In Vitro Simulation of the Fasted Gastric Conditions and Their Variability to Elucidate Contrasting Properties of the Marketed Dabigatran Etexilate Pellet-Filled Capsules and Loose Pellets.

Variability of the gastrointestinal tract is rarely reflected in in vitro test protocols but often turns out to be crucial for the oral dosage form performance. In this study, we present a generation method of dissolution profiles accounting for the variability of fasted gastric conditions. The workflow featured 20 biopredictive tests within the physiological variability. The experimental array was constructed with the use of the design of experiments, based on three parameters: gastric pH and timings of the intragastric stress event and gastric emptying. Then, the resulting dissolution profiles served as a training data set for the dissolution process modeling with the machine learning algorithms. This allowed us to generate individual dissolution profiles under a customizable gastric pH and motility patterns. For the first time ever, we used the method to successfully elucidate dissolution properties of two dosage forms: pellet-filled capsules and bare pellets of the marketed dabigatran etexilate product Pradaxa. We showed that the dissolution of capsules was triggered by mechanical stresses and thus was characterized by higher variability and a longer dissolution onset than observed for pellets. Hence, we proved the applicability of the method for the in vitro and in silico characterization of immediate-release dosage forms and, potentially, for the improvement of in vitro-in vivo extrapolation.

Assessment of the Potential for Veverimer Drug-Drug Interactions.

Veverimer is a polymer being developed as a potential treatment of metabolic acidosis in patients with chronic kidney disease. Veverimer selectively binds and removes hydrochloric acid from the gastrointestinal tract, resulting in an increase in serum bicarbonate. Veverimer is not systemically absorbed, so potential drug-drug interactions (DDIs) are limited to effects on the absorption of other oral drugs through binding to veverimer in the gastrointestinal tract or increases in gastric pH caused by veverimer binding to hydrochloric acid. In in vitro binding experiments using a panel of 16 test drugs, no positively charged, neutral, or zwitterionic drugs bound to veverimer. Three negatively charged drugs (furosemide, aspirin, ethacrynic acid) bound to veverimer; however, this binding was reduced or eliminated in the presence of normal physiologic concentrations (100-170 mM) of chloride. Veverimer increased gastric pH in vivo by 1.5-3 pH units. This pH elevation peaked within 1 hour and had returned to baseline after 1.5-3 hours. Omeprazole did not alter the effect of veverimer on gastric pH. The clinical relevance of in vitro binding and the transient increase in gastric pH was evaluated in human DDI studies using two drugs with the most binding to veverimer (furosemide, aspirin) and two additional drugs with pH-dependent solubility effecting absorption (dabigatran, warfarin). None of the four drugs showed clinically meaningful DDI with veverimer in human studies. Based on the physicochemical characteristics of veverimer and results from in vitro and human studies, veverimer is unlikely to have significant DDIs. SIGNIFICANCE STATEMENT: Patients with chronic kidney disease, who are usually on many drugs, are vulnerable to drug-drug interactions (DDIs). The potential for DDIs with veverimer was evaluated based on the known site of action and physicochemical structure of the polymer, which restricts the compound to the gastrointestinal tract. Based on the findings from in vitro and human studies, we conclude that veverimer is unlikely to have clinically significant DDIs.

Physiologically-based pharmacokinetics modeling to investigate formulation factors influencing the generic substitution of dabigatran etexilate.

The exposure-response relationship of direct acting oral anti-coagulants (DOACs) for bleeding risk is steep relative to ischemic stroke reduction. As a result, small changes in exposure may lead to bleeding events. The overall goal of this project was to determine the effect of critical formulation parameters on the pharmacokinetics (PKs) and thus safety and efficacy of generic DOACs. In this first installment of our overall finding, we developed and verified a physiologically-based PK (PBPK) model for dabigatran etexilate (DABE) and its metabolites. The model was developed following a middle out approach leveraging available in vitro and in vivo data. External validity of the model was confirmed by overlapping predicted and observed PK profiles for DABE as well as free and total dabigatran for a dataset not used during model development. The verified model was applied to interrogate the impact of modulating the microenvironment pH on DABE systemic exposure. The PBPK exploratory analyses highlighted the high sensitivity of DABE exposure to supersaturation ratio and precipitation kinetics.

A composite nanocarrier to inhibit precipitation of the weakly basic drug in the gastrointestinal tract.

For weakly basic drugs, the sharp decrease of drug solubility and the following drug precipitation after drugs transferring from the gastric fluid to the intestinal fluid in the gastrointestinal (GI) tract is a main reason for the poor oral bioavailability of drugs. Here, an anticoagulant dabigatran etexilate (DE) was used as a model drug, and a composite nanocarrier system of DE was developed to improve the drug dissolution by decreasing the drug leakage in the stomach and inhibiting the drug precipitation in the intestinal tract. With the encapsulation of drugs in nanocarriers, the precipitation percentage of DE in composite nanocarriers was 22.25 ± 3.88% in simulated intestinal fluid, which was far below that of the commercial formulation. Moreover, the relative bioavailability of DE-loaded composite nanocarriers (456.58%) was greatly enhanced and the peak of its activated partial thromboplastin time was also significantly prolonged (p < .01) compared with the commercial formulation, indicating that the anticoagulant effect of DE was effectively improved. Therefore, the designed composite nanocarrier system of DE presents great potentials in improving the therapeutic efficiency and expanding the clinical applications of poorly water-soluble weakly basic drugs.

Computer-aid drug design, synthesis, and anticoagulant activity evaluation of novel dabigatran derivatives as thrombin inhibitors.

In this study, computer-aided drug design techniques were adopted to explore the structural and chemical features for dabigatran and design novel derivatives. The built 3D-QSAR models demonstrated significant statistical quality and excellent predictive ability by internal and external validation. Based on QSAR information, 11 novel dabigatran derivatives (12a-12k) were designed and predicted, then ADME prediction and molecular docking were performed. Furthermore, all designed compounds were synthesized and characterized by 1H NMR, 13C NMR and HR-MS. Finally, they were evaluated for anticoagulant activity in vitro. The activity results showed that the 10 obtained compounds exhibited comparable activity to the reference dabigatran (IC50 = 9.99 ±â€¯1.48 nM), except for compound 12i. Further analysis on molecular docking was performed on three compounds (12a, 12c and 12g) with better activity (IC50 = 11.19 ±â€¯1.70 nM, IC50 = 10.94 ±â€¯1.85 nM and IC50 = 11.19 ±â€¯1.70 nM). MD simulations (10 ns) were carried out, and their binding free energies were calculated, which showed strong hydrogen bond and pi-pi stacking interactions with key residues Gly219, Asp189 and Trp60D. The 10 novel dabigatran derivatives obtained can be further studied as anticoagulant candidate compounds.

In Vitro Selection of Specific DNA Aptamers Against the Anti-Coagulant Dabigatran Etexilate.

Dabigatran Etexilate (PRADAXA) is a new oral anticoagulant increasingly used for a number of blood thrombosis conditions, prevention of strokes and systemic emboli among patients with atrial fibrillation. It provides safe and adequate anticoagulation for prevention and treatment of thrombus in several clinical settings. However, anticoagulation therapy can be associated with an increased risk of bleeding. There is a lack of specific laboratory tests to determine the level of this drug in blood. This is considered the most important obstacles of using this medication, particularly for patients with trauma, drug toxicity, in urgent need for surgical interventions or uncontrolled bleeding. In this work, we performed Systematic evolution of ligands by exponential enrichment (SELEX) to select specific DNA aptamers against dabigatran etexilate. Following multiple rounds of selection and enrichment with a randomized 60-mer DNA library, specific DNA aptamers for dabigatran were selected. We investigated the affinity and specificity of generated aptamers to the drug showing dissociation constants (Kd) ranging from 46.8-208 nM. The most sensitive aptamer sequence was selected and applied in an electrochemical biosensor to successfully achieve 0. 01 ng/ml level of detection of the target drug. With further improvement of the assay and optimization, these aptamers would replace conventional antibodies for developing detection assays in the near future.

Effects of dabigatran regulates no­reflow phenomenon in acute myocardial infarction mice through anti­inflammatory and anti­oxidative activities and connective tissue growth factor expression.

Pradaxa is a novel oral anticoagulant, which was originally used to prevent thrombosis following joint replacement surgery. The aim of the current study was to investigate the effect dabigatran on acute myocardial infarction through regulating no­reflow phenomenon and oxidative stress, neutrophil intraplaque infiltration and apoptosis. In the present study, dabigatran significantly inhibited the infarct size, increased arterial pressure and reduced no­reflow phenomenon in acute myocardial infarction (AMI) vehicle rabbits. Treatment with dabigatran significantly inhibited the P65 of nuclear factor κB, tumor necrosis factor α, interleukin (IL)­1ß and IL­6 activities and significantly enhanced the catalase and superoxide dismutase activities in the AMI rabbits. In addition, dabigatran significantly suppressed inducible nitric oxide synthase (iNOS), collagen I, transforming growth factor ß1 (TGF­ß1), α­smooth muscle actin (α­SMA) and connective tissue growth factor (CTGF) protein expression in AMI rabbits. Taken together, these results suggest that the effects of dabigatran inhibit no­reflow phenomenon, infarct size and enhance arterial pressure in AMI through anti­inflammatory and anti­oxidative activity, and regulating iNOS, collagen I, TGF­ß1, α­SMA and CTGF protein expression in AMI rabbits.

Novel dabigatran etexilate hemisuccinate-loaded polycap: Physicochemical characterisation and in vivo evaluation in beagle dogs.

The purpose of this study was to develop a novel dabigatran etexilate hemisuccinate (DEH) salt-loaded polycap with bioequivalence to the dabigatran etexilate mesylate (DEM)-loaded commercial product. DEH prepared with dabigatran etexilate base (DE) and succinic acid was less hygroscopic but less soluble than DEM. Numerous micronized DEHs and DEH-loaded solid dispersions were prepared employing the spiral jet-milling and spray-drying techniques, respectively. Among the formulations prepared, a micronized DEH prepared with the injection air at 1.5bar and the grinding air at 2bar, and a DEH-loaded solid dispersion prepared with 6g HPMC most improved the drug solubility, respectively. Moreover, the micronized DEH provided more increased drug solubility and dissolution compared with the solid dispersion, even though its drug solubility was still lower than that of DEM. Unlike the situation in other studies, the enhanced solubility and dissolution of DEH was more due to particle size reduction than to a change to the amorphous form. The micronized DEH prepared with Myrj 52S had greater drug solubility than preparations with other surfactants. Among the organic acids investigated, only fumaric acid (128.8mg) showed a similar pattern in pH changes to the DEM-loaded commercial product. Furthermore, in order to make the environment acidic while preventing the direct contact of the drug with fumaric acid, the polycap was composed of a tablet containing the micronized DEH, Myrj 52S and other ingredients, and separate fumaric acid. This micronized DEH-loaded polycap was dissolution- and bio-equivalent to the DEM-loaded commercial product in beagle dogs. Thus, the novel micronized DEH-loaded polycap would be a promising alternative to the DEM-loaded commercial product.

Improved oral bioavailability and therapeutic efficacy of dabigatran etexilate via Soluplus-TPGS binary mixed micelles system.

The clinical use of dabigatran etexilate (DABE) is limited by its poor absorption and relatively low bioavailability. Our study aimed to explore the potential of a mixed micelle system composed of Soluplus® and D-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS) to improve the oral absorption and bioavailability of DBAE. DBAE was first encapsulated into Soluplus/TPGS mixed micelles by a simple thin film hydration method. The DBAE loaded micelles displayed an average size distribution of around 83.13 nm. The cellular uptake of DBAE loaded micelles in Caco-2 cell monolayer was significantly enhanced by 2-2.6 fold over time as compared with DBAE suspension. Both lipid raft/caveolae and macropinocytosis-mediated the cell uptake of DBAE loaded micelles through P-glycoprotein (P-gp)-independent pathway. Compared with the DBAE suspension, the intestinal absorption of DBAE from DBAE mixed micelles in rats was significantly improved by 8 and 5-fold in ileum at 2 h and 4 h, respectively. Moreover, DBAE mixed micelles were absorbed into systemic circulation via both portal vein and lymphatic pathway. The oral bioavailability of DBAE mixed micelles in rats was 3.37 fold higher than that of DBAE suspension. DBAE mixed micelles exhibited a comparable anti-thrombolytic activity with a thrombosis inhibition rate of 63.18% compared with DBAE suspension in vivo. Thus, our study provides a promising drug delivery system to enhance the oral bioavailability and therapeutic efficacy of DBAE.

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.

The impact of polymeric excipients on the particle size of poorly soluble drugs after pH-induced precipitation.

Active pharmaceutical ingredients (APIs) with strongly pH-dependent aqueous solubility can face the problem of precipitating from solution when the pH changes from acidic in the stomach to neutral in the intestine. The present work investigates the effect of two polymeric excipients - polyvinylpyrrolidone (PVP) and Soluplus - on the ability to either prevent precipitation, or to control the size distribution of precipitated particles when precipitation cannot be prevented. Two different APIs were compared, Dabigatran etexilate mesylate and Rilpivirine hydrochloride. The effect of excipient concentration on the precipitation behaviour during pH titration was systematically investigated and qualitatively different trends were observed: in case of Soluplus, which forms a micellar solution when critical micelle concentration is exceeded, precipitation was inhibited in the case of Dabigatran etexilate, which partitioned into the micelles. On the other hand, Rilpivirine precipitated independently of Soluplus concentration. In the case of PVP, which does not form micelles, precipitation could not be avoided. Increased polymer concentration, however prevented the aggregation of precipitated particles into larger cluster. The observed effect of PVP was especially pronounced for Rilpivirine. The main conclusion of this study is that a suitably chosen polymeric excipient can either prevent precipitation altogether or reduce the size of the resulting particles. The mechanism of action, however, seems-specific to a given molecule. It was also shown that the polymer-stabilised particles have a potential to redissolve.

A solid self-nanoemulsifying system of the BCS class IIb drug dabigatran etexilate to improve oral bioavailability.

AIM: To develop dabigatran etexilate (DE)-loaded self-nanoemulsifying drug delivery systems (SNEDDS) for the prevention of stroke and thromboembolism. MATERIALS & METHODS: SNEDDS were optimized by ternary phase diagrams and then further solidified into dispersible tablets. In vitro dissolution was analyzed by a phase distribution study. In situ absorption and in vivo pharmacokinetic studies were tested in male Sprague-Dawley rats. RESULTS: The phase distribution study showed that more than 60% of DE was retained in the oil phase. Dissolution rate was dramatically enhanced without significant precipitation (<30%) in simulated intestinal fluid. Optimized SNEDDS had 531.80% relative bioavailability compared with Pradaxa(®) capsules (a commercial DE product). CONCLUSION: The developed SNEDDS are promising materials for improving the dissolution and oral bioavailability of BCS class IIb drugs.

Carbon-13 and carbon-14 labeled dabigatran etexilate and tritium labeled dabigatran.

Dabigatran etexilate or pradaxa, a novel oral anticoagulant, is a reversible, competitive, direct thrombin inhibitor. It is used to prevent strokes in patients with atrial fibrillation and the formation of blood clots in the veins (deep venous thrombosis) in adults who have had an operation to replace a hip or a knee. Pradaxa is the only novel oral anticoagulant available with both proven superiority to warfarin and a specific reversal agent for use in rare emergency situations. The detailed description of the synthesis of carbon-13 and carbon-14 labeled dabigatran etexilate, and tritium labeled dabigatran is described. The synthesis of carbon-13 dabigatran etexilate was accomplished in eight steps and in 6% overall yield starting from aniline-13 C6 . Ethyl bromoacetate-1-14 C was the reagent of choice in the synthesis of carbon-14 labeled dabigatran etexilate in six steps and 17% overall yield. Tritium labeled dabigatran was prepared using either direct tritium incorporation under Crabtree's catalytic conditions or tritium-dehalogenation of a diiodo-precursor of dabigatran.

The laboratory's 2015 perspective on direct oral anticoagulant testing.

The introduction of direct oral anticoagulant (DOAC) therapy into clinical use in the past 5 years has had significant impact on the clinical laboratory. Clinicians' desire to determine plasma drug presence or measure drug concentration, and more recent observations regarding the limitations and utility of coagulation testing in the setting of DOAC treatment, suggest that early published recommendations regarding laboratory testing should be reassessed. These initial recommendations, furthermore, were often based on drug-spiked plasma studies, rather than samples from patients receiving DOAC therapy. We have demonstrated that reagent sensitivity varies significantly whether drug-spiked samples or samples from DOAC-treated patients are tested. Data from drug-enriched samples must therefore be interpreted with caution or be used as a guide only. We present laboratory assays that can be used to determine drug presence and to measure drug concentration, and provide recommended testing algorithms. As DOAC therapy may significantly impact on specialty coagulation assays, we review those tests with the potential to give false-positive and false-negative results.

Molecular modeling studies, synthesis and biological evaluation of dabigatran analogues as thrombin inhibitors.

In this work, 48 thrombin inhibitors based on the structural scaffold of dabigatran were analyzed using a combination of molecular modeling techniques. We generated three-dimensional quantitative structure-activity relationship (3D-QSAR) models based on three alignments for both comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) to highlight the structural requirements for thrombin protein inhibition. In addition to the 3D-QSAR study, Topomer CoMFA model also was established with a higher leave-one-out cross-validation q(2) and a non-cross-validation r(2), which suggest that the three models have good predictive ability. The results indicated that the steric, hydrophobic and electrostatic fields play key roles in QSAR model. Furthermore, we employed molecular docking and re-docking simulation explored the binding relationship of the ligand and the receptor protein in detail. Molecular docking simulations identified several key interactions that were also indicated through 3D-QSAR analysis. On the basis of the obtained results, two compounds were designed and predicted by three models, the biological evaluation in vitro (IC50) demonstrated that these molecular models were effective for the development of novel potent thrombin inhibitors.

Design, synthesis and antithrombotic evaluation of novel dabigatran etexilate analogs, a new series of non-peptides thrombin inhibitors.

Thrombin is a serine protease that plays a key role in blood clotting, which makes it a promising target for the treatment of thrombotic diseases. Dabigatran is direct potent thrombin inhibitor. Based on bioisosteric and scaffold hopping principle, two dabigatran mimics (I-1 and II-1) in which the benzamidine moiety of dabigatran was replaced by a tricyclic fused scaffold were designed, synthesized and evaluated for their in vitro activities for inhibiting thrombin. The results reveal that compounds I-1 (IC50=9.20nM) and II-1 (IC50=7.48nM) are potent direct thrombin inhibitors and the activity is in the range of reference drug. On this basis, twenty-two ester and carbamate derivatives of I-1 or II-1 were prepared and evaluated for their anticoagulant activity. Prodrugs I-4a (IC50=0.73µM), I-4b (IC50=0.75µM), II-2a (IC50=1.44µM) and II-2b (IC50=0.91µM) display excellent effects of inhibiting thrombin induced-platelet aggregation. Moreover, compounds I-9 and II-4, which contain a cleavable moiety with anti-platelet activity, show the best anticoagulant efficacy among the tested compounds in the rat venous thrombosis model. The compounds which have better in vitro and in vivo activity were subjected to rat tail bleeding test, and the result demonstrates that compound I-9 is less likely to have bleeding risk than dabigatran etexilate.

Dabigatran for the treatment of venous thromboembolism.

Venous thromboembolism (VTE) is a major cause of morbidity and mortality, and individuals with a first VTE are at risk of recurrent VTE. VTE treatment is divided into three phases: a first short phase of acute (traditionally parenteral) anticoagulation, followed by a second maintenance phase with an oral anticoagulant, which may be continued into a third extended maintenance phase in patients considered to be at increased risk of recurrent VTE. Vitamin K antagonists are effective oral anticoagulants but have well-known limitations; non-vitamin K oral anticoagulants including dabigatran etexilate (DE) were therefore developed. DE was approved for VTE treatment on the basis of an extensive clinical trial program that evaluated DE during both the maintenance phase and the extended maintenance phase of VTE treatment. This article provides a comprehensive overview of DE in VTE treatment, from its preclinical characteristics and pharmacokinetic properties to its efficacy and safety in major clinical trials.

Quantification of dabigatran and indirect quantification of dabigatran acylglucuronides in human plasma by LC-MS/MS.

BACKGROUND: An assay for the quantification of dabigatran and its active metabolites, dabigatran acylglucuronides, has not previously been described in detail. RESULTS: For the quantification of total dabigatran concentration (free dabigatran and acylglucuronides), samples were subjected to alkaline hydrolysis. For the quantification of free dabigatran, samples were acidified with ammonium formate. Following acetonitrile protein precipitation, the samples were analyzed by LC-MS/MS using gradient elution to ensure separation of dabigatran from dabigatran acylglucuronides. Mean recoveries ≥98% were achieved. The assay was validated over the range 2.5-1000 ng/ml dabigatran, imprecision was <9% CV (<15% at LLOQ) and accuracy was 101-114%. CONCLUSION: An assay for dabigatran with indirect quantification of dabigatran acylglucuronides in plasma was developed, validated and applied.

Design, synthesis and structural exploration of novel fluorinated dabigatran derivatives as direct thrombin inhibitors.

Twenty-one fluorinated dabigatran derivatives were designed based on the bioisosteric principle. All derivatives were synthesised and evaluated for their thrombin inhibitory activity in vitro. Among these compounds, 14h, 14m, 14s and 14t were potent and the activity was in the range of reference drug, dabigatran. Three structural changes were introduced in these 21 compounds to elucidate the structure-activity relationship of the drugs. In addition, prodrugs of compounds 14h and 14s were developed to investigate their anticoagulant activities in vivo. In these experiments, compound 16 showed a fairly strong inhibitory effect on thrombin-induced platelet aggregation, and demonstrated potent activity for inhibiting arteriovenous thrombosis with an inhibition rate of (73 ± 6) %, which was comparable to that of dabigatran etexilate (76 ± 2) %. Moreover, molecular docking studies were performed to understand the binding interactions of active compounds 14h, 14s and 14t with thrombin protein (PDB ID:1KTS). Contour maps obtained from the 3D-QSAR model are meaningful in designing more active molecules to act as direct inhibitors of thrombin.