribution. Depression of blood clotting activity (Thrombotest) was observed only after administration of R-AC. The inactivity of S-AC as a vitamin K antagonist must be ascribed to its short MRT. Cimetidine did not affect the acute oral kinetics of R- and S-AC, nor did it affect the anticlotting activity of R-AC. The urinary excretion pattern of the 6- and 7-hydroxylated AC metabolites was not altered during cimetidine treatment. Although the present studies showed no effect of cimetidine on the pharmacokinetics and dynamics of acenocoumarol, the findings of Serlin et al. suggest that cimetidine should not be administered during acenocoumarol therapy.

In two different studies, the relation between plasma concentrations of acenocoumarol and apparent prothrombin levels (Quick test) was investigated, using a new specific analytical method for the assay of the drug. The prothrombin level appeared independent of the plasma concentration of the drug, which is higher in old patients (over 70 years) than in younger patients (under 51 years).

The effect of benazepril (CGS 14824), 20 mg/day orally, on the steady-state pharmaco-dynamics and plasma levels of the anticoagulants, warfarin and acenocoumarol, was studied in healthy volunteers. The anticoagulant activity of acenocoumarol was not affected by benazepril; there was an apparent slight but statistically significant reduction of the anticoagulant effect of warfarin. The magnitude of the inhibitory effect was considered not to be clinically important. There was no effect of benazepril on plasma steady-state levels of either anticoagulants.

A simple, rapid, and sensitive method for the determination fo the short-acting coumarin anticoagulant acenocoumarin in human serum is described. This drug is estracted from the acidified biological matrix together with the internal standard 5-methoxypsoralen. Separation and quantitation are performed on a high-performance liquid chromatograph with a reversed-phase column and an ultraviolet detector operating at 308 nm. Accuracy and precision are good. The lowest limit of detection is 15 microgram/L, which means that acenocoumarin concentrations can be measured in serum from the subtherapeutic to the toxic range. This method can also be used for rapid measurement of warfarin serum concentrations.

Stereoselective binding of rac-acenocoumarol to human serum albumin (HSA) and alpha 1-acid glycoprotein (alpha 1-AGP) was investigated by affinity chromatography and by combined ultrafiltration (UF) and circular dichroism (CD) methods. For HSA, the ratio of the enantiomeric constants was KR/KS = 2, while for alpha 1-AGP, KS/KR = 3.

The four sulphonamides studied--furosemide, tolbutamide, sulfafurazole and sulfonamidochlorobenzoic acid--bind to human albumin at the same sites but with decreasing affinity. These sites are also common to other drugs, namely acenocoumarin, chlorophenoxyisobutyric acid, phenylbutazone and warfarin. In plasma, the four sulphonamides considered bind mainly to albumin, but also, at higher concentrations, to globulins, to an extent that increases as their affinity for albumin lessens.

Acenocoumarin and its acetamido metabolite, after extraction at pH 4.4, were analysed by isocratic reversed-phase high-performance liquid chromatography using aqueous acetonitrile (pH 4.90) as eluent. Warfarin was used as internal standard. The amino metabolite, after back-extraction into 0.5 N HCl, was derivatized by diazotization and heat treatment. The resulting product was analysed by the same reversed-phase system. The sensitivity of the method for acenocoumarin and its amino metabolite was in the range of 20 ng/ml. To achieve this sensitivity for the analysis of the acetamido compound, the acetonitrile content of the eluent had to be lowered. The assay was applied to the analysis of plasma samples of patients under acenocoumarin therapy. The disposition of the amino compound in rats was investigated.

Acenocoumarol, to which a 14C-labeled internal standard has been added, is extracted at pH 4 into ethyl acetate-heptane (20:80 v/v), back-extracted into aqueous sodium hydroxide solution after solvent washing with a pH 7 buffer, and reextracted after acidification in the solvent mixture. It is then acetylated with 3H-acetic anhydride. The acenocoumarol acetyl derivative is separated from the metabolite derivatives by TLC, and its radioactivity is measured. The method is specific and sensitive to a concentration of 8 ng/ml.

Reversed-phase liquid chromatography was performed on C18 column using a pH 4 buffered water-methanol 45:55 (v/v) mobile phase and ultraviolet detection (307 nm). Warfarin was used as internal standard and dichloro-1,2 ethane as extraction solvent. The analytical recovery was greater than 98 p. cent. The within-day coefficient of variation (CV) was 5 p. cent, while the between day CV was 6.4 p. cent. The limit of detection was 0.02 mg/l.