The Distribution of the Genotypes of ABCB1 and CES1 Polymorphisms in Kazakhstani Patients with Atrial Fibrillation Treated with DOAC.

Nowadays, direct oral anticoagulants (DOACs) are the first-line anticoagulant strategy in patients with non-valvular atrial fibrillation (NVAF). We aimed to identify the influence of polymorphisms of the genes encoding P-glycoprotein (ABCB1) and carboxylesterase 1 (CES1) on the variability of plasma concentrations of DOACs in Kazakhstani patients with NVAF. We analyzed polymorphisms rs4148738, rs1045642, rs2032582 and rs1128503 in ABCB1 and rs8192935, rs2244613 and rs71647871 CES1 genes and measured the plasma concentrations of dabigatran/apixaban and biochemical parameters in 150 Kazakhstani NVAF patients. Polymorphism rs8192935 in the CES1 gene (p = 0.04), BMI (p = 0.01) and APTT level (p = 0.01) were statistically significant independent factors of trough plasma concentration of dabigatran. In contrast, polymorphisms rs4148738, rs1045642, rs2032582 and rs1128503 in ABCB1 and rs8192935, rs2244613 and rs71647871 CES1 genes did not show significant influence on plasma concentrations of dabigatran/apixaban drugs (p > 0.05). Patients with GG genotype (138.8 ± 100.1 ng/mL) had higher peak plasma concentration of dabigatran than with AA genotype (100.9 ± 59.6 ng/mL) and AG genotype (98.7 ± 72.3 ng/mL) (Kruskal-Wallis test, p = 0.25). Thus, CES1 rs8192935 is significantly associated with plasma concentrations of dabigatran in Kazakhstani NVAF patients (p < 0.05). The level of the plasma concentration shows that biotransformation of the dabigatran processed faster in individual carriers of GG genotype rs8192935 in the CES1 gene than with AA genotype.

Carboxylesterase-2 plays a critical role in dabigatran etexilate active metabolite formation.

Dabigatran etexilate (DABE), an oral anticoagulant prodrug, is nearly completely metabolized to the dabigatran (DAB) active metabolite by carboxylesterase-1 (CES1) and carboxylesterase-2 (CES2). The high interpatient variation in DAB plasma concentrations, coupled with its low therapeutic index, emphasizes the need to understand how CES1 and CES2 impact active metabolite formation. Previous work focused on CES1 enzyme activity but the contributions of CES2 remain unclear. The purpose of this study was to determine how CES2 activity influences DAB active metabolite formation. We compared the efficiency of DAB formation from DABE when exposed sequentially to human intestinal and then human hepatic microsomes (mimicking the normal metabolic sequence) with the reverse metabolic sequence in which DABE is exposed to hepatic and then intestinal microsomes. The poor efficiency of DAB formation with reverse sequential hydrolysis indicates that CES2 activity is crucial for active metabolite formation. Thus, the decrease in DAB formation with normal sequential hydrolysis was more sensitive to CES2 inhibition by verapamil (CES2 IC50 = 3.4 µM) than CES1 inhibition by diltiazem (CES2 IC50 = 9.1 µM). These results show CES2 activity plays a crucial role in DAB formation and that variability in its activity is an important determinant of therapeutic response.

[The role of direct oral anticoagulants in the management of cancer-associated thrombosis in 2020]. / Place des anticoagulants oraux directs dans la prise en charge de la maladie thromboembolique veineuse associée au cancer en 2020.

Direct oral anticoagulants, anti-IIa or anti-Xa, are widely used in the treatment and prevention of venous thromboembolic disease as well as in nonvalvular atrial fibrillation. Direct oral anticoagulants are characterized by a rapid onset of activity, a predictable response and a relatively wide therapeutic window. Nevertheless, theoretical drug interactions exist since direct oral anticoagulants are substrates of the transport protein P-glycoprotein and/or of isoforms of cytochromes P450 pathway. Direct oral anticoagulants do not have a marketing authorization for the treatment of cancer-associated thrombosis unlike low-molecular-weight heparins which remain the gold standard treatment today. However, recent studies have compared low-molecular-weight heparins to direct oral anticoagulants in the treatment of cancer-associated thrombosis. Results of these studies showed a non-inferiority of direct oral anticoagulants in the prevention of recurrent thromboembolic events but at the cost of an increased hemorrhagic risk, in particular for patients with gastrointestinal and urogenital cancers. Thus, international guidelines, unlike French guidelines, integrate them in first line of the therapeutic strategy of cancer patients. We are certainly entering an era of personalized therapy for cancer-associated thrombosis, considering cancer type and also the theoretical risk of drug interactions with anti-cancer treatments or supportive care.

Interruption of platelets and thrombin function as a new approach against liver fibrosis induced experimentally in rats.

AIM: Liver fibrosis is a serious health problem which is a critical cause of morbidity and mortality worldwide. It is the main complication of untreated chronic inflammatory liver diseases which can progress to liver cirrhosis, hepatocellular carcinoma, and finally death. Coagulation cascade plays a mechanistic role in the pathogenesis of different chronic inflammatory disease including atherosclerosis, stroke, and tissue fibrosis. The current study was designed to investigate the effect of inhibition of coagulation cascade on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. MATERIAL AND METHODS: The study was conducted in rats. Rats were treated with CCl4 subcutaneously for 6 consecutive weeks to determine the onset of coagulation system activation in relation to development of fibrosis. To investigate the effects of coagulation system inhibition in CCl4-induced liver fibrosis, the anticoagulants drugs dabigatran and clopidogrel were administrated orally concurrently with CCl4 treatment. KEY FINDINGS: The results of our study revealed that during the first week, there were significant elevations of fibrin, tissue factor expressions, and prothrombin time (PT) coupled with neutropenia without significant changes in liver fibrosis markers such as TGF-ß, α-SMA and collagen deposition. Starting from the second week, tissue injury markers including the oxidative, inflammatory and fibrosis markers as well as histopathological changes became evident progressively. Intriguingly, dabigatran and clopidogrel significantly normalized the biochemical and pathological changes. SIGNIFICANCE: In conclusion, activation of coagulation cascade is a triggering stimulus in the initiation of CCl4-induced liver fibrosis and the anticoagulant drugs may exert promising anti-fibrotic effect.

Catalytic Reaction Mechanism for Drug Metabolism in Human Carboxylesterase-1: Cocaine Hydrolysis Pathway.

Carboxylesterase-1 (CE-1) is a crucial enzyme responsible for metabolism/activation/inactivation of xenobiotics (therapeutic agents, prodrugs, abused drugs, and organophosphorus nerve agents etc.) and also involved in many other biological processes. In this study, we performed extensive computational modeling and simulations to understand the fundamental reaction mechanism of cocaine hydrolysis catalyzed by CE-1, revealing that CE-1-catalyzed cocaine hydrolysis follows a novel reaction pathway with only two reaction steps: a single-step acylation process and a single-step deacylation process. In the transition states of both single-step processes, the cocaine NH group joins the oxyanion hole to form an additional hydrogen bond with the negatively charged carbonyl oxygen atom of the cocaine. Thus, the transition states are stabilized by both intermolecular and intramolecular hydrogen bonds with the methyl ester of cocaine, specifically the carbonyl oxygen atom. The rate-limiting transition state is associated with the acylation process, and the activation free energy barrier was predicted to be 20.1 kcal/mol. Further, in vitro experimental kinetic analysis was performed for human CE-1-catalyzed cocaine hydrolysis. For CE-1-catalyzed cocaine hydrolysis, the computationally predicted free energy barrier (20.1 kcal/mol) is reasonably close to the experimentally derived turnover number ( kcat = 0.058 min-1), indicating the reasonability of the computational results. The obtained novel mechanistic insights are expected to benefit not only CE-1 related rational drug discovery but also future research on the catalytic mechanism of other esterases.

The Renal Elimination Pathways of the Dabigatran Reversal Agent Idarucizumab and its Impact on Dabigatran Elimination.

Idarucizumab, a humanized monoclonal antibody fragment (Fab), provides rapid and sustained reversal of dabigatran-mediated anticoagulation. Idarucizumab and dabigatran are mainly eliminated via the kidneys. This analysis aimed to characterize the renal elimination of idarucizumab and investigate the influence of idarucizumab on the pharmacokinetics (PK) of dabigatran and vice versa. Studies were conducted in 5/6 nephrectomized rats, in human volunteers with and without renal impairment, and in a porcine liver trauma model. In both rats and humans, renal impairment increased idarucizumab exposure and initial half-life but did not affect its terminal half-life. Urinary excretion of unchanged idarucizumab increased with increasing idarucizumab dose, suggesting saturation of renal tubular reuptake processes at higher doses. The PK of idarucizumab was unaffected by dabigatran. In contrast, idarucizumab administration resulted in redistribution of dabigatran to the plasma, where it was bound and inactivated by idarucizumab. Urinary excretion of dabigatran after administration of idarucizumab was delayed, but total dabigatran excreted in urine was unaffected. Idarucizumab and dabigatran were eliminated together via renal pathways.

Different Involvement of OAT in Renal Disposition of Oral Anticoagulants Rivaroxaban, Dabigatran, and Apixaban.

This study aimed to investigate the interactions of 3 anticoagulants, rivaroxaban, apixaban, and dabigatran, with 5 human solute carrier transporters, hOAT1, hOAT3, hOCT2, hOATP1B1, and hOATP1B3. Apixaban inhibited hOAT3, hOATP1B1, and hOATP1B3, and rivaroxaban inhibited hOAT3 and hOATP1B3, with IC50 values of >20 and >5 µM, respectively. The effect of dabigatran was negligible or very weak, so significant drug interactions at therapeutic doses are unlikely. Specific uptake of rivaroxaban was observed only in human and mouse OAT3-expressing cells. The Km for mouse Oat3 (mOat3) was 1.01 ± 0.70 µM. A defect in mOat3 reduced the kidney-to-plasma concentration ratio of rivaroxaban by 38% in mice. Probenecid treatment also reduced the kidney-to-plasma concentration ratio of rivaroxaban in rats by 73%. Neither mOat3 defect nor probenecid administration in rats reduced the renal clearance of rivaroxaban. The uptake of rivaroxaban by monkey kidney slices was temperature dependent and inhibited by probenecid but not by tetraethylammonium. Taken together, organic anion transporters, mainly OAT3, may mediate basolateral uptake of rivaroxaban in kidneys. hOAT3 could be an additional factor that differentiates the potential drug-drug interactions of the 3 anticoagulants in the urinary excretion process in clinical settings.

Real life dabigatran and metabolite concentrations, focused on inter-patient variability and assay differences in patients with atrial fibrillation.

BACKGROUND: Dabigatran is prescribed to increasing numbers of patients with atrial fibrillation (AF). Although routine monitoring is not considered to be useful, measuring drug concentrations can be clinically relevant in specific situations. The aim of this study was the comparison of different functional and non-functional assays for determination of dabigatran concentrations at different timepoints in a real-life patient population with AF. We focused on the differences between assays in identifying patients with low drug concentrations. Furthermore, we studied the effect of glucuronidation on the established concentration as determined with different assays. METHODS: This study established dabigatran concentration ranges in 40 real-life AF patients by an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) reference method and compared these with results from coagulation assays (Hemoclot dTT, LD-dTT and ECA). Samples were taken just before and 2 and 4 h after taking the drug. RESULTS: A wide range of concentrations at different time points was found in this patient group. Coagulation assays correlate best with UPLC-MS/MS results that include the glucuronidated metabolites, showing that the pharmacologically active glucuronides are also measured in coagulation testing. The LD-dTT has the best agreement with UPLC-MS/MS and combines good sensitivity with high specificity. Several patients show consistently low or high drug concentrations, implying that drug exposure differs between patients. CONCLUSIONS: Based on the association of dabigatran concentrations with bleeding and thromboembolic risk, we believe that dabigatran monitoring could be beneficial for further optimizing anticoagulation therapy in AF.

Novel dabigatran derivatives with a fluorine atom at the C-2 position of the terminal benzene ring: Design, synthesis and anticoagulant activity evaluation.

This manuscript describes the preparation of dabigatran derivatives and their inhibitory potentials toward human thrombin. Among the tested compounds, 7c, 7k, 7m and 7o, with IC50 values of 1.54, 0.84, 1.18 and 1.42 nM, exhibited comparable inhibitory activity to dabigatran (IC50 = 1.20 nM). The in vivo anti-thrombotic activity of compounds 7c and 7o in SD rats was studied. Results showed that intravenously administering the two compounds significantly inhibited the growth of thrombus with an inhibition rate of (84.24 ± 1.53)% and (84.57 ± 0.45)%, which were comparable to that of dabigatran (85.07 ± 0.61)%. Furthermore, the docking simulation of active compounds (7k and 7m) provided a potential binding model. Results indicated that these compounds could be further investigated to determine their anticoagulant activities.

Dabigatran etexilate activation is affected by the CES1 genetic polymorphism G143E (rs71647871) and gender.

The oral anticoagulant prodrug dabigatran etexilate (DABE) is sequentially metabolized by intestinal carboxylesterase 2 (CES2) and hepatic carboxylesterase 1 (CES1) to form its active metabolite dabigatran (DAB). A recent genome-wide association study reported that the CES1 single nucleotide polymorphisms (SNPs) rs2244613 and rs8192935 were associated with lower DAB plasma concentrations in the Randomized Evaluation of Long-term Anticoagulation Therapy (RE-LY) study participants. In addition, gender differences in exposure to DAB were observed in clinical studies. The aim of this study was to examine the effect of CES1 genetic polymorphisms and gender on DABE activation using several in vitro approaches. The genotypes of the CES1 SNPs rs2244613, rs8192935, and the known loss-of-function CES1 variant rs71647871 (G143E), and the activation of DABE and its intermediate metabolites M1 and M2 were determined in 104 normal human liver samples. DABE, M1, and M2 activations were found to be impaired in human livers carrying the G143E variant. However, neither rs2244613 nor rs8192935 was associated with the activation in human livers. The incubation study of DABE with supernatant fractions (S9) prepared from the G143E-transfected cells showed that the G143E is a loss-of-function variant for DABE metabolism. Moreover, hepatic CES1 activity on M2 activation was significantly higher in female liver samples than male. Our data suggest that CES1 genetic variants and gender are important contributing factors to variability in DABE activation in humans. A personalized DABE treatment approach based on patient-specific CES1 genotypes and sex may have the potential to improve the efficacy and safety of DABE pharmacotherapy.

Pharmacogenetics of dabigatran etexilate interindividual variability.

INTRODUCTION: Dabigatran etexilate is given in fixed doses without coagulation monitoring for the prevention of blood clots in at risk adults. A high inter-individual variability in blood concentrations of the active metabolite of dabigatran has been reported. ABCB1 and CES1 exert an important effect in the metabolism of dabigatran etexilate and allele variants at these two loci are likely to play a pivotal role. To investigate whether screening for polymorphisms within the ABCB1 and the CES1 genes would explain a portion of the inter-individual variability in blood concentrations of the active metabolite of dabigatran. MATERIAL AND METHODS: In a cohort of patients who had atrial fibrillation and on anticoagulant prophylaxis with dabigatran etexilate, we investigated whether genotypes at rs4148738 (ABCB1), rs8192935 (CES1), and rs2244613 (CES1) loci would affect plasma dabigatran trough and peak concentrations. RESULTS AND DISCUSSION: Among 92 patients (median age: 72.0years, range: 52-92) analyzed, no clinical variable or genotype was associated with a significant difference in dabigatran peak concentrations. As for trough concentrations, in addition to creatinine clearance, and sex a significant association with the CES1 SNP rs8192935 (p=0.023) was detected. The mean adjusted plasma levels were higher among patients with the CC genotype (86.3ng/dl) than in those carrying the T allele (62.1ng/dl). No significant effect was found for the ABCB1 SNP rs4148738. The CES1 SNP rs8192935 significantly influenced the dabigatran trough concentrations and carriers of the T allele showed significantly lower concentrations than did carriers of the CC genotype.

Novel oral anticoagulants in plastic surgery.

Novel oral anticoagulants (NOACs) have emerged as a good alternative to warfarin in the prevention of stroke for patients with atrial fibrillation. NOAC use is increasing rapidly; therefore, greater understanding of their use in the perioperative period is important for optimal care. Studies and reviews that reported on the use of NOACs were identified, with particular focus on the perioperative period. PubMed was searched for relevant articles published between January 2000 and August 2015. The inevitable rise in the use of NOACs such as rivaroxaban (Xarelto™), apixaban (Eliquis™), edoxaban (Lixiana™) and dabigatran (Pradaxa™) may present a simplified approach to perioperative anticoagulant management due to fewer drug interactions, rapidity of onset of action and relatively short half-lives. Coagulation status, however, cannot reliably be monitored and no antidotes are currently available. When planning for discontinuation of NOACs, special consideration of renal function is required. Advice regarding the management of bleeding complications is provided for consideration in emergency surgery. In extreme circumstances, haemodialysis may be considered for bleeding with the use of dabigatran. NOACs will increasingly affect operative planning in plastic surgery. In order to reduce the incidence of complications associated with anticoagulation, the management of NOACs in the perioperative period requires knowledge of the time of last dose, renal function and the bleeding risk of the planned procedure. Consideration of these factors will allow appropriate interpretation of the current guidelines.

Histones Differentially Modulate the Anticoagulant and Profibrinolytic Activities of Heparin, Heparin Derivatives, and Dabigatran.

The antithrombin activity of unfractionated heparin (UFH) is offset by extracellular histones, which, along with DNA, represent a novel mediator of thrombosis and a structural component of thrombi. Here, we systematically evaluated the effect of histones, DNA, and histone-DNA complexes on the anticoagulant and profibrinolytic activities of UFH, its derivatives enoxaparin and fondaparinux, and the direct thrombin inhibitor dabigatran. Thrombin generation was assessed by calibrated automated thrombinography, inhibition of factor Xa and thrombin by synthetic substrates, tissue plasminogen activator-mediated clot lysis by turbidimetry, and thrombin-activatable fibrinolysis inhibitor (TAFI) activation by a functional assay. Histones alone delayed coagulation and slightly stimulated fibrinolysis. The anticoagulant activity of UFH and enoxaparin was markedly inhibited by histones, whereas that of fondaparinux was enhanced. Histones neutralized both the anti-Xa and anti-IIa activities of UFH and preferentially blocked the anti-IIa activity of enoxaparin. The anti-Xa activity of fondaparinux was not influenced by histones when analyzed by chromogenic substrates, but was potentiated in a plasma prothrombinase assay. Histones inhibited the profibrinolytic activity of UFH and enoxaparin and enhanced that of fondaparinux by acting on the modulation of TAFI activation by anticoagulants. Histone H1 was mainly responsible for these effects. Histone-DNA complexes, as well as intact neutrophil extracellular traps, impaired the activities of UFH, enoxaparin, and fondaparinux. Dabigatran was not noticeably affected by histones and/or DNA, whatever the assay performed. In conclusion, histones and DNA present in the forming clot may variably influence the antithrombotic activities of anticoagulants, suggesting a potential therapeutic advantage of dabigatran and fondaparinux over heparins.

In vitro and in vivo evaluation of drug-drug interaction between dabigatran and proton pump inhibitors.

To quantify the drug-drug interactions between dabigatran etexilate (DE) and proton pump inhibitors (PPI) and in particular the role of P-gp activity modulation. In the first part of the study, efflux ratios of DE were evaluated using the caco-2 cell line in the presence of pantoprazole, omeprazole, rabeprazole, lansoprazole and ciclosporin A (positive control). The two PPI that reduced the efflux ratio of dabigatran to the greatest and least extent, respectively, were used during the second part of the study, comprising a single-centre, randomised, open-label study with an incomplete Latin square design. Nine healthy volunteers received DE (150 mg) alone, DE (150 mg) with the first PPI and DE (150 mg) with the second PPI in randomised sequence. Dabigatran plasma concentration and thrombin time were measured in blood samples withdrawn at 11 time points after each treatment. Models were built using a nonlinear mixed-effect modelling approach. Omeprazole and rabeprazole were the two PPI that reduced the efflux ratio of DE least and most, respectively. The PK model was based on an inverse Gaussian absorption process with one compartment. The relationship between dabigatran concentration and thrombin time was considered linear. Some PK profiles had dramatically low concentration values due to poor absorption. These profiles were clustered using a between subject model mixture with interoccasion variability. The concomitant administration of PPI did not significantly change dabigatran pharmacokinetics. DE is subject to high absorption variability, precluding evaluation of the effect of PPI on its pharmacokinetics.

Design, Synthesis, and Biological Evaluation of Dabigatran Etexilate Mimics, a Novel Class of Thrombin Inhibitors.

Human α-thrombin is a particularly promising target for anticoagulant therapy, and identification of oral small-molecular inhibitors of thrombin remains a research focus. On the basis of the X-ray crystal structure of human α-thrombin and its inhibitor dabigatran, we designed and synthesized a series of dabigatran etexilate mimics containing a novel tricyclic fused scaffold. The biological evaluations reveal that all of the compounds possess moderate activity of antiplatelet aggregation induced by thrombin in vitro. Moreover, compound I-8, which contains 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP), a cleavable moiety with antiplatelet activity, shows the best anticoagulant effect among the tested compounds in vivo. Those synthesized compounds that have better in vitro activity were subjected to bleeding complication tests, and the results demonstrate that the novel compounds are less likely to have bleeding risk than dabigatran etexilate.

Development and validation of LC-MSMS assay for the determination of the prodrug dabigatran etexilate and its active metabolites in human plasma.

Dabigatran etexilate (DABE) is a low-molecular-weight prodrug that is converted after oral administration to dabigatran (DAB)-a directly acting oral anticoagulant. In this study, an LC-MSMS assay was developed and validated for the determination of DABE, free DAB and its equipotent O-glucuronide conjugates in plasma. Owing to the susceptibility of DABE and DAB to chemical hydrolysis, cleavage of the O-glucuronide moiety was carried out using ß-glucuronidase enzyme. Free and total plasma concentrations of DAB were determined in incurred plasma samples before and after enzymatic cleavage (50°C and 3 h), respectively. RP-HPLC separation was carried out using acetonitrile: water (30:70, v/v), adjusted to pH 3.0 using formic acid. Tandem mass spectrometric detection at positive electrospray ionization in the MRM mode was then employed for the determination of DABE and DAB. The analysis was carried out within 5.0 min over a linear concentration range of 1.00-600.00 ng/mL for the prodrug and its active metabolite. Validation was carried out according to FDA guidelines for bioanalytical method. The recoveries were higher than 89.48%, the accuracy was within 98.33-110.12% and the RSD was below 10% for the studied compounds in both incurred plasma and quality control samples. Results of incurred sample re-analysis and incurred sample stability revealed less than 10% variability. This indicated good assay precision and sufficient stability of target analytes in their real matrix at the employed experimental conditions. The applicability of the assay for therapeutic drug monitoring and the determination of the pharmacokinetic parameters were demonstrated.

[Companion Diagnostics for Thrombotic Disease].

In recent years, thrombotic disease, including myocardial infarction and ischemic stroke, has rapidly increased in Japan. To treat and prevent thromboembolism, warfarin has been commonly prescribed for a long period as an oral anticoagulant. However, it is difficult to define an appropriate warfarin dose because of large inter-individual variability in dose requirements and the narrow therapeutic range. Recent pharmacogenomic (PGx) studies have shown that several single nucleotide polymorphisms (SNPs) in CYP2C9 (warfarin metabolic enzyme) and VKORC1 (warfarin target enzyme) are responsible for an individual's warfarin sensitivity. In order to realize personalized warfarin treatment, algorithms to estimate the required warfarin dose based on PGx are under consideration, including the cost-effectiveness. Recently, novel oral anticoagulants (NOACs; dabigatran, rivaroxaban, apixaban, and edoxaban) have become available as well as alternatives to warfarin treatment for the prevention of ischemic stroke in non-valvular atrial fibrillation. Although NOACs are prescribed at a fixed-dose without frequent monitoring of blood coagulability, it has been reported that there is inter-individual variability in the blood concentration of dabigatran caused by gene polymorphisms. Further studies are needed to perform more effective and safer anticoagulant therapy using NOACs. Progress in PGx studies and the realization of personalized anticoagulant therapy are expected in the future. [Review].