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Reliable detection of anticoagulation status in patients treated with non-vitamin K antagonist oral anticoagulants (NOACs) is challenging but of importance especially in the emergency setting. This study evaluated the potential of a whole-blood clotting time assay based on Surface Acoustic Waves (SAW-CT) in stroke-patients. The SAW-technology was used for quick and homogenous recalcification of whole blood inducing a surface-activated clotting reaction quantified and visualised by real-time fluorescence microscopy with automatic imaging processing. In 20 stroke or transient ischaemic attack (TIA)-patients taking NOACs kinetics of SAW-CT were assessed and correlated to other coagulation parameters (PT, aPTT) and NOAC-plasma concentration measured by tandem mass spectrometry (LC-MS/MS). In 225 emergency patients with suspicion of acute stroke or TIA, SAW-CT values were assessed. Mean (± SD) SAW-CT in non-anticoagulated stroke patients (n=180) was 124 s (± 21). In patients on dabigatran or rivaroxaban, SAW-CT values were significantly higher 2 and 8 hours (h) after intake rising up to 267 seconds (s) (dabigatran, 2 h after intake) and 250 s (rivaroxaban, 8 h after intake). In patients on apixaban, SAW-CT values were only moderately increased 2 h after intake (SAW-CT 153 s). In emergency patients, SAW-CT values were significantly higher in NOAC and vitamin K antagonist (VKA)-treated as compared to non-anticoagulated patients. In conclusion, the SAW-CT assay is capable to monitor anticoagulant level and effect in patients receiving dabigatran, rivaroxaban and the VKA phenprocoumon. It has a limited sensitivity for apixaban-detection. If specific SAW-CT results were used as cut-offs, SAW-CT yields high diagnostic accuracy to exclude relevant rivaroxaban and dabigatran concentrations in stroke-patients.

Background: This investigation was carried out with already available point-of-care testing (POCT) systems for coagulation parameters to evaluate the qualitative and semiquantitative determination of the time- and concentration-dependent anticoagulant effects of the direct oral anticoagulants rivaroxaban and dabigatran., Methods: The whole blood prothrombin time (PT), activated partial thromboplastin time (aPTT), and activated clotting time (ACT) were determined using the GEM PCL Plus coagulation system. Whole blood PT was also measured on the CoaguCheck XS instrument. In addition, PT and aPTT values were obtained in citrated plasma using the PT reagent Neoplastin Plus and the STA APTT reagent. Drug concentrations of rivaroxaban and dabigatran were determined with a chromogenic anti-Xa assay and the hemoclot assay, which are reported to have good agreement with liquid chromatography coupled with tandem mass spectrometry measurements. POCT was performed in 27 consecutive patients who received rivaroxaban 10, 15, or 20 mg once daily and in 15 patients receiving dabigatran 110 or 150 mg twice daily. Blood samples were collected predose and 2 hours after observed drug intake at steady state., Results: Two hours after observed rivaroxaban administration, the whole blood PT measured on the GEM PCL Plus was prolonged by an average of 64.5% in comparison with predose levels. Less differentiation was observed for rivaroxaban when the PT was measured on the CoaguCheck XS instrument or in plasma (prolongation of 24.1% and 36.8%, respectively). After 2 hours observed dabigatran administration, the whole blood aPTT was comparable with plasma values and was prolonged by 23.5% in comparison with trough values. Significant concentration-dependent prolongations of the activated clotting time were observed to different extents for both direct anticoagulants., Conclusions: Direct oral anticoagulants display variable ex vivo effects on different POCT-assays. POCT for aPTT is sensitive to increased concentrations of dabigatran, whereas the PT-POCT assessed with test systems such as the GEM PCL Plus may be helpful to measure the pharmacodynamic anticoagulant effects of rivaroxaban in emergency clinical situations.

Unlabelled: A recently developed multiparameter computer-aided expert system (TheMa) for guiding anticoagulation with phenprocoumon (PPC) was validated by a prospective investigation in 22 patients. The PPC-INR-response curve resulting from physician guided dosage was compared to INR values calculated by "twin calculation" from TheMa recommended dosage. Additionally, TheMa was used to predict the optimal time to perform surgery or invasive procedures after interruption of anticogulation therapy., Results: Comparison of physician and TheMa guided anticoagulation showed almost identical accuracy by three quantitative measures: Polygon integration method (area around INR target) 616.17 vs. 607.86, INR hits in the target range 166 vs. 161, and TTR (time in therapeutic range) 63.91 vs. 62.40 %. After discontinuation of anticoagulation therapy, calculating the INR phase-out curve with TheMa INR prognosis of 1.8 was possible with a standard deviation of 0.50 ± 0.59 days., Conclusion: Guiding anticoagulation with TheMa was as accurate as Physician guided therapy. After interruption of anticoagulant therapy, TheMa may be used for calculating the optimal time performing operations or initiating bridging therapy.

Purpose: The anticoagulation response to vitamin K antagonists is characterised by high inter-individual variability. The impact of single nucleotide polymorphisms (SNPs) in several genes of enzymes involved in the vitamin K cycle on phenprocoumon dose variability and phenprocoumon plasma concentrations is still under investigation., Methods: We assessed the influence of VKORC1 c.-1639G>A, CYP2C9*2, CYP2C9*3, CYP4F2 c.1297G>A, CALU c.*4A>G, EPHX1 c.337T>C, GGCX c.214+597G>A, F7 c.-402G>A, F7 c.-401G>T, PROC c.-228C>T and PROC c.-215G>A along with clinical and demographic parameters on steady-state phenprocoumon therapy in 75 patients. A prediction model was developed for total phenprocoumon plasma concentrations and daily phenprocoumon doses required for therapeutic anticoagulation., Results: The VKORC1 c.-1639 genotype was the main predictor of the phenprocoumon daily dose (adjusted R(2) = 37.6%) and the total phenprocoumon concentration (adjusted R(2) = 38.3%). CYP2C9 affected the phenprocoumon concentration, but not the dose requirements. SNPs in the other genes of the vitamin K cycle, concomitant medication, nicotine use and alcohol consumption did not predict phenprocoumon concentrations and phenprocoumon dose requirements in a multiple linear regression model. Phenprocoumon concentrations were predicted by VKORC1 c.-1639, CYP2C9 genotype, age and BMI. The final prediction model for the daily phenprocoumon dose requirements comprised VKORC1 c.-1639 genotype, age and height accounting for 48.6% of the inter-individual variability., Conclusions: A rough prediction of phenprocoumon maintenance doses can be achieved by a limited set of parameters (VKORC1, age, height). The investigated SNPs in CYP4F2, CALU, EPHX1, GGCX, F7, and PROC did not improve the predictive value of a pharmacogenetic-based dosing equation for phenprocoumon.

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.

The present prospective study was designed to evaluate the effectiveness and safety of prothrombin complex concentrate (PCC) for emergency reversal of oral anticoagulation with phenprocoumon, a long-acting coumarin. Patients were eligible for study entry if they required emergency reversal of phenprocoumon anticoagulation because they needed invasive surgical or diagnostic procedures or were actively bleeding. Patients received one or more infusions of pasteurized nanofiltered PCC (Beriplex P/N). Primary study endpoints were changes in International Normalized Ratio, Quick value, factors II, VII, IX and X, and protein C 10, 30 and 60 min following PCC infusion. Eight adult patients were enrolled, seven requiring urgent invasive procedures and one experiencing intracranial bleeding. In the first infusion, patients received a median 3600 IU PCC at median infusion rate 17.0 ml/min. Mean (SD) baseline International Normalized Ratio was 3.4 (1.2). The International Normalized Ratio 10 min after PCC infusion declined to 1.3 or less in seven of eight patients and to 1.4 in one patient. After PCC infusion, the Quick value increased by a mean of 57% [confidence interval (CI), 45-69%], circulating factor II concentration by 85% (CI, 68-103%), factor VII by 51% (CI, 40-62%), factor IX by 61% (CI, 47-76%), factor X by 115% (CI, 95-135%) and protein C by 100% (CI, 82-117%). Clinical effectiveness of PCC treatment was rated 'very good' in seven patients and 'satisfactory' in one. No thromboembolic or other adverse events occurred. PCC treatment rapidly, effectively and safely reversed phenprocoumon anticoagulation in patients undergoing urgent invasive procedures or actively bleeding.

A 78-year-old man was treated with coumarin derivatives following myocardial infarction. The international normalised ratio was not increased by using standard loading doses and dose adjustments for acenocoumarol and phenprocoumon. The desired level of anticoagulation was achieved with a high dosage of phenprocoumon (18-21 mg daily). This dose was associated with a phenprocoumon serum concentration that was ten times higher than the normal therapeutic concentration. The serum concentration of vitamin K1 was low. After exclusion of alternative causes, we concluded that the exceptionally high dose of phenprocoumon needed was due to partial resistance to coumarin derivatives. Partial resistance is related to a polymorphism of the gene coding for the enzyme vitamin K epoxide reductase. The patient was successfully treated with chronic high-dose phenprocoumon. Resistance to coumarin derivatives caused by a congenital polymorphism in the vitamin K reductase gene is a rare phenomenon. Resistance is seldom absolute. The desired anticoagulation effect can be achieved with doses that are 10-20 times higher than standard doses. Phenprocoumon is advantageous in this situation because it requires fewer tablets than acenocoumarol. Determination of serum concentrations of acenocoumarol and phenprocoumon can be used to exclude other causes of treatment resistance.

This study was designed to develop methods for monitoring of the selective factor Xa inhibitor fondaparinux sodium (ARIXTRA) based on standard laboratory methods for the chromogenic determination of the anti-factor Xa activity of low molecular weight heparin. To examine the biologic activity of fondaparinux in comparison to its plasma concentration, 2 methods were investigated: 1 working with the addition of antithrombin (AT), the other without exogenous AT. Both methods showed a linear relationship of fondaparinux concentration and OD/min on a log-lin scale in the range from 0.1 to 2 microg/mL. Inter- and intra-assay variability was <6% in all cases. The results of spiked samples from patients on vitamin K antagonists (VKA) were in good agreement with both methods, and the determination of the fondaparinux concentration was not influenced by reduced levels of factor X in plasma caused by VKA-intake. Ex vivo samples from orthopedic patients (n=18) on prophylactic treatment with fondaparinux showed concentrations between 0.2 to 0.7 microg/mL 3 hours after s.c. injection. No significant differences were detected between both methods with these samples. The presented methods are suitable tools for monitoring of fondaparinux. The linear calibration curve in the range 0.1 to 2 microg/mL is suitable for determination of prophylactic and therapeutic application of fondaparinux. Both methods, with and without addition of AT, can be performed fully automated in clinical routine on an automated coagulation analyzer (STA coagulation analyzer). No significant differences were detected between both methods with these samples.

Phenprocoumon belongs to the group of vitamin K antagonists (VKAs), for example warfarin and acenocoumarol. It is widely used for therapeutic anticoagulation and clinically administered as a racemate. Both enantiomers are partially metabolized by the polymorphic CYP2C9 enzyme. The pharmacokinetics are, however, substantially less dependent on CYP2C9 activity or genotype than for other CYP2C9-metabolised VKAs, and pharmacokinetic differences for the enantiomers are only minor. We have investigated the stereospecific pharmacokinetics of the monohydroxylated phenprocoumon metabolites in human plasma by achiral-chiral LC-LC-MS-MS coupling. In addition to the known metabolites, 4'-, 6-, and 7-hydroxyphenprocoumon, two other monohydroxylated metabolites (M1 and M2) were detected in plasma and human liver microsomal incubations. One of these was identified as 2'-hydroxyphenprocoumon by comparison with synthetic standards; the other seemed to be a side-chain-hydroxylated derivative. Analysis of enantiomeric metabolite ratios after a single oral dose of phenprocoumon revealed changes over time with an overall preponderance of the respective (R) enantiomers. The minor role of CYP2C9 in 4'-hydroxy-PPC formation and the effect of CYP2C9 genotype for (S)-6- and (S)-7-hydroxy-PPC were confirmed. M1 and M2 are formed highly stereoselectively, without dependence on CYP2C9 genotype. These may be interpreted as alternative metabolic pathways that render phenprocoumon less dependent on CYP2C9 activity or genotype.

Many physiological processes show a high degree of stereoselectivity, including the metabolism of xenobiotics as catalyzed by cytochrome P450 enzymes. An analysis of these chiral discrimination effects in drug metabolism is essential for an in-depth understanding of metabolic pathways that differ between enantiomers of a given chiral drug or metabolite thereof. Achiral chromatographic separation and structural identification followed by chiral analysis of metabolites from blood specimens usually requires a time-consuming multistage analytical technique. In an effort to optimize such a complicated analytical scheme, a novel two-dimensional online achiral-chiral liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) coupling method was developed by using a peak parking technique in combination with a makeup flow system. Metabolites were separated in the first dimension using a C18 reversed-phase system. A makeup eluent of water/methanol (95/5) was split into the flow before storing the metabolites separately on chiral cartridges. Subsequently, the metabolite enantiomers were eluted backward onto the analytical chiral column and separated, and the ratio of enantiomers was determined. The method was successfully validated with respect to limit of detection, linearity, intra- and interday accuracy, and precision. In the course of a human volunteer study investigating the influence of CYP (cytochrome) 2C9 genetic polymorphism on phenprocoumon (PPC) metabolism, we used this new two-dimensional online analytical technique for the analysis of PPC metabolites in plasma. The enantiomeric forms of 4'-, 6-, and 7-hydroxy-PPC metabolites as well as two novel metabolites were identified, and the ratio of the enantiomers was calculated. We found that the enantiomeric ratio for the different metabolites in the plasma sample of each measured individual differs markedly from a nearly 100% chiral discrimination for the two new putative metabolites. This new analytical coupling method possesses general utility in the analysis of chiral discrimination effects, particularly as it relates to pharmacokinetics and dynamics, a scientific field that is rapidly becoming an area of concern and interest.

A high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for the quantification of phenprocoumon, warfarin, and their known monohydroxylated metabolites in human plasma and urine was developed using a simple, selective solid-phase extraction scheme. Chromatographic separation was achieved on a reversed-phase Luna C18 column and step gradient elution resulted in a total run time of about 13 min. Limits of quantification (LOQ) were < or = 40 nM for the parent compounds and < or = 25 nM for the metabolites and the limit of detection (LOD) was < or = 2.5 nM for all analytes. Average recovery was 84% (+/- 3.7) and 74% (+/- 13.2) in plasma and urine, respectively. Intra- and inter-day coefficients of variation were < or = 8.6 and < or = 10.6% in plasma and urine, respectively. The method was successfully applied to the analysis of phenprocoumon samples from four healthy volunteers and should prove useful for future comparative studies of warfarin and phenprocoumon pharmacokinetics., (Copyright 2004 Elsevier B.V.)

Phenprocoumon is a commonly used oral anticoagulant of the coumarin type, and has found extensive clinical use in the treatment of thrombophlebitis, pulmonary embolism and atrial fibrillation. In the course of a clinical study to investigate the influence of genetic polymorphisms of the CYP2C9 enzyme on phenprocoumon metabolism, we developed a new enantioselective liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/MS/MS) method to quantify (R)- and (S)-phenprocoumon in human plasma. HPLC separation of the enantiomers was achieved on a Chira-Grom-2 column under isocratic conditions using a water/acetonitrile/formic acid eluent. For detection and quantification a triple-quadrupole MS system was used in the selected reaction monitoring (SRM) mode. As an internal standard the structurally homologous compound warfarin was chosen. The detector response was linear with a correlation coefficient of 0.988-0.999 for (R)-phenprocoumon and 0.989-0.999 for (S)-phenprocoumon in the investigated concentration range between 62.5 and 1000 ng/mL (per enantiomer). The limit of detection (LOD) was 12.5 ng/mL., (Copyright 2004 John Wiley & Sons, Ltd.)

CYP2C9 catalyses the biotransformation of the oral anticoagulants S-warfarin and R- and S-acenocoumarol. According to data obtained in vitro, phenprocoumon is also metabolized by CYP2C9 but the impact of the CYP2C9 polymorphism on phenprocoumon pharmacokinetics has not been studied. Twenty-six healthy heterozygous and homozygous carriers of the CYP2C9 alleles *1 (wild-type), *2 (Arg144Cys), and *3 (Ile359Leu) received a single oral dose of 12 mg of racemic phenprocoumon. Plasma and 12 h urine concentrations of both enantiomers and their monohydroxylated metabolites were measured by high-performance liquid chromatography with mass spectrometry detection. No significant effect of the CYP2C9 variants *2 and *3 on R-phenprocoumon pharmacokinetic parameters was detected, but S-phenprocoumon clearance tended to decrease with increasing number of CYP2C9*2 and *3 alleles. The ratios of S- to R-phenprocoumon plasma clearances were higher with a median of 0.95 in carriers of *1/*1 versus 0.65 in *3/*3 (P < 0.001 for trend). Plasma and urine concentrations of 4'-, 6- and 7-hydroxyphenprocoumon were significantly lower in homozygous carriers of the CYP2C9*2 and *3 variants compared to CYP2C9*1/*1. Carriers of CYP2C9*3/*3 had a median AUC of (R,S) 7-OH-phenprocoumon of only approximately 25% compared to the wild-type genotype. The AUC of (R,S) 6-OH-phenprocoumon was only approximately 50% in CYP2C9*3/*3 compared to the homozygous wild-type genotype. In conclusion, carriers of CYP2C9*2 and *3 alleles had a lower metabolic capacity regarding phenprocoumon hydroxylation than those with CYP2C9*1/*1. However, regarding phenprocoumon hydroxylation CYP2C9 genotypes had only marginal effects on S- and R-phenprocoumon total clearance in healthy volunteers.

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%.

A 46-year old nurse complaining of multiple hematomas including bleeding into the tongue was referred for hemostasis evaluation. A very low Quick percentage value, i.e. a severely prolonged prothrombin time with severely depressed vitamin K-dependent coagulation factors (FII:C, FVII:C, FX:C) and normal FV:C and fibrinogen level was found. In the absence of cholestasis, malabsorption and broad-spectrum antibiotic therapy, ingestion of vitamin K antagonists was suspected. Three years previously, she had been on oral anticoagulant treatment with phenprocoumon (Marcoumar) for postoperative pulmonary embolism. She denied having voluntarily ingested anticoagulant drugs. A high plasma level of coumarins was found. To exclude accidental ingestion, the patient's son living in the same household was tested as well. Surprisingly, a low level of coumarin was found also in his plasma. We suspect that the patient voluntarily intoxicated herself and gave a low dose of coumarin anticoagulant to her son as well.

The use of capillary electrophoresis (CE) for the analysis of biological samples is rather problematic because of the large number of interferences present in the matrix. One of the possibilities to solve such problems is to couple solid-phase extraction (SPE) at-line with CE, a technique developed in our laboratory. In this study at-line SPE-CE is performed for the determination of the anticoagulant phenprocoumon in biological fluids. Plasma samples are injected after the addition of 1 vol.% of formic acid to release the drug from binding proteins, while urine samples can be directly injected. The procedure is linear between 0.2 and 30 microg ml(-1) with a correlation coefficient, r2, of 0.9996. The detection limit in plasma is 0.1 microg ml(-1), which is fully adequate in view of the concentrations, that have to be dealt with in practice. The phenprocoumon concentration in a plasma sample of a patient treated with the anticoagulant was 3.8 microg ml(-1).

We investigated various pharmacokinetic and pharmacodynamic parameters in a 63-year-old man, resistant to warfarin, fluindione, acenocoumarol and phenprocoumon. Daily doses of up to 30 mg of the long-acting phenprocoumon yielded a drug concentration of 85 mg/l (usual range 1-5 mg/l) but the international normalized ratio remained around 1. The plasma half-life of phenprocoumon was approximately 350 h (normal 120-150 h). Thus, the resistance was not due to malabsorption or to an accelerated metabolism of the drug. The level of vitamin K1 (1,202 ng/l) was insufficient to induce resistance. Decarboxyprothrombin concentrations were low, demonstrating that the gamma-carboxylation of the precursors of the vitamin K-dependent coagulation factors was not effectively reduced. The concentration of vitamin K epoxide, normally increased under oral anticoagulation, correlated to the vitamin K concentration (r2 = 0.77) but the quotient epoxide/vitamin K remained 4-fold lower than that of 22 warfarin-sensitive patients, suggesting an absence of blockade of the vitamin K reductase by phenprocoumon. This resistance to all the molecular forms of the vitamin K antagonists is most likely due to a reduced affinity of the drugs to a mutant vitamin K reductase.

The influence of flupirtine, a non-opioid, centrally acting analgesic agent on phenprocoumon plasma levels and protein binding as well as prolongation of prothrombin time has been investigated in 12 healthy male volunteers. Subjects received phenprocoumon 1.5 mg od over 28 days. From day 15 to 28 oral flupirtine 100mg tid was added. Phenprocoumon plasma levels and prothrombin time (Quick time), measured at trough before the morning drug intake, were chosen as primary pharmacokinetic and pharmacodynamic variables. In addition, phenprocoumon protein binding and the eudismic ratio of phenprocoumon were determined. The mean values from data obtained on day 12 to 15 (i.e. measurements under phenprocoumon alone) and the mean values from those data obtained from day 25 to 28 (i.e. under comedication with flupirtine) were subject to subsequent statistical procedures. Phenprocoumon plasma concentrations came to 1.38 +/- 0.28 micrograms/ml on day 12 to 15 and were not significantly altered under flupirtine coadministration with 1.48 +/- 0.36 micrograms/ml on day 25 to 28 (95% confidence interval: 1.02-1.11, point estimator: 1.06). The average Quick time came to 68 +/- 10% on day 12-15 and 73 +/- 15% on day 25-28. The nonparametric 95%-confidence interval for the ratio ranged between 0.96 and 1.14, the point estimator was determined to 1.04. Protein binding of phenprocoumon was determined to 88.8% +/- 0.5 on day 14 and to 88.9 +/- 0.5% on day 28. The ratio of S/R-phenprocoumon was 1:0.84 on day 14 and 1:0.84 on day 28. These results do not provide any evidence for a pharmacokinetic and/or pharmacodynamic interaction between flupirtine and phenprocoumon.

Thrombotic occlusion is a major complication limiting the application of stents in coronary arteries. In an in vitro model we investigated the thrombogenicity of different stent materials and several medical regimens to prevent thrombotic occlusion. Experiments were conducted in a closed system of silicon tubing with circulating citrated platelet rich plasma of healthy volunteers (n = 7) and of patients (n = 7 for each condition). Patients were either treated with phenprocoumon or with high or low dose heparin in combination with aspirin alone (100 mg) or aspirin (990 mg) plus dipyridamole (225 mg). After placement of tantalum wire stents into the system platelet aggregates were visible after 13.5 +/- 3.0 min, and occlusion occurred after 15.0 +/- 3.5 min. Similarly, with implanted stainless steel stents aggregation was seen after 13.0 +/- 3.5 min and thrombosis occurred after 14.5 +/- 3.5 min (p < 0.001 vs control without stent). Microscopic examination revealed combined platelet fibrin thrombi occluding the lumen. Platelet components predominately covered stent wires, particularly at crossing points. In all experiments high-dose heparin prevented platelet aggregate formation and stent occlusion independently of additional aspirin or aspirin plus dipyridamole; perfusion time > 60 min (p < 0.001 vs no heparin). Low-dose heparin could not prevent clotting. With aspirin alone aggregates were visible after 16.0 +/- 4.0 min and clotting occurred after 23.0 +/- 5.0 min. In combination with dipyridamole aggregates were visible after 15.5 +/- 5.0 min and clotting after 21.0 +/- 4.0 min (NS vs aspirin alone). Phenprocoumon prevented platelet aggregate formation and stent occlusion; perfusion time > 60 min.(ABSTRACT TRUNCATED AT 250 WORDS)

The coumarin anticoagulant phenprocoumon (PH) and metabolites (6-, 7- and 4'-hydroxyphenprocoumon) were analysed in plasma and urine samples from anticoagulated patients using solid-phase extraction and high-performance liquid chromatography with reversed-phase columns and ultraviolet and fluorescence detection; a simpler handling of samples, higher selectivity, precision, accuracy and analytical recovery were obtained compared to analysis using liquid-liquid extraction. Similarly, a method for the analysis of PH in human breast milk was developed to assess the passage of anticoagulants into breast milk in anticoagulated lactating women.

The pharmacokinetics of phenprocoumon was studied in 24 healthy volunteers between the ages of 23 and 28 years and a mean body weight of 72 kg by intraindividual comparison of the plasma level after i.v. and oral administration of 9 mg phenprocoumon (PPC) or by the evaluation of the total plasma clearance of PPC by simultaneous measurement of the urinary excretion and of the plasma concentration after the administration of 9 mg PPC. The following mean data were obtained after i.v. injection:t1/2 alpha 0.432h, t1/2 beta 128 h, co 0.651 microgram/ml, Vd 14.41,AUCo-omega 121 micrograms x h/ml. After intake of 9 mg PPC the following mean values were measured: tmax 2.25 h, Cmax 1.01 micrograms/ml, tabs 0.553 h, co 0.865 micrograms/ml, t1/2 beta 132 h, AUC0-infinity 164 micrograms x h/ml. By comparison of the PPC plasma level with the corresponding urinary excretion, a total mean PPC clearance of 20.0 (i.v.) and 15.1 (per os) ml/h was calculated within the first 8 h post dose, while the values measured did not differ between 8 and 48 h post dose (14.8 vs 15.3 ml/h). The steep decline in plasma level after i.v. injection of PPC might be caused by an enhanced renal and hepatic elimination of the free drug to a higher degree than after oral intake, while no differences existed between both modes of administration during the phase of elimination. A nearly total absorption of PPC from the tablet formulation is suggested.

Two convenient high-performance liquid chromatography procedures are proposed for the determination of phenprocoumon (Marcumar) in serum (S-PPC) and its unbound fraction in serum water (SW-PPC). S-PPC and SW-PPC were determined in samples from 53 patients undergoing oral anticoagulant therapy who were within the therapeutic interval of the thrombotest. S-PPC concentrations ranged from 0.6 to 5.0 mg/l, SW-PPC from 1.4 to 21.1 micrograms/l. A linear correlation (r = 0.714) was found between S-PPC and SW-PPC concentrations in the patient samples, the mean free fraction being 0.30%. S-PPC and SW-PPC concentrations were intra-individually stable during therapy. A linear correlation (r = 0.710) was observed between S-PPC concentrations and dosage.

The binding equilibria of warfarin and phenprocoumon with defatted human serum albumin were studied by equilibrium dialysis in 33 mM sodium phosphate buffer, pH 7.4, 37 degrees C. The binding isotherms for both ligands were consistent with binding to two similar and independent sites in the albumin molecule. The binding affinity of warfarin was markedly increased on adding palmitic acid up to palmitate 4 mol per mol albumin and then it decreased. The binding affinity of phenprocoumon varied similarly but to a lesser degree. Serum samples were obtained from 14 patients undergoing knee joint surgery, six consecutive samples from each patient. The binding affinity of warfarin and phenprocoumon added in low concentrations to the serum samples was consistently less than to purified albumin. The binding affinity for warfarin increased slightly with increasing fatty acid concentrations during surgery, but the increase was much less than expected from the in vitro studies. The binding of phenprocoumon in the serum samples was not influenced by changing fatty acid concentration. The binding affinity for both drugs decreased markedly during the three days following surgery.

The possibility of a pharmacokinetic interaction between carvedilol and digitoxin (Study I) or phenprocoumon (Study II) has been evaluated in groups of 12 healthy volunteers. The bioavailability (Cmax, tmax, AUC) of digitoxin and phenprocoumon were assessed after a single dose, given once alone and once on day 6 of treatment with carvedilol 25 mg o.d. Cmax, tmax, AUC and Ut of carvedilol and desmethylcarvedilol were also investigated after the fifth dose of carvedilol and after the sixth dose given concomitantly with digitoxin or phenprocoumon. In Study I, the 95% confidence intervals of the ratio test versus the reference findings were; digitoxin Cmax 0.80-1.20, tmax 0.56-1.14, AUC 0.97-1.33, and for carvedilol Cmax 0.81-1.22; tmax 0.66-1.23; AUC 0.91-1.17. Formation of the active metabolite desmethylcarvedilol and the urinary recovery of carvedilol and desmethylcarvedilol were not influenced by digitoxin. In Study II Cmax and AUC of phenprocoumon were not changed after carvedilol. Cmax of carvedilol was decreased after phenprocoumon. The kinetic parameters of phenprocoumon were Cmax 0.80-1.05, tmax 0.47-2.00, AUC 0.78-1.05, and for carvedilol Cmax 0.59-1.06, tmax 0.71-1.73; AUC 0.80-1.08, respectively. The plasma levels of desmethylcarvedilol and the urinary recovery of carvedilol and desmethylcarvedilol were not influenced by phenprocoumon. The blood pressure and heart rate after carvedilol alone were not affected by concomitant administration of digitoxin or phenprocoumon.

The anticoagulant phenprocoumon (PH) and its metabolites 6-hydroxy-, 7-hydroxy- and 4'hydroxy-phenprocoumon (6-OH-PH, 7-OH-PH and 4'-OH-PH, respectively) were separately administered intravenously as racemates to rats in order to measure the potential effects of the major metabolites of PH on coagulation. Plasma samples were assayed for total concentrations of the administered compounds and the corresponding prothrombin times; kinetic parameters and anticoagulant activities were estimated using a pharmacodynamic model based on the rate of synthesis of clotting factors. The relative potencies were in the order PH > 4'-OH-PH > 6-OH-PH > 7-OH-PH the latter showing no activity. Patients on PH therapy showed lower plasma concentrations of metabolites than of PH; in humans the metabolites of PH will not contribute significantly to the overall anticoagulant activity of the drug.

Positive ion mass spectra were obtained from several coumarin oral anticoagulants (phenprocoumon, warfarin, acenocoumarol and dicoumarol) and derivatives by liquid chromatography-thermospray mass spectrometry (LC-TSP-MS) and liquid chromatography-electron impact mass spectrometry (LC-EI-MS) to assess the use of LC-MS methods for the determination of these compounds in biological materials. LC-TSP mass spectra showed a single [M + 1]+ ion with no fragmentation; LC-EI mass spectra showed fragment ions which were similar in mass and relative intensities to those obtained by conventional EI-MS. These data should serve as a basis for the development of LC-MS methods for the qualitative and quantitative analysis of coumarin anticoagulants in biological samples. LC-TSP-MS was applied to the determination of phenprocoumon in a plasma extract from an anticoagulated patient.

We have studied the pharmacokinetics of phenprocoumon with and without co-administration of frusemide and probenecid in two groups of 17 healthy volunteers. Frusemide 40 mg b.i.d. for 7 days did not interact with phenprocoumon to a significant extent. Probenecid 500 mg q.i.d. for 7 days significantly accelerated the overall elimination of phenprocoumon, as indicated by a decrease in AUC from 295 to 157 micrograms.h.ml-1, and a reduction in the fraction of the dose excreted by the kidneys. The data are consistent with inhibition of the glucuronidation of phenprocoumon by probenecid. Its accelerated elimination may be a consequence of the increased formation of hydroxylated metabolites.

The determination of the anticoagulant phenprocoumon in plasma, after acidification and extraction with 1,2-dichloroethane was effected through isocratic high-performance liquid chromatography; a C18 reversed-phase column was used as stationary phase using aqueous acetonitrile as eluent and UV detection at 313 nm; p-chlorophenprocoumon was used as internal standard. A high proportion of phenprocoumon in urine is eliminated as the glucuronide and must be hydrolyzed enzymatically before extraction; the same column and detector as for plasma were used, but with gradient elution. The method was used in the range 0.1-5 mg/1, the sensitivity was 0.1 mg/1 for plasma and 0.02 mg/1 for urine, the precision was in the range 3-5% and the absence of interference due to other anticoagulants, drugs or endogenous compounds allows the specific determination of phenprocoumon in plasma and urine from patients and volunteers in clinical relevant cases, drug interaction, compliance, toxicological and pharmacokinetic studies.

Observations of a variable anticoagulatory response in patients being treated by phenprocoumon and wheat bran resulted in a study of seven healthy volunteers. Up to 168 h after the administration of phenprocoumon, both in the fasting state and following the ingestion of 35 g wheat bran in a crossover design, blood samples were taken and urine was collected. The usual pharmacokinetic parameters were calculated from the concentrations measured. Following the ingestion of wheat bran, a decreased absorption rate for phenprocoumon but no decrease in overall bioavailability was observed. In addition, a decrease in total body clearance of phenprocoumon and an increase in the free plasma fraction of phenprocoumon, resulting in decreased free drug clearance, was seen after wheat bran administration. No differences were observed in the urinary excretion of either phenprocoumon or its metabolite. These findings cannot be completely explained by our present knowledge and need further investigation.

In 23 female and male healthy volunteers the pharmacokinetic behaviour of [3H]phenprocoumon and [3H]phytomenadion was investigated. The time-dependent changes in plasma radioactivity and the amount of urinary excretion were measured. Under simultaneous administration of phytomenadion (PHY) in daily doses of 10 mg, the elimination of phenprocoumon (PPC) was not altered (t0.5 beta 141.4 h, CUE50 20.4% of administered dose) as compared with data of previous investigations, while its anticoagulant effect was inhibited. PHY in doses of 10 mg was absorbed quickly and eliminated with a mean half-life of 60.6 h at a total body clearance (Cltot) of 9.3 ml/min and a renal clearance (Clren) of 1.9 ml/min. Under the co-administration of PPC the vitamin was eliminated with a half-life time of 23.1 h (Cltot 16.7 ml/min) and the amount of excreted radioactivity was enhanced (26.3% of administered dose; Clren 4.5 ml/min) as compared with the data of PHY without co-medication. From the experiments it is concluded that (1) PHY inhibits the action of PPC without influencing its pharmacokinetics, and (2) that PPC distinctly changes the pharmacokinetics of PHY and diminishes its pharmacodynamic action as measured by changes in the prothrombin complex activity.

The construction of two ultrafiltration devices which, under application of high-pressure liquid chromatographic (HPLC) technique, allow the determination of unbound portions of drugs in albumin solutions is described. The usefulness of this method and its advantages compared with prevailing methods are demonstrated by means of determination of the unbound portion of carbamazepine, chlorpromazine, phenobarbital and phenprocoumon in buffered bovine serum albumin solutions (4%). The binding of each drug to albumin was characterised by determining the overall binding constant (K1), the apparent binding constant (k+), the free reaction energy (delta F0), the number of binding sites per albumin molecule (n), the rate of taking up of drug by albumin (V) and the unbound portions of the drug (alpha).

The interaction of benzodiazepine derivatives with phenprocoumon (Marcumar) was investigated after a single dose of the combination of phenprocoumon/diazepoxide (Librium), phenprocoumon/diazepam (Valium) and phenprocoumon/nitrazepam (Mogadan) had been applied to rats. By means of the high pressure liquid chromatography (HPLC) the change of concentration of these pharmaceutical agents per unit time in serum was determined and related to the corresponding change of prothrombin-time. Hence it can be concluded that the benzodiazepine derivatives influence the distribution of phenprocoumon in the organism.

A specific thin-layer chromatographic assay for phenprocoumon has been developed with a sensitivity of 5 ng/ml of plasma, using only 0.2 ml. This sensitivity is more than 20 times higher than that of the published methods. The drug is extracted from acidified plasma, an aliquot of the extract is applied to a silica-gel thin-layer plate and separated from interfering substances. The quantity of phenprocoumon is determined by fluorescence densitometry in situ. The standard deviation of the whole procedure is less than +/- 3%. The new procedure permits pharmacokinetic studies with low doses of phenprocoumon to be performed on volunteers. Furthermore, due to the high sensitivity of the method, it is possible to determine the free drug fraction of this highly protein-bound substance in the plasma of patients. It was shown that, in the therapeutic concentration range, phenprocoumon is bound by about 99.5% to the plasma proteins. Since the assay is simple and quick to perform, a large series of plasma samples can be analysed without any problems.

Pooled plasma from patients receiving phenprocoumon anticoagulant therapy was extracted and the following substances were characterized: phenprocoumon, and its 7-hydroxy,4'-hydroxy and 6-hydroxy derivatives; they were identified by HPLC and after methylation by quartz capillary GC-MS using the electron impact and selective ion monitoring modes. This is the first occasion when phenprocoumon metabolites have been identified in plasma; they were unconjugated and in much lower concentrations (43.2 and 2 ng/ml for the 7,4' and 6-hydroxy derivatives, respectively) than the original compound (2000 ng/ml).

Phenprocoumon was given orally to 9 patients with biopsy proven liver cirrhosis (dose range 0.12-0.25 mg/kg) and to 7 healthy volunteers (0.23 mg/kg). Concentrations of phenprocoumon were determined using HPLC in plasma and urine samples obtained for 6-7 days after drug administration. The binding of [3H]-phenprocoumon in plasma from all subjects was determined by equilibrium dialysis. Antipyrine plasma concentrations were determined spectrophotometrically following oral administration of antipyrine (1200 mg). The total body clearance of phenprocoumon was higher in the cirrhotic patients (1.64 +/- 0.16 ml/h/kg mean +/- SEM) than in the healthy volunteers (0.90 +/- 0.07 ml/h/kg), however the free drug clearance was not significantly different in the patients (144 +/- 14 ml/h/kg) compared with normal (113 +/- 11 ml/h/kg). In contrast the clearance of antipyrine was much reduced in the cirrhotic group (17.5 +/- 2.9 ml/h/kg) compared with normal (35.6 +/- 3.9 ml/h/kg). The metabolic clearance of phenprocoumon via glucuronidation, is relatively unaffected during cirrhosis compared with antipyrine clearance via oxidation.

The enantiomers of the chiral coumarin-type anticoagulants phenprocoumon, warfarin and p-chlorophenprocoumon were separated by high-performance liquid chromatography on a chiral stationary phase (Nucleosil-Chiral 2) and normal-phase conditions. Chromatographic peak identification was performed with authentic reference compounds of the enantiomers and on-line UV spectra comparison. This method was applied to the determination of the enantiomeric ratio of phenprocoumon in plasma and urine extracts from patients under racemic drug therapy. The limit of detection (50 and 80 ng/ml) and precision (less than 5%) of the method are adequate for pharmacokinetic and enantioselective disposition studies, respectively, of phenprocoumon. No racemization was detected during the extraction procedures.

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.

13 mammalian species are classified into 3 clearcut groups with respect to the stereospecific serum protein-binding of phenprocoumon: 2 groups showing opposed stereospecific binding characteristics and a 3rd group exhibiting no stereospecific binding. Structural differences in the albumin molecule account for these stereospecific differences in serum protein-binding.

The percentual binding of furosemide (5 micrograms/ml) was slightly but significantly lower in serum from elderly patients than in serum from normal blood donors (96.5 +/- 0.7 versus 97.9 +/- 0.3). A significant positive correlation was demonstrated between protein binding and albumin concentration in serum. The reduced binding in the elderly could be explained by an observed decrease in the concentration of albumin in the elderly. The percentual binding did not change in samples obtained during the first hour after the intravenous administration of 40 mg furosemide. Of 4-chloro-5-sulfamoyl antranilic acid (CSA) and antranilic acid (A) about 60% were bound to serum proteins in vitro and none of these two compounds affected the protein binding of furosemide. The addition of phenprocoumon (PPC) caused significant decreases in the percentual binding of furosemide in serum (5 microgram/ml) at PPC values of 10 microgram/ml and more. The effect of PPC on the binding of furosemide was studied in vivo in 7 patients receiving 40 mg furosemide intravenously without and with the simultaneous intravenous administration of PPC (3 mg per 10 kg body weight). In good accordance with the in vitro experiments the modest average decrease in furosemide binding, caused by concentrations of PPC not exceeding 6.2 micrograms/ml, was not significant.