Factor Xa

The first novel anticoagulant that targets factor Xa specifically is fondaparinux sodium. The IC50 value (anti-Xa IU/ml) for fondaparinux is 0.59±0.05 for activated monocytes (ac-M) and 0.17±0.03 for MMPs (monocyte-derived microparticles) [2].

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Effect of Fondaparinux, low molecular weight heparin and unfractionated heparin on thrombin generation supported by activated monocytes [2]
Values obtained at baseline were 2.1 ± 0.1 min for lag time, 181 ± 6 nmol/l for peak, 1624 ± 84 nmol/l min for ETP and 59 ± 4 nmol/l/min for rate index. Fondaparinux, enoxaparin and UFH inhibited ac-M-supported thrombin generation in a concentration-dependent manner (Fig. 1). Except for the lag time, the inhibitory effect of UFH was superior to enoxaparin, which was superior to fondaparinux, from 0.3 to 1.0 anti-Xa IU/ml. Whereas comparable inhibitory effect of fondaparinux and enoxaparin on peak and ETP was noted at the lowest anti-Xa concentration (0.1 IU/ml), the inhibitory effect on rate index of fondaparinux was more pronounced than that of enoxaparin at 0.1 IU/ml anti-Xa concentration.

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Calculation of IC50 [2]
The inhibitory effect on thrombin generation was evaluated by calculating the inhibitory concentration (IC)50, which was defined for peak, ETP and rate index as the drug concentration yielding 50% reduction, and for lag time as the drug concentration resulting in doubling it. UFH was the only drug for which IC50 was calculated for all thrombin generation parameters in the two models. In contrast, enoxaparin IC50 values were calculated for ETP, peak and rate index, but not for lag time, in the two models. Furthermore, IC50 for Fondaparinux was reached only for rate index in the two models and for peak and ETP only in the MMP model. Rate index was the only parameter for which IC50 was reached in both models for the three drugs; UFH was the most effective, followed by enoxaparin and fondaparinux (Table 1).

The pharmacokinetics of fondaparinux sodium are linear and dose-dependent, resulting in a very predictable response. With a quick start of action, a half-life of 14 to 16 hours, and continuous antithrombotic activity for 24 hours, fondaparinux sodium is 100% bioavailable. The medication has no effect on platelet function or aggregation, prothrombin time, or activated partial thromboplastin time [1].

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Compared with the ex-vivo anti-Xa levels reported in Organization to Assess Strategies in Acute Ischemic Syndromes (OASIS)-5 substudy, our results demonstrate that anti-Xa values obtained in patients are either similar or even higher than those required in our in-vitro models used to reach rate index IC50. Fondaparinux anti-Xa level of 0.52 (±0.22) IU/ml in OASIS-5 study was sufficient to inhibit rate index in both in-vitro models tested. For enoxaparin, the anti-Xa level measured in OASIS-5 substudy patients was 1.2 (±0.45) IU/ml, which was markedly more superior than anti-Xa concentrations required to inhibit rate index in the two models of thrombin generation tested here (ac-M IC50: 0.27 ± 0.03 IU/ml, and MMPs IC50 0.19 ± 0.02 IU/ml).

The OASIS-5 substudy reported less inhibition of ETP in Fondaparinux-treated patients versus enoxaparin. Our in-vitro ETP results are in agreement with these data, taking into consideration that the Oasis-5 substudy ETP was performed in cell-free assay. Fondaparinux yielded lower inhibition than enoxaparin in both models, with anti-Xa concentrations ranging from 0.3 to 1.0 IU/ml. Fondaparinux ETP IC50 values were not always reached despite increased anti-Xa levels (fondaparinux tested at concentrations ranging from 0.1 to 1.0 anti-Xa IU/ml), and its ETP IC50 was reached only in the MMPs model. In contrast, enoxaparin reached ETP IC50 in the two in-vitro models tested here. As for rate index, the levels of anticoagulation in OASIS-5 enoxaparin-treated patients are much more superior to those required to inhibit ETP in our cell models.

Fondaparinux at 2.5 mg daily (corresponding to anti-Xa of 0.52 ± 0.22 IU/ml) in the Oasis-5 study, showed an optimal efficacy safety in ACS patients. This anti-Xa level for fondaparinux corresponded to the rate index IC50 of the ac-M model and to the ETP IC50 value of the MMP model. At this anti-Xa concentration, rate index was inhibited in the MMP model and ETP IC50 was not attained in ac-M model. For enoxaparin, rate index IC50 in ac-M model was similar to ETP IC50 in MMP model and was closely equal to 0.3 IU/ml. At this anti-Xa level, rate index in MMP model was largely inhibited and ETP IC50 in ac-M was not reached. Given this, it is assumed that an anti-Xa level of about 0.3 IU/ml for enoxaparin is sufficient in ACS for an optimal efficacy-safety profile. It should be noted that, of the three agents analysed here, fondaparinux is the only agent for which the daily dose has been rationally determined, as evidenced by the use of 2.5 mg dose on the basis of previous dose-finding studies [2].

Monocyte activation [2]
Purified monocytes were washed in RPMI-1640 medium and then adjusted to 1.5 106 cells/ml in RPMI-1640 containing 5% (v/v) heat-inactivated foetal calf serum, 2 mmol/l L-glutamine and 100 ng/ml LPS (Escherichia coli serotype O55:B5, Sigma). Following incubation of the monocytes for 5 h at 37°C in a 5% CO2 humidified atmosphere, supernatants were removed by centrifugation for 5 min at 400g. The ac-M-containing pellet was resuspended in 150 μl phosphate-buffered saline.

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Monocyte-derived microparticles preparation and quantification [2]
Monocytes were incubated for 18 h, as described above, and supernatants were collected by centrifugation for 5 min at 2200g. MMPs were recovered following an additional centrifugation at 17000g for 30 min and quantified by flow cytometry [5]. MMPs (100 μl) were incubated for 15 min in the dark with 1 μl annexin V-FITC, according to manufacturer's recommendations, followed by the addition of 400 μl annexin V-binding buffer and 100 μl flow-count Fluorospheres. Fluorescence was acquired for 60 s on Epics XL-MCL flow cytometer, using System 2 software. MMPs were quantified using the flow-count Fluorospheres and expressed as MMPs/μl.

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Fluorogenic measurement of thrombin generation [2]
Thrombin generation test was performed according to the assay described by Hemker and modified by Poitevin; ac-Ms and MMPs being defined as cell models. PPP used for all experimental conditions was systematically supplemented with aprotinin at 200 kallikrein inhibitory units (KUI)/ml final concentration. PPP was spiked with increasing concentrations of Fondaparinux, LMWH or UFH; final concentrations being 0.0, 0.1, 0.3, 0.6 and 1.0 anti-FXa IU/ml. We previously optimized the concentrations of ac-Ms and MMPs included in the assay. Thus, 20 μl of ac-M (0.2 × 106 ac-M per well) or of MMPs (160 000 MMPs per well) was added to 80 μl of PPP. Results were expressed as percentage, taking the baseline measurements as 100%.

Fluorometric determination of thrombin generation was done on Fluoroskan Ascent plate reader, following addition of the fluorogenic substrate, Z-Gly-Gly-Arg-AMC; Thrombinoscope software used for calculating thrombin generation. Four parameters of the thrombin generation curve were analysed: lag time (min), thrombin peak (peak, nmol/l), endogenous thrombin potential (ETP, nmol/l min), rate index of propagation phase defined by the formula: peak/(time to peak – lag-time) (rate index, nmol/l/min).

Platelet-poor plasma preparation [2]
Venous blood samples were obtained from five healthy volunteers (mean age 25 ± 2 years). Volunteers were laboratory staff members and did not receive any medication for the last 2 weeks. Informed consents were obtained from all of them. Blood was withdrawn by antecubital venipuncture into Monovette tubes (0.106 mol/l citrate). A three-step centrifugation procedure including 10 min at 190g, 10 min at 1750g and 30 min at 13 000g was used. Platelet-poor plasma (PPP) supernatants were pooled, stored at −80°C and thawed immediately before use.

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Monocyte purification [2]
Cytapheresis material was obtained from six healthy volunteers who were admitted for platelet donation in the blood transfusion unit of CHU Robert Debré. Informed consent was obtained from the participants. Monocytes were purified from cytapheresis residues by elutriation, as previously described. Monocyte purity was assessed by CD14 staining of isolated cells (>95% CD14-positive). Cell viability (>98%) was determined by the trypan blue exclusion principle. Purified monocytes were used for preparing ac-Ms and MMPs.

The pharmacology and mechanism of action of fondaparinux sodium are described. Fondaparinux sodium is the first agent of a new class of anticoagulants that selectively target factor Xa. It has a linear, dose-dependent pharmacokinetic profile, which provides a highly predictable response. It is 100% bioavailable, has a rapid onset of action, and has a half-life of 14 to 16 hours, allowing for sustained antithrombotic activity over a 24-hour period. The drug does not affect prothrombin time or activated partial thromboplastin time, nor does it affect platelet function or aggregation. Studies in patients with confirmed heparin-induced thrombocytopenia demonstrate that the drug is not associated with in vitro cross-reactivity to heparin antibodies. Fondaparinux sodium appears to meet the criteria for an ideal antithrombotic agent: equal or better effectiveness than currently available agents, a low bleeding risk, no need for laboratory monitoring, and once-daily administration.[1]

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Fondaparinux is considered to be acceptable to use during breastfeeding.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[1]. Fondaparinux sodium: a selective inhibitor of factor Xa. Am J Health Syst Pharm. 2001 Nov 1;58 Suppl 2:S14-7.

[2]. Differential coagulation inhibitory effect of fondaparinux, enoxaparin and unfractionated heparin in cell models of thrombin generation. Blood Coagul Fibrinolysis. 2011 Jul;22(5):369-73.

Fondaparinux Sodium is the sodium salt form of fondaparinux, a synthetic glucopyranoside with antithrombotic activity. Fondaparinux sodium selectively binds to antithrombin III, thereby potentiating the innate neutralization of activated factor X (Factor Xa) by antithrombin. Neutralization of Factor Xa inhibits its activity and interrupts the blood coagulation cascade, thereby preventing thrombin formation and thrombus development. (NCI05)
Synthetic pentasaccharide that mediates the interaction of HEPARIN with ANTITHROMBINS and inhibits FACTOR Xa; it is used for prevention of VENOUS THROMBOEMBOLISM after surgery.
See also: Fondaparinux (has active moiety).

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Drug Indication
1. 5-mg/0. 3-ml and 2. 5-mg/0. 5-ml solution for injectionPrevention of venous thromboembolic events (VTE) in adults undergoing major orthopaedic surgery of the lower limbs such as hip fracture, major knee surgery or hip-replacement surgery. Prevention of VTE in adults undergoing abdominal surgery who are judged to be at high risk of thromboembolic complications, such as patients undergoing abdominal cancer surgery. Prevention of VTE in adult medical patients who are judged to be at high risk for VTE and who are immobilised due to acute illness such as cardiac insufficiency and / or acute respiratory disorders, and / or acute infectious or inflammatory disease. Treatment of adults with acute symptomatic spontaneous superficial-vein thrombosis of the lower limbs without concomitant deep-vein thrombosis. 2. 5-mg/0. 5-ml solution for injectionTreatment of unstable angina or non-ST-segment-elevation myocardial infarction (UA/NSTEMI) in adult patients for whom urgent (< 120 mins) invasive management (PCI) is not indicated. infarction (STEMI) in adult patients who are managed with thrombolytics or who initially are to receive no other form of reperfusion therapy. 5-mg/0. 4-ml, 7. 5-mg/0. 6-ml and 10-mg/0. 8-ml solution for injectionTreatment of adults with acute deep-vein thrombosis (DVT) and treatment of acute pulmonary embolism (PE), except in haemodynamically unstable patients or patients who require thrombolysis or pulmonary embolectomy.
1. 5 mg/0. 3 ml and 2. 5 mg/0. 5 ml, solution for injection: , Prevention of Venous Thromboembolic Events (VTE) in patients undergoing major orthopaedic surgery of the lower limbs such as hip fracture, major knee surgery or hip replacement surgery. , Prevention of Venous Thromboembolic Events (VTE) in patients undergoing abdominal surgery who are judged to be at high risk of thromboembolic complications, such as patients undergoing abdominal cancer surgery (see section 5. 1). , Prevention of Venous Thromboembolic Events (VTE) in medical patients who are judged to be at high risk for VTE and who are immobilised due to acute illness such as cardiac insufficiency and/or acute respiratory disorders, and/or acute infectious or inflammatory disease. , , 2. 5 mg/0. 5 ml, solution for injection: , Treatment of unstable angina or non-ST segment elevation myocardial infarction (UA/NSTEMI) in patients for whom urgent (< 120 mins) invasive management (PCI) is not indicated (see sections 4. 4 and 5. 1). , Treatment of ST segment elevation myocardial infarction (STEMI) in patients who are managed with thrombolytics or who initially are to receive no other form of reperfusion therapy. , , 5 mg/0. 4 ml, 7. 5 mg/0. 6 ml and 10 mg/0. 8 ml solution for injection: , Treatment of acute Deep Vein Thrombosis (DVT) and treatment of acute Pulmonary Embolism (PE), except in haemodynamically unstable patients or patients who require thrombolysis or pulmonary embolectomy.

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Anticoagulants, including unfractionated heparin (UFH), enoxaparin and fondaparinux, are approved drugs in acute coronary syndrome (ACS). Monocytes and monocyte-derived microparticles (MMPs) play an important procoagulant role in ACS by expressing high tissue factor (TF) levels, which in turn triggers thrombin generation. The objective of our study is to compare the in-vitro inhibitory effect of UFH, enoxaparin and fondaparinux in monocytes and MMP models. Human-elutriated monocytes were activated for 5 and 18 h by lipopolysaccharide to obtain activated monocytes (ac-M) or MMPs, respectively. Thrombin generation inhibition was assessed using ac-M or MMPs mixed with platelet-poor plasma containing increased concentrations of anticoagulants. Thrombin generation inhibition was dose-dependent with a differential effect according to the drug: the highest for UFH, the lowest for fondaparinux. Rate index was the most sensitive parameter. For fondaparinux, its IC50 values (anti-Xa IU/ml) were 0.59±0.05 for ac-M and 0.17±0.03 for MMPs. For enoxaparin, rate index IC50 values were 0.27±0.03 for ac-M and 0.19±0.02 for MMPs. Our data support the notion that cell-induced thrombin generation assay may be a reliable alternative to anti-Xa assessment in determining patient anticoagulation level. [2]

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In conclusion, our study demonstrates a differential inhibitory effect of the three anticoagulants approved in ACS, the evaluation being performed using cell-induced thrombin generation models. Our results support the conclusion reached in OASIS-5 substudy that lower anticoagulation levels should be considered. Our results clearly show the limitation to answer the effect of an anticoagulant molecule using anti-Xa assays. Thrombin generation is an informative and sensitive assay, which remains difficult to standardize, although improvement of standardization is ongoing. Thrombin generation could be used to evaluate the pharmacodynamic effect of new anticoagulant drugs. Furthermore, thrombin generation assay could be a helpful tool to assess more accurately the level to reach in phase I and II studies.[2]

C31H43N3NA10O49S8

1728.0815

1726.77

C, 21.55; H, 2.51; N, 2.43; Na, 13.30; O, 45.37; S, 14.84

114870-03-0

104993-28-4;114870-03-0 (sodium);

636380

White to off-white solid powder

9

52

20

101

3330

25

CO[C@@H]1[C@@H]([C@H]([C@@H]([C@H](O1)COS(=O)(=O)[O-])O[C@H]2[C@@H]([C@H]([C@@H]([C@@H](O2)C(=O)[O-])O[C@@H]3[C@@H]([C@H]([C@@H]([C@H](O3)COS(=O)(=O)[O-])O[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)C(=O)[O-])O[C@@H]5[C@@H]([C@H]([C@@H]([C@H](O5)COS(=O)(=O)[O-])O)O)NS(=O)(=O)[O-])O)O)OS(=O)(=O)[O-])NS(=O)(=O)[O-])O)OS(=O)(=O)[O-])O)NS(=O)(=O)[O-].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+]

XEKSTYNIJLDDAZ-JASSWCPGSA-D

InChI=1S/C31H53N3O49S8.10Na/c1-69-27-9(33-85(48,49)50)13(37)17(6(74-27)3-71-88(57,58)59)76-31-22(83-91(66,67)68)16(40)21(24(81-31)26(43)44)79-29-10(34-86(51,52)53)19(82-90(63,64)65)18(7(75-29)4-72-89(60,61)62)77-30-15(39)14(38)20(23(80-30)25(41)42)78-28-8(32-84(45,46)47)12(36)11(35)5(73-28)2-70-87(54,55)56;;;;;;;;;;/h5-24,27-40H,2-4H2,1H3,(H,41,42)(H,43,44)(H,45,46,47)(H,48,49,50)(H,51,52,53)(H,54,55,56)(H,57,58,59)(H,60,61,62)(H,63,64,65)(H,66,67,68);;;;;;;;;;/q;10*+1/p-10/t5-,6-,7-,8-,9-,10-,11-,12-,13-,14-,15-,16+,17-,18-,19-,20+,21+,22-,23+,24-,27+,28-,29-,30-,31-;;;;;;;;;;/m1........../s1

decasodium;(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5R,6R)-6-[(2R,3S,4S,5R,6R)-2-carboxylato-4-hydroxy-6-[(2R,3S,4R,5R,6S)-4-hydroxy-6-methoxy-5-(sulfonatoamino)-2-(sulfonatooxymethyl)oxan-3-yl]oxy-5-sulfonatooxyoxan-3-yl]oxy-5-(sulfonatoamino)-4-sulfonatooxy-2-(sulfonatooxymethyl)oxan-3-yl]oxy-3-[(2R,3R,4R,5S,6R)-4,5-dihydroxy-3-(sulfonatoamino)-6-(sulfonatooxymethyl)oxan-2-yl]oxy-4,5-dihydroxyoxane-2-carboxylate

Fondaparinux sodium; 114870-03-0; Arixtra; Quixidar; Fondaparin sodium; Xantidar; Fondaparinux sodium for assay; SR 90107A;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~125 mg/mL (~72.33 mM)

Solubility in Formulation 1: 100 mg/mL (57.87 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)