Development of a New Automated Assay for Measurement of the Oral Direct Factor Xa Inhibitor Edoxaban Plasma Concentration

Abstract Edoxaban is a reversible orally active factor Xa inhibitor approved in Japan for Venous ThromboEmbolism (VTE) prophylaxis in major orthopedic surgery and submitted for approval in multiple markets for Stroke Prevention in non-valvular Atrial Fibrillation (SPAF) and VTE treatment and recurrence prevention. Although routine monitoring is not required, determination of anti-Xa activity with results expressed in edoxaban plasma concentration may be helpful in some special clinical settings such as urgent invasive procedures or in cases of bleeding. We have developed a specific, automated, user-friendly assay for measuring plasma edoxaban-related anti-Xa activity using the STA® Liquid Anti-Xa with an edoxaban dedicated test set-up, along with specific edoxaban calibrator and control sets, namely STA® Edoxaban Calibrator and STA® Edoxaban Control, on the STA® line analyzers. These calibrator and control sets are freeze-dried in vitro edoxaban spiked citrated plasmas. Test results are expressed in ng/mL of edoxaban. Assay performances including Limit of Blank (LOB), Lower Limit of Detection (LLOD) both according to CLSI EP-17-A guideline, Lower Limit of Quantification (LLOQ), Upper Limit of Quantification (ULOQ) according to CLSI EP6-A guideline, with and without automated re-dilution of plasma sample, and within and between-run reproducibility have been determined. Anti-Xa assay results were compared to those obtained with Mass Spectrometry Liquid Chromatography (LC-MS) reference method to evaluate assay recovery. All study assays were performed using freeze-dried of frozen in vitro edoxaban-spiked citrated plasma samples. Main potential interferences, i.e., hemoglobin, non-conjugated bilirubin, and lipemia, have been assessed. Assay performance results are summarized in Table I. Table I: Main Edoxaban assay performances as determined during test development Parameter Results obtained with prototype reagent batch and test set-up LOB 10 ng/mL LLOD 15 ng/mL LLOQ (preliminary estimation) 20 ng/mL ULOQ Without sample re-dilution 150 ng/mL With sample re-dilution 450 ng/mL Reproducibility Within run (n = 21) Freeze-dried controls 40 ng/mL ≤ 4.5% 120 ng/mL ≤ 6.0% Frozen spiked samples 50 ng/mL ≤ 7.1% 100 ng/mL ≤ 4.9% 200 ng/mL ≤ 4.3% 350 ng/mL ≤ 4.0% Between run (n = 10) Freeze-dried controls 40 ng/mL ≤ 7.0% 120 ng/mL ≤ 3.6% Frozen spiked samples 50 ng/mL ≤ 5.0% 100 ng/mL ≤ 4.0% 350 ng/mL ≤ 4.6% Recovery(freeze-dried samples) 40 ng/mL 87.4% 120 ng/mL 101.9% Edoxaban calibrator and control stability Calibrators Onboard 4 hours Controls Onboard 24 hours +2 – 8°C 7 days Interferences Hemoglobin None up to 1 g/L Non-conjugated bilirubin None up to 200 µM Lipemia None up to 2.5 g/L (as Intralipid® concentration) In conclusion, the proposed edoxaban assay developed using STA® Liquid Anti-Xa reagent with a dedicated test set-up and specific STA® Edoxaban Calibrator and STA® Edoxaban Control sets allows an accurate, reproducible, automated, and user-friendly, edoxaban plasma concentration determination. Further studies are required to confirm assay performance in ex vivo samples. Disclosures Herve: Diagnostica Stago: Employment. Beaufils:Diagnostica Stago: Employment. Kochan:Daiichi Sankyo: Employment. He:Daiichi Sankyo: Employment. Depasse:Diagnostica Stago: Employment.

Download Full-text

IN VITRO ANTHELMINTIC AND ANTI-AMYLASE PROPERTIES OF GARCINIA PEDUNCULATA ROXB. ETHANOLIC EXTRACT

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2017v9i1.15395 ◽

2016 ◽

Vol 9 (1) ◽

pp. 189

Author(s):

Pinky Sarmah ◽

Nako Kobing ◽

Jyotchna Gogoi ◽

Ananta Madhab Dutta

Keyword(s):

Anthelmintic Activity ◽

Ethanolic Extract ◽

Ethanol Extract ◽

Biological Properties ◽

Death Time ◽

Control Sets ◽

Point Analysis ◽

Soxhlet Apparatus ◽

And Control

Objective: The present study aims to investigate in vitro anthelmintic and anti-amylase properties of the ethanolic fruit extract of Garcinia pedunculata.Methods: For the study, mature fruits of G. pedunculata were collected from local markets during April-May. Fleshy pericarps of fruits were chopped into small pieces, dried and extracted by using a Soxhlet apparatus. Ethanol extract of G. pedunculata was used for evaluation of in vitro anthelmintic and anti-amylase activities. In vitro anthelmintic activity was evaluated in animal models, Pheretima posthuma, an earthworm species. In vitro anti-amylase activity was evaluated by using zymographic, achromic point analysis (Starch-Iodine method) and spectrophotometric method [Di Nitro Salicylic acid (DNS)–Maltose method].Results: Ethanolic extract of G. pedunculata showed anthelmintic activity at a concentration of 75 mg/ml, paralysis and death timing was reported at 0.62±0.26 min and 1.42±0.07 min, respectively. The reference standard (Albendazole) showed paralysis time: 2.13±0.28 min and death time: 5.12±0.29 min. In the anti-amylase study, a zymographic density analysis of G. pedunculata showed significant variation in band intensity as compared to Starch–Iodine achromic point analysis and DNS–Maltose method. A concentration of 1.5 mg/ml of extract showed inhibition of amylase: 67.65±1.53 % as compared to other concentrations and control sets.Conclusion: It could be concluded that ethanolic extract of G. pedunculatahas biological properties which could be utilised in medicine by characterising its bioactive components.

Download Full-text

Investigating Eremophila glabra as a bioactive agent for preventing lactic acidosis in sheep

Animal Production Science ◽

10.1071/an09191 ◽

2010 ◽

Vol 50 (6) ◽

pp. 449 ◽

Cited By ~ 7

Author(s):

P. G. Hutton ◽

Z. Durmic ◽

P. E. Vercoe

Keyword(s):

Lactic Acidosis ◽

Volatile Fatty Acids ◽

Native Plant ◽

Rumen Ph ◽

Freeze Dried ◽

Australian Native Plant ◽

And Control ◽

Higher Doses

The Australian native plant Eremophila glabra was tested as a potential agent for preventing lactic acidosis in sheep after it was observed to be effective against acidosis in vitro. Ruminally fistulated wethers were infused via rumen cannula with single doses of kibbled wheat (14 g/kg bodyweight) and either virginiamycin (Eskalin500; AB, 80 mg/kg of wheat plus 100 g milled oaten hay/kg of wheat, n = 6), E. glabra (EG, 100 g freeze-dried and milled leaf material per kg of wheat, n = 10) or milled oaten hay (Control, 100 g milled oaten hay/kg of wheat, n = 16). Rumen samples were collected immediately before infusion and then 2, 4, 6, 8, 12, 16 and 24 h after the infusion. The samples were analysed for pH, D-lactate, volatile fatty acids (VFA) and osmolality. Rumen pH and D-lactate values indicative of acidosis were detected in the Control and EG groups. The pH nadir of the rumen was 12 h after the wheat infusion, at which time the values in the EG (pH = 4.87) and Control (pH = 5.09) groups were lower (P < 0.05) than in the AB group (pH = 5.63) and the D-lactate concentrations were higher (P < 0.05) in the EG and Control groups (24 mmol/L and 15 mmol/L, respectively) than in the AB group (0.9 mmol/L). At the same time, total VFA concentration was higher (P < 0.05) in the AB group (102 mmol/L) than in the Control (65 mmol/L) and the EG (14 mmol/L) groups. Rumen osmolality did not differ between groups. Virginiamycin was effective at preventing lactic acidosis. However, the inclusion of dried leaves from E. glabra at a similar level that was effective in vitro did not prevent lactic acidosis in vivo, and the reasons behind this remain unclear. The study demonstrates the difficulty in converting in vitro results to in vivo and highlights the need to test the plant at higher doses in vivo.

Download Full-text

Inhibition of Prothrombinase and Not Soluble Factor Xa (fXa) Predicts Efficacy of fXa Inhibitors in an In Vivo Model of Deep Vein Thrombosis.

Blood ◽

10.1182/blood.v108.11.902.902 ◽

2006 ◽

Vol 108 (11) ◽

pp. 902-902

Author(s):

Suzanne Delaney ◽

Ann Arfsten ◽

Sherin Halfon ◽

Gail Siu ◽

John Malinowski ◽

...

Keyword(s):

Deep Vein Thrombosis ◽

Plasma Concentration ◽

Plasma Concentrations ◽

Factor Xa ◽

Vein Thrombosis ◽

Prothrombinase Complex ◽

Antithrombotic Efficacy ◽

Deep Vein

Abstract Factor Xa (fXa) inhibitors are being tested in the clinic for the prevention and treatment of deep vein thrombosis (DVT) following orthopedic surgery. The antithrombotic efficacy of these drug candidates has traditionally been established in animal models as it is not known whether fXa amidolytic activity, activated partial thromboplastin time (aPTT) or prothrombin time (PT) predict efficacious doses. The present study was designed to test the hypothesis that the potency of fXa inhibitors against fXa incorporated into the prothrombinase complex would predict in vivo antithrombotic efficacy. Eight fXa inhibitors from four structurally distinct chemical series with a range of activities against fXa were tested for their ability to inhibit the prothrombinase complex in human plasma. Thrombin generation and subsequent cleavage of a specific thrombin substrate was used as a measure of prothrombinase activity, inhibitory activity being defined by the concentration of inhibitor required to produce a 2-fold extension in the time to maximal thrombin production (2x lag). In vitro rabbit PTs were also determined. Inhibition in the rabbit DVT model was assessed as previously described (Thromb Haemost1994; 71:357) and related to plasma concentrations of drug. Agent fXa IC50 (nM) Prothrombinase 2x lag (μM) Plasma concentration in DVT (μM) Thrombosis inhibition (%) Rabbit PT 2x change (μM) PRT50034 0.5 0.18 0.06 94 7.0 PRT54681 1.3 0.22 1.14 37 2.7 PRT54676 0.7 0.24 1.65 47 1.7 PRT54004 0.4 0.25 1.04 47 1.0 PRT54456 0.8 0.34 3.39 41 1.5 PRT56848 4.4 0.92 5.2 11 4.7 PRT54955 3.5 1.35 4.6 19 8.8 PRT57106 8.2 1.66 9.2 0 64 All compounds inhibited soluble fXa by 50 % at concentrations less than 10 nM. However, the rank order of potencies for inhibition of soluble fXa differed from that required to inhibit the prothrombinase complex. There was also poor correlation between the 2x lag value for prothrombinase inhibition and the concentration required to achieve a 2x change in rabbit PT (r2 = 0.57). Neither the activities of fXa inhibition nor the change in rabbit PT predicted activity in the DVT model. In contrast, compounds could be broadly divided into 3 levels of efficacy for inhibition of in vivo thrombus growth depending on their potency in the in vitro prothrombinase assay. PRT50034 had the lowest 2x lag value of 0.18 μM and was the most potent inhibitor of in vivo thrombosis with 94 % inhibition at a plasma concentration of 65 nM. The second group of compounds, with 2x lag values in the prothrombinase assay ranging from 0.22 to 0.34 μM, inhibited in vivo thrombus formation by 37 to 47 % at plasma concentrations ranging from 1.04 to 3.39 μM. Compounds in the third category were the least potent prothrombinase inhibitors (2x lag values greater than 0.92 μM) and were unable to significantly inhibit in vivo thrombosis even at plasma concentrations of 9.2 μM. These data show that the 2x lag value obtained in the prothrombinase assay, and not inhibition of soluble fXa or extension of rabbit PT, is capable of predicting fXa inhibitor efficacy in the in vivo rabbit DVT model.

Download Full-text

Impact of Idarucizumab and Andexanet Alfa on DOAC Plasma Concentration and ClotPro® Clotting Time: An Ex Vivo Spiking Study in A Cohort of Trauma Patients

Journal of Clinical Medicine ◽

10.3390/jcm10163476 ◽

2021 ◽

Vol 10 (16) ◽

pp. 3476

Author(s):

Daniel Oberladstätter ◽

Christoph J. Schlimp ◽

Johannes Zipperle ◽

Marcin F. Osuchowski ◽

Wolfgang Voelckel ◽

...

Keyword(s):

Plasma Concentration ◽

Healthy Volunteers ◽

Oral Anticoagulants ◽

Direct Oral Anticoagulants ◽

Factor Xa ◽

Factor Xa Inhibitors ◽

Clotting Time ◽

Andexanet Alfa ◽

The Impact

Specific antagonists have been developed for the reversal of direct oral anticoagulants (DOAC). We investigated the impact of these reversal agents on the plasma concentration and visco-elastic test results of dabigatran and factor Xa inhibitors. After baseline measurements of dabigatran, the plasma concentration, and the visco-elastic ClotPro® ecarin clotting time (ECA-CT), we added the reversal agent Idarucizumab in vitro and these two analyses were repeated. Likewise, the baseline plasma concentration of apixaban, edoxaban, and rivaroxaban as well as ClotPro® Russell’s viper venom test clotting time (RVV-CT) were measured and reanalyzed following Andexanet alfa spiking. We analyzed fifty blood samples from 37 patients and 10 healthy volunteers. Idarucizumab decreased the measured dabigatran plasma concentration from 323.9 ± 185.4 ng/mL to 5.9 ± 2.3 ng/mL and ECA-CT from 706.2 ± 344.6 s to 70.6 ± 20.2 s, (all, p < 0.001). Andexanet alfa decreased the apixaban concentration from 165.1 ± 65.5 ng/mL to 9.8 ± 8.1 ng/mL, edoxaban from 152.4 ± 79.0 ng/mL to 36.4 ± 19.2 ng/mL, and rivaroxaban from 153.2 ± 111.8 ng/mL to 18.1 ± 9.1 ng/mL (all p < 0.001). Andexanet alfa shortened the RVV-CT of patients with apixaban from 239.2 ± 71.7 s to 151.1 ± 30.2 s, edoxaban from 288.2 ± 65.0 s to 122.7 ± 37.1 s, and rivaroxaban from 225.9 ± 49.3 s to 103.7 ± 12.1 s (all p < 0.001). In vitro spiking of dabigatran-containing blood with Idarucizumab substantially reduced the plasma concentration and ecarin-test clotting time. Andexanet alfa lowered the concentration of the investigated factor Xa-inhibitors but did not normalize the RVV-CT. In healthy volunteers’ blood, Idarucizumab spiking had no impact on ECA-CT. Andexanet alfa spiking of non-anticoagulated blood prolonged RVV-CT (p = 0.001), potentially as a consequence of a competitive antagonism with human factor Xa.

Download Full-text

Structuring ENIAC

ENIAC in Action ◽

10.7551/mitpress/9780262033985.003.0003 ◽

2016 ◽

Author(s):

Thomas Haigh ◽

Mark Priestley ◽

Crispin Rope

Keyword(s):

Control Method ◽

Automatic Computation ◽

Control Sets ◽

Nested Loops ◽

Set Up ◽

And Control ◽

Distinct Approach ◽

Conditional Branch ◽

Design And Construction

This chapter explores the development of ENIAC’s overall architecture and control method. This was shaped, to a degree that has not previously been recognized, by an early and very detailed exploration led by Arthur W. Burks of how the machine could be “set up” to calculate shell trajectories, the task for which it was commissioned. Programming ENIAC was not, as has often been asserted, an “afterthought” to its design and construction. Discussion is focused in particular on the development of its master programmer unit, used to control sets of nested loops. Although it is widely believed that the ability to change the course of a computation based on results so far obtained (later be conceptualized as a conditional branch) was added to ENIAC late in its development, we show that this capability was planned for early on and that its eventual implementation as a capability of the master programmer reflected a distinct approach to the structuring of automatic computation shaped by the team’s work on the trajectory computation problem.

Download Full-text

An In-vitro Test Set-Up for Evaluation of a Voice-Producing Element Under Physiologic Acoustic Conditions

Annals of Biomedical Engineering ◽

10.1007/s10439-006-9083-y ◽

2006 ◽

Vol 34 (5) ◽

pp. 893-900 ◽

Cited By ~ 6

Author(s):

Arjan van der Plaats ◽

Harm K. Schutte ◽

Frits J. M. van der Eerden ◽

Martin P. de Vries ◽

Hans F. Mahieu ◽

...

Keyword(s):

In Vitro Test ◽

Test Set ◽

Vitro Test ◽

Set Up ◽

Acoustic Conditions

Download Full-text

The Effects of Drying Techniques on Clay-Rich Soil Texture

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100052341 ◽

1974 ◽

Vol 32 ◽

pp. 466-467

Author(s):

T. G. Naymik

Keyword(s):

Critical Point ◽

Liquid Nitrogen ◽

Liquid Nitrogen Temperature ◽

Drying Methods ◽

Air Drying ◽

Freeze Dried ◽

Critical Point Drying ◽

Set Up ◽

The Relationship ◽

Quartz Grains

Three techniques were incorporated for drying clay-rich specimens: air-drying, freeze-drying and critical point drying. In air-drying, the specimens were set out for several days to dry or were placed in an oven (80°F) for several hours. The freeze-dried specimens were frozen by immersion in liquid nitrogen or in isopentane at near liquid nitrogen temperature and then were immediately placed in the freeze-dry vacuum chamber. The critical point specimens were molded in agar immediately after sampling. When the agar had set up the dehydration series, water-alcohol-amyl acetate-CO2 was carried out. The objectives were to compare the fabric plasmas (clays and precipitates), fabricskeletons (quartz grains) and the relationship between them for each drying technique. The three drying methods are not only applicable to the study of treated soils, but can be incorporated into all SEM clay soil studies.

Download Full-text

Preparation of cultured astrocytes for x-ray microanalysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156924 ◽

1989 ◽

Vol 47 ◽

pp. 988-989

Author(s):

N.K.R. Smith ◽

K.E. Hunter ◽

P. Mobley ◽

L.P. Felpel

Keyword(s):

Cultured Cells ◽

Elemental Content ◽

Elemental Distribution ◽

Sprague Dawley ◽

X Ray ◽

Cultured Astrocytes ◽

Freeze Dried ◽

Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

Download Full-text

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615117 ◽

1998 ◽

Vol 79 (05) ◽

pp. 1041-1047 ◽

Cited By ~ 20

Author(s):

Kathleen M. Donnelly ◽

Michael E. Bromberg ◽

Aaron Milstone ◽

Jennifer Madison McNiff ◽

Gordon Terwilliger ◽

...

Keyword(s):

Active Site ◽

Thrombin Generation ◽

Factor Xa ◽

Coagulation Factor ◽

Melanoma Cells ◽

Factor V ◽

Ancylostoma Caninum ◽

Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

Download Full-text

A Heparin-coated Circuit Maintains Platelet Aggregability in Response to Shear Stress in an In Vitro Model of Cardiopulmonary Bypass

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615226 ◽

1998 ◽

Vol 80 (09) ◽

pp. 437-442 ◽

Cited By ~ 2

Author(s):

I. Hioki ◽

K. Onoda ◽

T. Shimono ◽

H. Shimpo ◽

K. Tanaka ◽

...

Keyword(s):

Shear Stress ◽

Cardiopulmonary Bypass ◽

Membrane Skeleton ◽

Platelet Glycoprotein ◽

Platelet Surface ◽

Platelet Aggregability ◽

Vitro Model ◽

Platelet Defects ◽

Set Up

SummaryAlterations in platelet aggregability may play a role in the pathogenesis of qualitative platelet defects associated with cardiopulmonary bypass (CPB). We circulated fresh heparinized whole blood through tubing sets coated with heparin (C group, n = 10) and through non-coated sets (N group, n = 10) as a simulated CPB circuit. Shear stress (108 dyne/cm2)-induced platelet aggregation (hSIPA), plasma von Willebrand factor (vWF) activity and platelet glycoprotein (GP) Ib expression were measured, before, during, and after this in vitro set up of circulation. In the two groups, the extent of hSIPA significantly decreased during circulation and was partially restored after circulation. Decreases in the extent of hSIPA were significantly less with use of heparin-coated circuits. There was an equivalent reduction in plasma vWF activity, in the two groups. Expression of platelet surface GP Ib decreased significantly during circulation and recovered after circulation. Reduction of surface GP Ib expression during circulation was significantly less in the C group than that in the N group. Decrease in surface GP Ib expression correlated (r = 0.88 in either group) with the magnitude of hSIPA, in the two groups. The progressive removal of surface GP Ib was mainly attributed to redistribution of GP Ib from the membrane skeleton into the cytoskeleton. Our observations suggest that use of heparin-coated circuits partly blocks the reduction of hSIPA, as a result of a lesser degree of redistribution of GP Ib.

Download Full-text