Warfarin Enantiomers Research Articles

Effects of sodium ferulate for injection on anticoagulation of warfarin in rats in vivo

BackgroundHerb-drug interactions may result in increased adverse drug reactions or diminished drug efficacy, especially for drugs with a narrow therapeutic index such as warfarin. The current study investigates the effects of sodium ferulate for injection (SFI) on anticoagulation of warfarin from aspects of pharmacodynamics and pharmacokinetics in rats and predicts the risk of the combination use.MethodsRats were randomly divided into different groups and administered single- or multiple-dose of warfarin (0.2 mg/kg) with or without SFI of low dose (8.93 mg/kg) or high dose (26.79 mg/kg). Prothrombin time (PT) and activated partial thromboplastin time (APTT) were detected by a blood coagulation analyzer, and international normalized ratio (INR) values were calculated. UPLC-MS/MS was conducted to measure concentrations of warfarin enantiomers and pharmacokinetic parameters were calculated by DAS2.0 software.ResultsThe single-dose study demonstrated that SFI alone had no effect on coagulation indices, but significantly decreased PT and INR values of warfarin when the two drugs were co-administered (P < 0.05 or P < 0.01), while APTT values unaffected (P > 0.05). Cmax and AUC of R/S-warfarin decreased but CL increased significantly in presence of SFI (P < 0.01). The multiple-dose study showed that PT, APTT, INR, and concentrations of R/S-warfarin decreased significantly when SFI was co-administered with warfarin (P < 0.01). Warfarin plasma protein binding rate was not significantly changed by SFI (P > 0.05).ConclusionsThe present study implied that SFI could accelerate warfarin metabolism and weaken its anticoagulation intensity in rats.

A Physiological-Based Pharmacokinetic Model Embedded with a Target-Mediated Drug Disposition Mechanism Can Characterize Single-Dose Warfarin Pharmacokinetic Profiles in Subjects with Various CYP2C9 Genotypes under Different Cotreatments.

Warfarin, a commonly prescribed oral anticoagulant medication, is highly effective in treating deep vein thrombosis and pulmonary embolism. However, the clinical dosing of warfarin is complicated by high interindividual variability in drug exposure and response and its narrow therapeutic index. CYP2C9 genetic polymorphism and drug-drug interactions (DDIs) are substantial contributors to this high variability of warfarin pharmacokinetics (PK), among numerous factors. Building a physiology-based pharmacokinetic (PBPK) model for warfarin is not only critical for a mechanistic characterization of warfarin PK but also useful for investigating the complicated dose-exposure relationship of warfarin. Thus, the objective of this study was to develop a PBPK model for warfarin that integrates information regarding CYP2C9 genetic polymorphisms and their impact on DDIs. Generic PBPK models for both S- and R-warfarin, the two enantiomers of warfarin, were constructed in R with the mrgsolve package. As expected, a generic PBPK model structure did not adequately characterize the warfarin PK profile collected up to 15 days following the administration of a single oral dose of warfarin, especially for S-warfarin. However, following the integration of an empirical target-mediated drug disposition (TMDD) component, the PBPK-TMDD model well characterized the PK profiles collected for both S- and R-warfarin in subjects with different CYP2C9 genotypes. Following the integration of enzyme inhibition and induction effects, the PBPK-TMDD model also characterized the PK profiles of both S- and R-warfarin in various DDI settings. The developed mathematic framework may be useful in building algorithms to better inform the clinical dosing of warfarin. SIGNIFICANCE STATEMENT: The present study found that a traditional physiology-based pharmacokinetic (PBPK) model cannot sufficiently characterize the pharmacokinetic profiles of warfarin enantiomers when warfarin is administered as a single dose, but a PBPK model with a target-mediated drug disposition mechanism can. After incorporating CYP2C9 genotypes and drug-drug interaction information, the developed model is anticipated to facilitate the understanding of warfarin disposition in subjects with different CYP2C9 genotypes in the absence and presence of both cytochrome P450 inhibitors and cytochrome P450 inducers.

A genome-wide association study of plasma concentrations of warfarin enantiomers and metabolites in sub-Saharan black-African patients.

Diversity in pharmacogenomic studies is poor, especially in relation to the inclusion of black African patients. Lack of funding and difficulties in recruitment, together with the requirement for large sample sizes because of the extensive genetic diversity in Africa, are amongst the factors which have hampered pharmacogenomic studies in Africa. Warfarin is widely used in sub-Saharan Africa, but as in other populations, dosing is highly variable due to genetic and non-genetic factors. In order to identify genetic factors determining warfarin response variability, we have conducted a genome-wide association study (GWAS) of plasma concentrations of warfarin enantiomers/metabolites in sub-Saharan black-Africans. This overcomes the issue of non-adherence and may have greater sensitivity at genome-wide level, to identify pharmacokinetic gene variants than focusing on mean weekly dose, the usual end-point used in previous studies. Participants recruited at 12 outpatient sites in Uganda and South Africa on stable warfarin dose were genotyped using the Illumina Infinium H3Africa Consortium Array v2. Imputation was conducted using the 1,000 Genomes Project phase III reference panel. Warfarin/metabolite plasma concentrations were determined by high-performance liquid chromatography with tandem mass spectrometry. Multivariable linear regression was undertaken, with adjustment made for five non-genetic covariates and ten principal components of genetic ancestry. After quality control procedures, 548 participants and 17,268,054 SNPs were retained. CYP2C9*8, CYP2C9*9, CYP2C9*11, and the CYP2C cluster SNP rs12777823 passed the Bonferroni-adjusted replication significance threshold (p < 3.21E-04) for warfarin/metabolite ratios. In an exploratory GWAS analysis, 373 unique SNPs in 13 genes, including CYP2C9*8, passed the Bonferroni-adjusted genome-wide significance threshold (p < 3.846E-9), with 325 (87%, all located on chromosome 10) SNPs being associated with the S-warfarin/R-warfarin outcome (top SNP rs11188082, CYP2C19 intron variant, p = 1.55E-17). Approximately 69% of these SNPs were in linkage disequilibrium (r 2 > 0.8) with CYP2C9*8 (n = 216) and rs12777823 (n = 8). Using a pharmacokinetic approach, we have shown that variants other than CYP2C9*2 and CYP2C9*3 are more important in sub-Saharan black-Africans, mainly due to the allele frequencies. In exploratory work, we conducted the first warfarin pharmacokinetics-related GWAS in sub-Saharan Africans and identified novel SNPs that will require external replication and functional characterization before they can be considered for inclusion in warfarin dosing algorithms.

Pharmacokinetic Modeling of Warfarin ІI - Model-based Analysis of Warfarin Metabolites following Warfarin Administered either Alone or Together with Fluconazole or Rifampin.

The objective of this study is to conduct a population pharmacokinetic (PK) model-based analysis on 10 warfarin metabolites (4'-, 6-, 7-, 8- and 10-hydroxylated (OH)-S- and R- warfarin), when warfarin is administered alone or together with either fluconazole or rifampin. One or two compartment PK models expanded from target mediated drug disposition (TMDD) models developed previously for warfarin enantiomers were able to sufficiently characterize the PK profiles of 10 warfarin metabolites in plasma and urine under different conditions. Model-based analysis shows CYP2C9 mediated metabolic elimination pathways are more inhibitable by fluconazole (% formation CL (CLf) of 6- and 7-OH-S-warfarin decrease: 73.2% and 74.8%) but less inducible by rifampin (% CLf of 6- and 7-OH-S-warfarin increase: 85% and 75%), compared with non-CYP2C9 mediated elimination pathways (% CLf of 10-OH-S-warfarin and CLR of S-warfarin decrease in the presence of fluconazole: 65.0% and 15.3%; % CLf of 4'- 8- and 10-OH-S-warfarin increase in the presence of rifampin: 260%, 127% and 355%), which potentially explains the CYP2C9 genotype-dependent DDIs exhibited by S-warfarin, when warfarin is administrated together with fluconazole or rifampin. Additionally, for subjects with CYP2C9 *2 and *3 variants, a model-based analysis of warfarin metabolite profiles in subjects with various CYP2C9 genotypes demonstrates CYP2C9 mediated elimination is less important and non-CYP2C9 mediated elimination is more important, compared with subjects without these variants. To our knowledge, this is so far one of the most comprehensive population-based PK analyses of warfarin metabolites in subjects with various CYP2C9 genotypes under different co-medications. Significance Statement The studies we wish to publish are potentially impactful. The need for a TMDD pharmacokinetic model and the demonstration of genotyped-dependent drug interactions may explain the extensive variability in dose-response relationships that are seen in the clinical dose adjustments of warfarin.

Simultaneous Characterization and Determination of Warfarin and Its Hydroxylation Metabolites in Rat Plasma by Chiral Liquid Chromatography-Tandem Mass Spectrometry.

Warfarin is extensively used for venous thromboembolism and other coagulopathies. In clinical settings, warfarin is administered as a mixture of S- and R-warfarin, and both enantiomers are metabolized by multiple cytochrome P450 enzymes into many hydroxylation metabolites. Due to the high degree of structural similarity of hydroxylation metabolites, their profile possesses significant challenges. The previous methods generally suffer from lacking baseline resolution and/or involving complex analysis processes. To overcome this limitation, a sensitive and specific chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously identify warfarin and hydroxywarfarins enantiomers. Chromatographic separation was achieved on a HYPERSIL CHIRAL-OT column. The mass spectrometric detection was carried out in negative ion MRM mode with electrospray ionization source. The optimized method exhibited satisfactory within-run and between-run accuracy and precision with lower limit of quantification (LLOQ) of 10.0 ng/mL and 1.0 ng/mL for warfarin and 7-, 10(R)-OH-warfarin enantiomers, respectively. Linear responses of warfarin enantiomers and 7-, and 10(R)-OH-warfarin enantiomers in rat plasma were observed over the range of 10.0–8000 ng/mL, and 1.00–800 ng/mL, respectively. The analytes were shown to be stable in various experimental conditions in rat plasma. Protein precipitation was used in sample preparation without a matrix effect. This method was successfully applied to pharmacokinetic study for quantitating the concentrations of S/R-warfarin, S/R-7-OH-warfarin, and S/R-10(R)-OH-warfarin and relatively quantitating 3′-, 4-, 6-, and 8-OH warfarin enantiomers in rat plasma.

A novel, rapid and simple UHPLC-MS/MS method for quantification of warfarin in dried blood spots

Warfarin is a common first line anticoagulant with a narrow therapeutic window. Because of the large blood volume needed, previous warfarin determination methods were not applicable to small animals, such as mice. To reduce the number of small animals used needed, we developed and validated a sensitive rapid assay for the simultaneous detection of warfarin enantiomers in mouse dried blood spot (DBS) samples. Analytes were extracted by tert-butyl methyl ether and then separated by a chiral Cellulose-1 column with a mobile phase of 75% acetonitrile (containing 0.1% formic acid). The total chromatographic run time was 3 min. Negative mode electrospray ionization was used for MS/MS detection, where the monitored ion transitions were m/z 307.1 → 161.0 and 341.1 → 284.0 for warfarin and coumachlor (internal standard) respectively. The calibration curves were linear with a correlation coefficient of ≥0.994 for both enantiomers over a concentration range of 10–1000 ng/mL. The satisfactory accuracy and adequate reproducibility of both warfarin enantiomers were validated in terms of intra- and interday precision with mouse DBS cards. The samples were stable at room temperature for at least 14 days. The validated method was applied to a pharmacokinetic study in mice.

Validation of warfarin enantiomer analysis method in plasma using high-performance liquid chromatography fluorescence detector.

Warfarin (WF) is an anticoagulant commonly used for thromboembolism-related diseases. This study aims to assess the pharmacokinetic profile of WF. The stereospecific interaction of S-and R-WF requires quantification of the enantiomer to determine the pharmacokinetic profile. The analysis method of the enantiomers in plasma is developed using an HPLC fluorescence detector with a Chiralcel OD-RH column (4.6 mm × 150 mm i.d., 5 m) and a Chiralcel OD-RH guard column (4.0 mm × 10 mm, 5 m). The separation is conducted using isocratic with acetonitrile mobile phase: Phosphate buffer, pH 2.00 (40:60 v/v), column temperature 40°C, flow rate 1 mL/min, injection volume 50 L. WF is measured at an excitation wavelength of 310 nm and emission of 350 nm. This method results in limit of detection (LOD) values of 18.6 ng/mL and limit of quantitation (LOQ) of 62.01 ng/mL for R-WF and LOD values of 18.61 ng/mL and LOQ of 62.04 ng/mL for S-WF. The results showed a linearity in concentration between 100 and 2500 ng/mL with r2 = 0.9969 and r2 = 0.9991 for R-and S-WF. The validation requirements of selectivity, accuracy, and precision for within and between run with a value of <15% for % relative standard deviation and % diff were achieved. This method can be used in the sample measurement of WF pharmacokinetic studies.

Effects of Herba Erigerontis injection on pharmacodynamics and pharmacokinetics of warfarin in rats in vivo.

Herba Erigerontis injection (HEI) is an aqueous solution derived from whole plants of Erigeron breviscapus, which may be co-administered with warfarin to treat cardiovascular and cerebrovascular disorders. This research was conducted to make sure whether HEI would affect anticoagulation of warfarin to guarantee reasonable medication. The pharmacodynamic study was designed to measure prothrombin time (PT) and activated partial thromboplastin time (APTT) values, and international normalized ratio (INR) values were calculated. For pharmacokinetic study, ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) technology was applied to measure plasma concentrations of warfarin enantiomers. The influence of HEI on plasma protein binding rate of warfarin was assessed by ultrafiltration. Pharmacodynamic study demonstrated that both HEI alone and co-administered with warfarin could increase PT and INR values significantly (P<.01), whereas the APTT values were unaffected (P>.05). Pharmacokinetic study manifested that Cmax , AUC and t1/2 prolonged significantly (P<.01) for R/S-warfarin in presence of HEI. Low (3.6mL/kg), medium (7.2mL/kg) and high (10.8mL/kg) doses of HEI could decrease plasma protein binding rate of warfarin significantly (P<.01). The results mean that HEI can potentiate the anticoagulant response of warfarin through both pharmacodynamics and pharmacokinetics.

A physiologically based pharmacokinetic/pharmacodynamic modeling approach for drug-drug interaction evaluation of warfarin enantiomers with sorafenib

Sorafenib was suggested to cause drug-drug interaction (DDI) with the common anticoagulant, warfarin based on published studies. The inhibition on CYP2C9 enzyme was thought to be the mechanism, but further studies are warranted. Thus, a mechanistic PBPK/PD model for warfarin enantiomers was developed to predict DDI potential with sorafenib, aiming at providing reference for the rational use of both drugs.PBPK models of warfarin enantiomers were constructed by Simcyp software. A mechanistic PK/PD model was built in NONMEM software. PBPK model of sorafenib was fitted via a top-down method. The final PBPK/PD model of warfarin enantiomers was verified and validated by different dosing regimens, ethnicities and genetic polymorphisms, and used to perform DDI simulations between warfarin racemate and sorafenib among general populations and sub-populations with various CYP2C9 and VKORC1 genotypes. Results suggested low DDI risk between warfarin and sorafenib for general populations. Potentially serious consequence was seen for those carrying both CYP2C9 ∗2 and ∗3 and VKORC1 A/A genotypes.This PBPK/PD modeling approach for warfarin enantiomers enabled DDI evaluation with sorafenib. Close monitoring and warfarin dosage adjustment were recommended for patients carrying mutant genotypes. The novel model could be applied to investigate other drugs that may interact with warfarin.

Effects of Xiangdan Injection on pharmacokinetics and pharmacodynamics of warfarin in rats in vivo

This study is to explore the effect of Xiangdan Injection on anticoagulation of warfarin in rats. Rats were randomly divided into different groups and then administered, subsequently the blood samples were collected at a set series of time points to measure PT(prothrombin time) and APTT(activated partial thromboplastin time) values, and INR(international normalized ratio) value was calculated. The plasma concentrations of warfarin enantiomers were determined by UPLC-MS/MS technology, and pharmacokinetic parameters were calculated by DAS 2.0 software. Statistical analysis was performed to compare differences between the groups. Single-dose study of warfarin showed that Xiangdan Injection alone had no effects on PT, APTT and INR, but when co-administrated with warfarin, PT and INR values were increased(P&lt;0.01), while APTT was unaffected; after co-administration of the two drugs, C_(max), AUC_(0-t), and AUC_(0-∞) of S-warfarin increased(P&lt;0.01), and t_(1/2) prolonged(P&lt;0.01), while the pharmacokinetic parameters of R-warfarin were not changed significantly. Steady-state study of warfarin showed that after co-administration of the two drugs, the PT and INR values increased(P&lt;0.05), and the plasma concentration of S-warfarin increased(P&lt;0.01), while the plasma concentration of R-warfarin was not changed significantly. The results suggest that Xiangdan Injection itself has no effect on coagulation index, but can enhance the anticoagulant effect of warfarin by slowing metabolism of S-warfarin.

Chiral voltammetric sensor for warfarin enantiomers based on carbon black paste electrode modified by 3,4,9,10-perylenetetracarboxylic acid

A chiral electrochemical sensor based on carbon black paste electrode (CBPE), consisting of graphitized carbon black powder Carboblack C modified by nanoclusters of 3,4,9,10-perylenetetracarboxylic acid (PTCA) for the selective recognition and determination of warfarin (WA) enantiomers is described in this work. The spontaneous appearance of two-dimensional space group during self-assembly by an external induction of PTCA nanoclusters provides their chirality. It was found that the CBPE modified by PTCA afforded specific interaction with WA enantiomers and acted as a chiral selector, which affected the current and peak potential change. CBPE modified by PTCA was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). The electrochemical and analytical characteristics of the sensor, and conditions of the voltammogram registration were studied by differential pulse voltammetry (DPV). The experimental results indicate a linear correlation between the peak current of warfarin enantiomers and their concentration in the range from 5 μM to 0.16 mM, leading to a detection limit of 3.09 μM and 4.02 μM for R- and S-WA, respectively. The potential ability of proposed electrode for WA enantiomer determinations has been evaluated by analyzing human urine and blood plasma with a recovery ranging from 97.1% to 102.9%. The present sensor can determine ratio of WA isomer's in enantiomeric mixture.

Lack of effect of Imrecoxib, an innovative and moderate COX-2 inhibitor, on pharmacokinetics and pharmacodynamics of warfarin in healthy volunteers

Imrecoxib is a registered treatment for osteoarthritis pain symptoms in China. This study aims to assess the effect of imrecoxib on the pharmacodynamics and pharmacokinetics of warfarin. 12 healthy male volunteers with CYP2C9*3 AA and VKORC1 AA genotypes took a 5 mg dose of warfarin both alone and concomitantly with steady-state imrecoxib. Both warfarin alone and concomitantly with imrecoxib have safey and good tolerance across the trial. Following warfarin and imrecoxib co-administration, neither Cmax, AUC0-t and t1/2 of warfarin enantiomers nor AUC of international normalized ratio (INR) were markedly different from those of warfarin alone. The geometric mean ratios (GMRs) (warfarin + imrecoxib: warfarin alone) of INR(AUC) was 1 (0.99, 1.01). The GMRs of warfarin AUC0-∞ (90% confidence interval, CIs) for warfarin + imrecoxib: warfarin alone were 1.12 (1.08, 1.16) for R-warfarin and 1.13 (1.07, 1.18) for S- warfarin. The 90% CIs of the GMRs of AUC0-∞, Cmax and INR (AUC) were all within a 0.8–1.25 interval. The combination of warfarin and imrecoxib did not impact the pharmacodynamics and pharmacokinetics of single-dose warfarin; therefore, when treating a patient with imrecoxib and warfarin, it is not required to adjust the dosage of warfarin.

Effects of sulfotanshinone sodium injection on the pharmacokinetics and pharmacodynamics of warfarin in rats in vivo

This study was to explore the effects of sulfotanshinone sodium injection (SSI) on the pharmacokinetics and pharmacodynamics of warfarin in rats.The studies of single dose and multiple dose of warfarin were designed to assess the interaction between warfarin and SSI. Rats were divided into different groups randomly and administered with warfarin in the absence or presence of SSI. Prothrombin time (PT) and activated partial thromboplastin time (APTT) values were detected by blood coagulation analyzer, and international normalized ratio (INR) values were calculated. Plasma concentrations of warfarin enantiomers were determined by UPLC–MS/MS method, pharmacokinetic parameters were calculated.The single-dose study demonstrated that the repeated doses of SSI alone had no effect on PT, APTT and INR values, but had a significant effect on PT and INR values produced by a single dose of warfarin, APTT values were unaffected. The Cmax, AUC of R-warfarin and S-warfarin were reduced, t1/2 were shortened. The multiple-dose study showed that PT, APTT, INR values, and the Cmax and AUC of R-warfarin and S-warfarin decreased significantly after administration of SSI.The finding implied that SSI could accelerate warfarin metabolism and weaken its anticoagulation. However, human SSI–warfarin interaction studies need to be conducted to confirm this finding.

Effect of genetic and patient factors on warfarin pharmacodynamics following warfarin withdrawal: Implications for patients undergoing surgery

IntroductionWarfarin therapy is stopped for a fixed period prior to surgery to minimise risk of perioperative bleeding. However, anticoagulation subsides at varying rates among different patients. We evaluated the influence of genetic (CYP2C9 and VKORC1), patient and clinical factors on warfarin clearance and the decline in INR following warfarin withdrawal. Materials and methods131 patients completing a course of warfarin provided blood samples over 9 days for initial genotyping, and measurement of INR and plasma warfarin enantiomer concentrations. ResultsS-warfarin clearance was significantly lower in patients with either CYP2C9 single (*2 or *3) or double (*2*2 or *2*3) variant alleles compared to those with wild-type genotype (P < 0.001). Regression analysis revealed that patient age (P = 0.037) and CYP2C9 *2*2 & *2*3 genotype (P = 0.005), but not VKORC1 genotype, significantly affected the time taken for the resumption of normal coagulation (INR value declining to ≤1.5). ConclusionsThe inter-individual variability in the time needed for normal coagulation to resume following warfarin withdrawal is influenced, in the main, by variance in S-warfarin clearance, which in turn is affected by CYP2C9 polymorphism and age. Cost-effectiveness of pharmacogenetics-based algorithms incorporating CYP2C9 genotype and patient age could be increased if used not only to guide dosing decisions but also estimation of the correct length of time needed for individual patients to stop taking warfarin prior to surgery.

Predicting the Prevalence of Alternative Warfarin Tautomers in Solution.

Warfarin, a widely used oral anticoagulant, is prescribed as a racemic mixture. Each enantiomer of neutral Warfarin can exist in 20 possible tautomeric states leading to complex pharmacokinetics and uncertainty as to the relevant species under different conditions. Here, the ability of alternative computational approaches to predict the preferred tautomeric form(s) of neutral Warfarin in different solvents is examined. It is shown that varying the method used to estimate the heat of formation in vacuum (direct or via homodesmic reactions), whether entropic corrections were included, and the method used to estimate the free enthalpy of solvation (i.e., PCM, COSMO, or SMD implicit models or explicit solvent) lead to large differences in the predicted rank and relative populations of the tautomers. In this case, only a combination of the enthalpy of formation using homodesmic reactions and explicit solvent to estimate the free enthalpy of solvation yielded results compatible with the available experimental data. The work also suggests that a small but significant subset of the possible Warfarin tautomers are likely to be physiologically relevant.

Role of Hepatic Organic Anion Transporter 2 in the Pharmacokinetics of R- and S-Warfarin: In Vitro Studies and Mechanistic Evaluation.

Interindividual variability in warfarin dose requirement demands personalized medicine approaches to balance its therapeutic benefits (anticoagulation) and bleeding risk. Cytochrome P450 2C9 ( CYP2C9) genotype-guided warfarin dosing is recommended in the clinic, given the more potent S-warfarin is primarily metabolized by CYP2C9. However, only about 20-30% of interpatient variability in S-warfarin clearance is associated with CYP2C9 genotype. We evaluated the role of hepatic uptake in the clearance of R- and S-warfarin. Using stably transfected HEK293 cells, both enantiomers were found to be substrates of organic anion transporter (OAT)2 with a Michaelis-Menten constant ( Km) of ∼7-12 μM but did not show substrate affinity for other major hepatic uptake transporters. Uptake of both enantiomers by primary human hepatocytes was saturable ( Km ≈ 7-10 μM) and inhibitable by OAT2 inhibitors (e.g., ketoprofen) but not by OATP1B1/1B3 inhibitors (e.g., cyclosporine). To further evaluate the potential role of hepatic uptake in R- and S-warfarin pharmacokinetics, mechanistic modeling and simulations were conducted. A "bottom-up" PBPK model, developed assuming that OAT2-CYPs interplay, well recovered clinical pharmacokinetics, drug-drug interactions, and CYP2C9 pharmacogenomics of R- and S-warfarin. Clinical data were not available to directly verify the impact of OAT2 modulation on warfarin pharmacokinetics; however, the bottom-up PBPK model simulations suggested a proportional change in clearance of both warfarin enantiomers with inhibition of OAT2 activity. These results suggest that variable hepatic OAT2 function, in conjunction with CYP2C, may contribute to the high population variability in warfarin pharmacokinetics and possibly anticoagulation end points and thus warrant further clinical investigation.

Stereospecific Metabolism of R- and S-Warfarin by Human Hepatic Cytosolic Reductases.

Coumadin (rac-warfarin) is the most commonly used anticoagulant in the world; however, its clinical use is often challenging because of its narrow therapeutic range and interindividual variations in response. A critical contributor to the uncertainty is variability in warfarin metabolism, which includes mostly oxidative but also reductive pathways. Reduction of each warfarin enantiomer yields two warfarin alcohol isomers, and the corresponding four alcohols retain varying levels of anticoagulant activity. Studies on the kinetics of warfarin reduction have often lacked resolution of parent-drug enantiomers and have suffered from coelution of pairs of alcohol metabolites; thus, those studies have not established the importance of individual stereospecific reductive pathways. We report the first steady-state analysis of R- and S-warfarin reduction in vitro by pooled human liver cytosol. As determined by authentic standards, the major metabolites were 9R,11S-warfarin alcohol for R-warfarin and 9S,11S-warfarin alcohol for S-warfarin. R-warfarin (Vmax 150 pmol/mg per minute, Km 0.67 mM) was reduced more efficiently than S-warfarin (Vmax 27 pmol/mg per minute, Km 1.7 mM). Based on inhibitor phenotyping, carbonyl reductase-1 dominated R-and S-warfarin reduction, followed by aldo-keto reductase-1C3 and then other members of that family. Overall, the carbonyl at position 11 undergoes stereospecific reduction by multiple enzymes to form the S alcohol for both drug enantiomers, yet R-warfarin undergoes reduction preferentially. This knowledge will aid in assessing the relative importance of reductive pathways for R- and S-warfarin and factors influencing levels of pharmacologically active parent drugs and metabolites, thus impacting patient dose responses.

Correlations between the enantio- and regio-selective metabolisms of warfarin.

To clarify whether the activities of multiple CYPs associated with warfarin metabolism would be correlated with each other. Oral clearances (CLpo) of warfarin enantiomers were estimated in 378 Chinese, Caucasians and African-Americans. The partial metabolic clearances (CLm) for 7-hydroxywarfarin enantiomers were also measured. In addition, CLpo and CLm were determined in a patient on warfarin and rifampicin. Correlations between CLpo for warfarin enantiomers existed across the three populations. In addition, there was a significant correlation between the CLm for 7-hydroxylation of warfarin enantiomers. Under induced conditions by rifampicin, there were significant correlations between the enantio- and regio-selective metabolisms of warfarin. Metabolic activities of CYP2C9, CYP1A2 and CYP3A4 may be regulated by common transcriptional mechanism(s).

Enantiomeric separation and quantitation of warfarin and its metabolites in human plasma by LC-MS/MS

The enantiomeric separation of warfarin (WAR) enantiomers and its hydroxy metabolites positional isomers is described in this work. The utilization of chiral chromatography coupled to Tandem mass spectrometry helps to achieve that. The developed method was able to separate R,S -warfarin and enantiomers of 4,7,10-hydroxy warfarin in human plasma. Plasma samples were processed with simple protein precipitation with acetonitrile. The chromatographic separation was done on chiral Astec Chirobiotic V column with gradient flow of 0.1% aqueous formic acid and 0.1% formic in acetonitrile:water (95:5, v : v ). The mass spectroscopic detection was done via negative mode multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer coupled with positive electrospray ionization (ESI). Linearity of the method was valid over a concentration range of 125.00-0.13 µg/mL for racemic WAR, 47.000-0.088 µg/mL for racemic 4’-OH-WAR, 56.00-0.18 µg/mL for racemic 7-OH-WAR and 56.00-0.02 µg/mL for racemic 10-OH-WAR. The LLOQs were found to be 0.13 µg/mL for racemic WAR, 0.088 µg/mL for racemic 4’-OH-WAR, 0.18 µg/mL for racemic 7-OH-WAR and 0.02 µg/mL for racemic 10-OH-WAR. The method was validated for matrix effect, intra- and inter-day precision, freeze/thaw and storage stability. Accuracy and precision were within acceptable range (<15%). Enantiomeric forms of WAR and its metabolites were separated by the chiral column. Standards for R - and S -WAR identified peak 2 as S -WAR while metabolite peaks could not be definitively identified. Peak 2 for 7-OH-WAR gave higher blood levels, while the opposite applied to 10-OH-WAR.

Mechanism of Drug-Drug Interactions Between Warfarin and Statins

The anticoagulant drug warfarin and the lipid-lowering statin drugs are commonly co-administered to patients with cardiovascular diseases. Clinically significant drug-drug interactions (DDIs) between these drugs have been recognized through case studies for many years, but the biochemical mechanisms causing these interactions have not been explained fully. Previous theories include kinetic alterations in cytochrome P-450–mediated drug metabolism or disturbances of drug-protein binding, leading to anticoagulant activity of warfarin; however, neither the enantioselective effects on warfarin metabolism nor the potential disruption of drug transporter function have been well investigated. This study investigated the etiology of the DDIs between warfarin and statins. Liquid chromatography–mass spectrometry methods were developed and validated to quantify racemic warfarin, 6 of its hydroxylated metabolites, and pure enantiomers of warfarin; these methods were applied to study the role of different absorption, distribution, metabolism, and excretion properties, leading to DDIs. Plasma protein binding displacement of warfarin was performed in the presence of statins using equilibrium dialysis method. Substrate kinetics of warfarin and pure enantiomers were performed with human liver microsomes to determine the kinetic parameters (Km and Vmax) for the formation of all 6 hydroxywarfarin metabolites, inhibition of warfarin metabolism in the presence of statins, was determined. Uptake transport studies of warfarin were performed using overexpressing HEK cell lines and efflux transport using human adenocarcinoma colonic cell line cells. Fluvastatin significantly displaced plasma protein binding of warfarin and pure enantiomers; no other statin resulted in significant displacement of warfarin. All the statins that inhibited the formation of 10-hydroxywarfarin, atorvastatin, pitavastatin, and simvastatin were highly potent compared to other statins; in contrast, only fluvastatin was found to be a potent inhibitor of formation of 7-hydroxy warfarin. Uptake and efflux drug transporters do not play any role in these DDIs. The results showed that DDIs between warfarin and statins are primarily caused by cytochrome P-450 inhibition.