Point-of-care coagulation testing for assessment of the pharmacodynamic anticoagulant effect of direct oral anticoagulant.

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.

Prediction of phenprocoumon maintenance dose and phenprocoumon plasma concentration by genetic and non-genetic parameters.

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.

Successful emergency reversal of phenprocoumon anticoagulation with prothrombin complex concentrate: a prospective clinical study.

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.

Microfluidic coagulation assay for monitoring anticoagulant therapy in acute stroke patients.

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.

[Partial resistance to acenocoumarol and phenprocoumon caused by enzyme polymorphism]. / Partiële acenocoumarol- en fenprocoumonresistentie door enzympolymorfisme.

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.

Phenylbutazone and sulfinpyrazone interaction with oral anticoagulant phenprocoumon.

To compare the marked hypoprothrombinemic augmentation in man of racemic warfarin sodium by the pyrazolons phenylbutazone and sulfinpyrazone with that of the coumarin anticoagulant phenprocoumon, these interactions were studied prospectively in six normal subjects. Large single doses of racemic phenprocoumon, 0.6 mg/kg orally, were administered with and without daily phenylbutazone, 300 mg, or sulfinpyrazone, 400 mg, beginning three days before phenprocoumon and continuing for 14 days. Daily blood samples were drawn for phenprocoumon content and one-stage prothrombin time. Phenylbutazone markedly increased both the phenprocoumon concentrations and prothrombin times, whereas sulfinpyrazone did not.

Effects of CYP2C9 polymorphisms on the pharmacokinetics of R- and S-phenprocoumon in healthy volunteers.

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.

The enantiomers of phenprocoumon: pharmacodynamic and pharmacokinetic studies.

The pharmacodynamics and pharmacokinetics of the optical enantiomers of phenprocoumon were studied in 5 normal subjects and compared to the racemic mixture. Each subject received a single oral dose of 0.6 mg/kg of racemic, S(-), and R(+) phenprocoumon. S(-) phenprocoumon was 1.6 to 2.6 times as a potent as R(+) phenprocoumon when the area under the effect/time curve was used to quantify the total anticoagulant effect per dose. Comparing the plasma concentrations that elicited the same anticoagulant effect, S(-) phenprocoumon was 1.5 to 2.5 times as potent as R(+) phenprocoumon. The anticoagulant activity of the racemic mixture was between that of the enantiomers. There was no distinct difference in the rate of elimination between the enantiomers. The apparent volume of distribution and the plasma clearance for S(-) phenprocoumon were less than those for R(+) phenprocoumon. When the binding of the enantiomers to human serum albumin was compared, S(-) phenprocoumon was more highly bound than R(+) phenprocoumon. The protein binding of racemic phenprocoumon was between that of the enantiomers. The results show that S(-) phenprocoumon is more potent anticoagulant than R(+) phenprocoumon and that the pharmacokinetic differences between the enantiomers are due mainly to differences in their distribution.

In vitro model to test the thrombogenicity of coronary stents.

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)

A gas chromatographic method for the estimation of phenprocoumon, 3-(1-phenyl-propyl)-4-hydroxycoumarin (Marcoumar, Liquamar), in human serum or plasma.

A rapid and reliable gas-chromatographic method is described for the estimation of phenprocoumon from human serum or plasma. No interference was found from other usual oral anticoagulant drugs. The method can be used for therapeutic and toxic levels. The appearance of two volatile derivatives of phenprocoumon (in constant ratio) is discussed.

Determination of phenprocoumon, warfarin and their monohydroxylated metabolites in human plasma and urine by liquid chromatography-mass spectrometry after solid-phase extraction.

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.

GLC determination of plasma concentrations of phenprocoumon.

A GLC method for the quantitative estimation of phenprocoumon from plasma is described. Plasma containing phenprocoumon, to which a known amount of phenytoin is added as the internal standard, is acidified and extracted with ethylene dichloride. The drug and the internal standard are then back-extracted into alkali, which is acidified and reextracted with ethylene dichloride. The organic extract is evaporated, and the evaporated residue is mixed with 50 mul of trimethylanilinium hydroxide in methanol. Aliquots (1-2 mul) are injected into a gas chromatography equipped with a flame-ionization detector in which the injection port is held at 325 degrees. The methyl derivatives of phenprocoumon and the internal standard give sharp, well-separated, symmetrical peaks. The method is of sufficient sensitivity to determine 0.125 mug/ml of the drug in plasma with a coefficient of variation of 7%.

Pharmacokinetics of phenprocoumon.

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.

Effects of flupirtine coadministration on phenprocoumon plasma concentrations and prothrombin time.

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.

Determination of phenprocoumon in plasma and urine using at-line solid-phase extraction-capillary electrophoresis.

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).

[46-year-old woman with multiple hematomas and bleeding of the base of the tongue: phenprocoumon poisoning]. / 46jährige Frau mit multiplen Hämatomen und Zungengrundblutung: Phenprocoumon-Intoxikation.

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.

[Computer aided dosage management of phenprocoumon anticoagulation therapy. Clinical validation]. / Rechnergestützte Dosierung in der Antikoagulationstherapie mit Phenprocoumon. Validierung für den klinischen Einsatz.

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.

Fatal intoxications by acenocoumarol, phenprocoumon and warfarin: method validation in blood using the total error approach.

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.