WO2013007762A1

WO2013007762A1 - Combination of an anticoagulant and avidin useful in surgical intervention and clinical procedure

Info

Publication number: WO2013007762A1
Application number: PCT/EP2012/063616
Authority: WO
Inventors: Eric Neuhart; Maurice Petitou
Original assignee: Endotis Pharma
Priority date: 2011-07-11
Filing date: 2012-07-11
Publication date: 2013-01-17

Abstract

The present invention concerns products containing: compound (I) of formula or a salt -thereof; and avidin, as a combination product for the separate and successive administration in prevention of blood clotting disorders during and after a surgical intervention or a clinical procedure, wherein compound (I) or a salt -thereof is administered just before the surgical intervention or clinical procedure and avidin is administered after the surgical intervention or clinical procedure.

Description

Combination of an anticoagulant and avidin useful in surgical intervention and clinical procedure

Technical Field

The present invention is concerned with use of an anticoagulant (i.e., a substance that prevents blood clotting) for preventing thrombosis during and after a surgical intervention or clinical procedure and of its antidote to prevent major bleeding complications after a surgical intervention or clinical procedure.

Background art

Surgical interventions, particularly cardiac surgical interventions include in particular coronary artery bypass grafting (CABG), valve replacement, congenital heart diseases, heart transplantation. Surgical interventions can be performed using open heart or beating heart techniques.

Clinical procedures include in particular percutaneous coronary intervention (PCI) such as coronary angioplasty and stenting, transcatheter aortic-valve implantation (TAVI) and other non-su rgical valve replacement/repair procedures.

Extracorporeal blood circulation (ECC) is used in particular during open-heart surgery (in this case ECC is synonym of cardiopulmonary bypass) for creating an artificial blood flow through a circuit constructed outside the body (including in particular pump, oxygenator and tubes). ECC is used for blood oxygenation in a heart lung apparatus to allow oxygen supply to all organs while the heart is arrested during open heart surgery.

ECC is also used during extracorporeal membrane oxygenation (ECMO), or for circulatory assistance such as artificial heart, ventricular assist device or the like. ECC is also used for blood purification in an artificial kidney during haemodialysis or haemofiltration. ECC can also be used for noncardiac surgical applications of cardiopulmonary bypass.

During these surgical interventions and clinical procedures blood coagulation is strongly activated, firstly by the contact with foreign materials used (catheter or tubes or surgical instruments, etc.) , secondly by various endogenous prothrombotic factors activated during such interventions. Such situations therefore requi re the use of potent anticoagulants . These anticoagulants are ideally neutralised at the end of the intervention to avoid a major bleeding complication.

Blood clotting is a complex process: coagulation factors (enzymes involved in blood coagulation) are present in blood in an inactive state. When needed, they are activated through partial proteolysis, in a cascade of reactions where the enzyme formed at one step acts to catalyse the next reaction, hence the common term "coagulation cascade". The final result of this series of reactions is the conversion of fibrinogen into fibrin, a major component of blood clots.

The nonbiological surfaces of the circuit used in ECC are known to have a strong procoagulant effect. When blood is in contact with these foreign surfaces, coagulation factor XII (contact factor) is converted into factor XI la, which activation triggers the whole coagulation cascade. The artificial materials used during other heart surgery interventions also activate the coagulation system. Thus, when blood is contact with nonbiological surfaces, it is rapidly coagulated and loses flowability.

Endogenous prothrombotic factors such as tissue factor a protein expressed by several cells under stress conditions also activates blood coagulation and contributes to the procoagulant state induced by surgical interventions or clinical procedures.

To offset this biological response, anticoagulants are routinely used during surgical or medical interventions. Historically, the reference anticoagulant unfractionated heparin (UFH) has been used in ECC for cardiac surgery since the origin of this technique, because it is a potent anticoagulant and because its anticoagulant effect can be neutralised by injection of a specific antidote protamine. UFH and protamine are still nowadays the only anticoagulant and antidote used in ECC during cardiac surgery.

Patients suffering from cardiac diseases are often treated with heparin and are therefore at risk of heparin-induced thrombocytopenia (HIT), a life- threatening condition of immunoallergic origin. In case of HIT, patients are at very high risk when cardiac surgery is required because of the need to re- administer UFH.

Protamine sulphate, the heparin antidote must be injected at the end of the procedure because the high level of anticoagulation required during ECC put the patient at risk of major bleeding at the end of the surgical procedure when the surgeon is to perform haemostasis of the operating field to be able to close the patients' chest.

Protamine is a highly cationic protein that is not always well tolerated . Protamine can cause in particular histamine release resulting in hypotension and bronchoconstriction. It can also cause pulmonary hypertension. At high doses, protamine itself has some anticoagulant effect.

The combined use of heparin and protamine is responsible for the so-called "rebound effect" that sometimes occurs and can result in bleeding and cardiac reintervention of the operated patient.

Therefore there is an unmet medical need for a more effective, safer and neutralizable anticoagulant agent that can be used for preventing blood clotting, particularly during extracorporeal blood circulation for cardiac surgery.

Compound 1 (see formula below) is an anticoagulant agent and was described in WO 2006/067173 as a new dual inhibitor of factor Xa and factor Ma that can be neutralised with avidin. In particular WO 2006/067173 describes its process of preparation and its anti-factor Xa and anti-factor Ma activities.

WO 2006/067173 indicates, among a lot of other therapeutic activities, its possible use as anticoagulants in extracorporeal blood circulation such as during surgery.

However no example of use is given . Furthermore the dosage is not indicated and cardiac surgery and in particular cardiopulmonary bypass is not mentioned.

1

Furthermore, while the Activated Clotting Time (ACT) test is used to monitor the UFH treatment during ECC, and while a high value in this test, typically above 400 seconds and between 400 and 500 seconds but even above 500 seconds depending on the country or center, is required by clinicians, compound 1 has a limited effect on the ACT test and for this mere reason its usefulness in ECC was not obvious. Indeed, at the highest dose tested in clinics in healthy subjects (12 mg), clinical trials have shown that the ACT value is hardly above 350 seconds, and the anticoagulant activity plateaus at this value. The inventors decided to test the use of this compound in a dog model of ECC with a circuit (pump, oxygenator and tubes) used in human clinics. Surprisingly the inventors have discovered that compound 1 , despite its low effect on factor Xlla (Ki > 10^"5 M) and its limited effect on the ACT test in human beings, was efficient in preventing clot formation in a dog model of ECC with a circuit (pump, oxygenator and tubes) used in human clinics. Moreover, it has been surprisingly observed that low doses that hardly affect the ACT (ACT around 200 seconds) are as efficient as higher doses in preventing clotting of the circuit.

Avidin is known to be efficient for a rapid elimination of biotinylated compounds like compound 1 . Avidin is a glycoprotein extracted from hen eggs. The glycosylation pattern of glycoprotei ns plays a major role in thei r pharmacokinetics. Glycoproteins are rapidly cleared from the blood circulation as soon as they display some previously hidden unique carbohydrates motifs. For instance, desialyiation of siaiyiated human glycoproteins uncovers galactose residues that are trapped by specific receptors in the liver (Ashwell & Morell, 1974) leading to elimination of the glycoprotein.

Avidin is a glycoprotein that contains mannose and N-acetyl-glucosamine residues. Glycoproteins displaying mannose and N-acetyl-glucosamine residues are cleared by a pathway comprising specialised liver cells, as well as cells present in the spleen, bones and elements of the reticuloendothelial system (Schlesinger et al., 1980). The mannose receptors, for examples, present on macrophages are involved in this process.

When avidin is injected into mammals, it is immediately cleared by this latter mechanism. The half-life of avidin is about 10 to 14 min in human.

Avidin forms an extraordinary tight equimolecular complex with biotin. This means that avidin can bind up to four biotin molecules. Binding of biotin to avidin has been reported to slightly influence the half-life of avidin, which is species dependent. For example, in mice, the biotin-avidin complex is cleared from blood even faster than free avidin (Schechter et al. , Eur. J. Biochem. 1990: 189, 327-331 ).

Based on the above rapid clearance of the biotin-avidin complex, biotinylation of anticoagulant oligosaccharides has been devised as a way to allow their neutralisation through injection of avidin. Indeed, the tight complex immediately formed between avidin and the biotinylated oligosaccharide is rapidly cleared from the circulation (Savi et al., 2008).

It was therefore believed that administration of avidin would lead to rapid elimination of compound 1 . The inventors have now surprisingly found out that the combined use of compound 1 , administered before a surgical intervention or clinical procedure, and avidin, administered after, results in an anticoagulant activity that can prevent post-operative ischemic events and can allow delaying the administration of the post-operative antithrombotic treatment in particular in case of major bleeding.

The authors have surprisingly found that the complex formed between compound 1 and avidin upon administration of the latter has an unexpectedly long half-life and that this complex exhibits anti-factor Xa activity that can be compared to the activity of a well acknowledged antithrombotic agent, fondaparinux, which is known to be sufficient to prevent blood clotting disorder.

Indeed, in human, injection of compound 1 resulted in a long-lasting anticoagulant effect. Following avidin injection, the anticoagulant activity was rapidly abolished, and using the ACT test, a return to the baseline was observed at the end of the avidin infusion period and no rebound of activity was noticed, as could be predicted from Savi et al. This observation is also in line with the widespread view that the biotin-avidin complex is cleared within minutes from the circulation by a mechanism involving trapping of the mannosylated protein by cells of the reticuloendothelial system.

However, using selective anticoagulant tests, particularly selective assessment of factor Xa inhibition, the authors have surprisingly detected significant long-lasting anti-factor Xa activity in human after injection of compound 1 followed by avidin. The authors have also surprisingly found that the half-life of the compound 1 -avidin complex is about three hours in rat, which is equivalent to the half-life of free compound 1 in the same species.

In human, the observed anti-factor Xa activity slowly disappears over 24 hou rs when compound 1 is i njected at a level suitable for ensuri ng anticoagulation during ECC and then neutralised by an appropriate amount of avidin.

Thus, the inventors have found that, using an appropriate dose of compound 1 before surgical intervention or clinical procedure and a successive appropriate molar ratio of avidin, the level of anti-factor Xa activity at the end of the treatment by compound 1 and avidin is such that it contributes to the post-operative antithrombotic treatment necessary in particular to prevent post-operative venous thromboembolism, to maintain coronary artery graft patency, and to prevent arterial ischemic embolism such as stroke or transient ischemic attack, particularly after valve replacement.

Indeed, conventional treatment after such surgical intervention or clinical procedure consists in administering aspirin and/or other platelet aggregation inhibitors on the very day, after the surgical intervention or clinical procedure and after it. Then, on the very day or typically from the following day onwards, an anticoagulant drug is administered, such as heparin, low molecular weight heparin or fondaparinux. Administering antiplatelet and/or anticoagulant drugs prevents post-operative venous thromboembolism, arterial ischemic embolism particularly after valve replacement, maintains coronary artery graft patency.

However, for a patient who had a surgical intervention or a clinical procedure and that presents a major bleeding complication after the surgery intervention or clinical procedure, it could be problematic to administer antiplatelet and/or anticoagulant drugs since these drugs might increase bleeding. Therefore, the physician needs to weight the pros and cons of antiplatelet and/or anticoagulant drugs administration, taking the risk of a post-operative major bleeding.

A major bleeding is defined as an above average chest tube drainage blood loss volume, and/or an overt bleeding requiring surgery or percutaneous intervention or intravenous inotropes for hemodynamic control, and/or an overt bleeding associated with hemoglobin drop > 30 g/L and or requiring any red blood cell transfusion and/or an intracranial or intraspinal bleeding, intraocular bleeding compromising vision or intrapericardial bleeding with cardiac tamponade.

Therefore, using compound 1 and avidin can still prevent post-operative thromboembolic events listed above while allowing the delay of antiplatelet and/or anticoagulant drugs in particular in case of major bleeding.

The authors have devised a new treatment scheme for anticoag during ECC using compound 1 and avidin. Description of the invention

Therefore, the present invention concerns products containing:

compound 1 of formula

1 ,

or a salt -thereof; and

avidin,

as a combination product for the separate and successive administration in prevention of both blood clotting disorder during and after a surgical intervention or a clinical procedure, wherein compound 1 or a salt thereof is administered just before and avidin is administered after the surgical intervention or the clinical procedure. Blood clotting disorder includes peri-operative bleedings and related transfusions, post surgical prophylaxis of deep venous thrombosis, thrombosis of the coronary grafts, coronary syndromes such as myocardial infarction, stroke and transient ischemic events, other thromboemebolic events and thrombosis of the ECC circuit.

The present invention deals particularly with cardiac surgical interventions comprising in particular coronary artery bypass grafting (CABG) performed using open or beating heart cardiac surgery, open-heart surgery (cardiopulmonary bypass) for valve replacement, congenital heart diseases or heart transplant, emergency cardiac surgery for patients with HIT, extra- and intra-corporeal artificial heart or ventricular assist device, noncardiac surgical applications of cardiopulmonary bypass.

The present invention could be as well applied to clinical procedures comprising in particular cardiac procedure such as PCI such as coronary angioplasty, TAVI and other non-surgical valve replacement/repair procedures, ECMO, and non-cardiac procedure such as artificial kidney during haemodialysis or haemofiltration.

Particularly, cardiac surgery on patient over 75 years old has become common and at least 50 % of all patients are considered at a high risk of complication. For example, severe bleeding occurs in more than 10 % of high- risk patient and is directly associated with a high rate of cardiac reintervention, massive transfusions, stroke, death and other complications (Fergusson D.A. et al. , N. Engl. J. Med., 2008, 358, 2319-2331 ).

"Before the surgical intervention or clinical procedure" means before any incision or other invasive intervention is performed. "After the surgical intervention or clinical procedure" means after the surgical intervention or clinical procedure is over and that no further invasive intervention is performed.

Advantageously, for a surgical intervention or a clinical procedure, compound 1 or a salt thereof is administered at the beginning thereof particularly between 5 to 30 minutes before the surgical intervention or the clinical procedure. Advantageously, compound 1 is administered as a bolus followed by a continuous infusion by intravenous route of administration to the patient undergoing a surgical intervention or clinical procedure. Avidin is administered just before the end of the surgical intervention or clinical procedure, i.e., between 30 minutes and several hours, advantageously 1 h 30 after the admi nistration of compound 1 and preferably by i nfusion of advantageously 30 min and potentially up to 60 min by intravenous infusion into the patient. The products are therefore advantageously in a form suitable for intravenous administration i.e., they are provided in a sterile buffer solution (1 to 20 mg/mL for compound 1 ; 5 to 30 mg/mL for avidin) or as a lyophilised powder (1 to 20 mg for compound 1 ; 5 to 300 mg for avidin), in which case a sterile buffer solution is provided for extemporaneous preparation of the solution to be injected.

In one embodiment, compound 1 and avidin of the combination product are separately and successively administered in prevention of blood clotting disorders during and after ECC.

For an ECC, compound 1 or a salt thereof is administered at the beginning of the ECC, particularly between 10-15 min before the beginning of the ECC. Advantageously, compound 1 is administered as a bolus followed by a continuous infusion by intravenous route of administration to the patient undergoing ECC or directly in the ECC circuit. Avidin is administered just after the end of ECC, advantageously on average 1 h30 after the administration of compound 1 and preferably by infusion of advantageously 30 min by intravenous infusion. The products are therefore advantageously in a form suitable for intravenous administration or administration directly in the ECC circuit.

Compound 1 provides stable and constant anticoagulant throughout cardiac surgery and ECC as opposed to UFH which needs to be frequently monitored and re-dosed. Compound 1 is also well tolerated by human subjects.

Compound 1 can be a pure compound or a mixture of two diastereoisomers, arising from epimerisation at the lysine connected to biotin. Both diastereoisomers arising from such epimerisation displayed similar in vitro pharmacodynamic (PD) effects on factor Xa and Ma inhibition and in vivo pharmacokinetic (PK) profiles in dogs and rats. Therefore, the configuration of the lysine motif in the structure of compound 1 has little influence, if any, on the PK/PD profile of the two separate isomers. In a particular embodiment, compound 1 is in the form of a pharmaceutical composition which is advantageously a clear, colourless and isotonic aqueous solution. It is in particular formulated in a 13 mM phosphate buffer at pH 6.0. It is supplied in a vial of 1 or 2 mL at a concentration of 10 mg/mL.

Avidin is in the form of a pharmaceutical composition which is advantageously a clear, colourless and isotonic aqueous solution. It is in particular formulated in phosphate buffer with 0.07% polysorbate 80 at pH 7. It is supplied in vials of 5 mL at a concentration of 20 mg/mL.

Compound 1 or its salt is administered as a bolus dose of 1 to 24 mg, preferably 4 to 16 mg/surgical interventions or clinical procedures, more preferably 8 to 16 mg/ surgical interventions or clinical procedures, still more preferably 12 to 16 mg/ surgical interventions or clinical procedures and might be followed by a continuous infusion of compound 1 of 4 to 20 mg/h, more preferably of 6 to 16 mg/h, still more preferably of 10 to 16 mg/h. Avidin is administered at a dose of 0.5 to 5 molar ratio over total administered compound 1 , preferably 1 to 3 molar ratio, more preferably 2 to 3 molar ratio.

These doses enable a clotting/bleeding risk balance management.

Surgical interventions or clinical procedures mean from the beginning to the end thereof as defined above. It does not include repetitive surgical interventions or clinical procedures, i.e. carried out at different and distinctive times. Surgical intervention can also mean the time during which ECC is undergoing, advantageously, from 10-15 min before the start of the ECC and until the end of the infusion of avidin.

Advantageously, the treatment with Compound 1 and avidin results in a residual anticoagulant anti-factor Xa activity that can prevent post-operative ischemic events and can allow delaying the administration of an antithrombotic treatment in particular in case of a major bleeding. This administration may be delayed by at least 12 h, preferably by at least 24 h or more preferably by at least 48 h.

In one particular embodiment, the blood clotting disorder after surgical intervention or clinical procedure can be thrombosis, such as post-operative venous tromboemolism, thrombosis of the coronary grafts, stroke, transient ischemic attack and other arterial thromboembolic events. Extracorporeal circulation can be any extracorporeal blood circuit including cardiopulmonary bypass, ECMO, haemodialysis, haemofiltration.

In all these embodiments, the patient may be at a high risk of bleeding and/or has heparin-induced thrombocytopenia.

The present invention also concerns a method for preventing or reducing blood clotting disorders during and after a surgical intervention or clinical procedure with the products as described above, wherein compound (I) or the salt, thereof, is administered just before the surgical intervention or clinical procedure and avidin is administered after the surgical intervention or clinical procedure to a patient in the need thereof.

It also concerns a method for preventing or reducing blood clotting disorders during and after extracorporeal circulation with the products as described above wherein compound (I) or the salts thereof is administered at the beginning of extracorporeal circulation and avidin is administered just after the end of extracorporeal circulation to a patient in the need thereof

Advantageously in the method of the invention the administration of postoperative antithrombotic treatment, i.e; antiplatelet and/or anticoagulant drugs after the administration of avidin can be postponed by at least 12 h, preferably by at least 24 h, more preferably by at least 48 h, in particular in case of a major bleeding.

The surgical intervention is preferably chosen among: open heart cardiac surgery, emergency cardiac surgery for patients with HIT, congenital heart diseases in newborns, children and adults, beating-heart (off-pump) surgery for CABG, extra- and intra-corporeal artificial heart/ventricular assisted devices. The clinical procedure is preferably chosen among PCI, TAVI, and ECMO in newborns and adults.

The ECC is chosen among cardiopulmonary bypass, extracorporeal membrane oxygenation and haemodialysis and haemofiltration. Definitions

The compounds of formula I of the invention exist in various diastereomeric forms. It will be understood that the invention comprehends the different diastereomers in isolation from each other as well as mixtures.

The counter-ions, which compensate the charged forms of the compounds of formula I of the present invention, are pharmaceutically acceptable counter- ions such as hydrogen, or more preferably alkali or alkali-earth metals ions, which include sodium, calcium, magnesium and potassium.

Other 'compound' group definitions will be readily understandable by the skilled person based on the previous definitions and the usual conventions of nomenclature.

It will be appreciated that any optional feature that has been described above in relation to any one aspect of the invention may also be applicable to any other aspect of the invention.

The compounds of formula I of the present invention may also be present in the form of pharmaceutically acceptable salts. For use in medicine, the salts of the compounds of this invention refer to non-toxic "pharmaceutically acceptable salts." FDA approved pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic/anionic or basic/cationic salts (Gould, P.L., International J. Pharm. , 1984, 33, 201 -271 ; Berge, S.M. et al. , J. Pharm. Sci., 1977, 66 (1 ), 1 -19).

Pharmaceutically acceptable salts of the acidic or basic compounds of the invention can of course be made by conventional procedures, such as by reacting the free base or acid with at least a stoichiometric amount of the desired salt-forming acid or base.

Pharmaceutically acceptable salts of the acidic compounds of invention include salts with inorganic cations such as sodium, potassium, calcium, magnesium, zinc, ammonium, and salts with organic bases. Suitable organic bases include N-methyl-D-glucamine, arginine, benzathine, diolamine, olamine, procaine and tromethamine.

Pharmaceutically acceptable salts of the basic compounds of the invention include salts derived from organic or inorganic acids. Suitable anions include acetate, adipate, besylate, bromide, camsylate, chloride, citrate, edisylate, estolate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hyclate, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactiobionate, maleate, mesylate, methylbromide, methylsulfate, napsylate, nitrate, oleate, pamoate, phosphate, polygalacturonate, stearate, succinate, sulfate, subsalicylate, tannate, tartrate, terephthalate, tosylate and triethiodide. Sulphate salts are particularly preferred.

Within the scope of the invention, "pharmaceutically acceptable" means all that is useful in the preparation of a pharmaceutical composition, that is generally safe and non toxic and that is no biologically or else non desirable and that is acceptable for veterinary and human pharmaceutical use.

The principal route of administration of the compounds (compound of formula I and avidin) of the invention is the parenteral route, in particular the intravenous route.

In case of the use of the compounds of the invention in extracorporeal circulation, the compounds (compound of formula I and avidin) are directly injected in the extracorporeal circuit or intravenously.

For parenteral use, including intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate. Modes of Administration

The compounds of the present invention will be administered in the patient (intravenously or intra-arterialy, possibly subcutaneously) or in the ECC circuit.

Compound 1 may be administered as a single intravenous bolus or as a single bolus followed by a continuous infusion or as a continuous infusion, i n a peripheral or central venous catheter or directly in the ECC circuit.

Avidin may be administered by infusion, preferably an intravenous infusion. Advantageously, avidin is infused during 5 to 60 min, preferably 30 min and 15 min to 6 h after compound 1 has been administered, preferably on average 1 .5 h after.

The present compositions may, if desired , be presented in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient. Such a pack or device may, for example, comprise metal or plastic foil, such as a blister pack, or glass and rubber stoppers such as in vials. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising an agent of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition.

The present invention also concerns a kit containing the products as described above and instructions for use during and after a surgical intervention or clinical procedure, the instructions are preferably for use in ECC during cardiac surgery. Presentation of the drawings

The following examples and figures illustrate the invention but are not limitative. Figure 1 represents the activity of compound 1 over time, after injection in rats, either alone or after complexation with avidin in two different molar ratios. Figures 2a to 2c represent results of another anticoagulant assay. The activated coagulation ti me (ACT) is measured at different times after successive injections of compound 1 and avidin into human for monitoring the tested anticoagulant treatment during ECC. Nine different combinations were studied with the following avidin/compound 1 weight (mg) ratios: 24/4, 48/4, 72/4 (figure 2a); 48/8, 96/8, 144/8 (figure 2b); 72/12, 144/12, 216/12 (figure 2c).

Figures 3a to 3c represent results of an anti-factor Xa assay. The anti-factor Xa activity, expressed as equivalent of fondaparinux, is measured at different times after successive injections of compound 1 and avidin into human. Nine different combinations were tested with the following avidin/compound 1 weight (mg) ratios: 24/4, 48/4, 72/4 (figure 3a); 48/8, 96/8, 144/8 (figure 3b); 72/12, 144/12, 216/12 (figure 3c). Example 1 - Pharmacokinetics Studies in Rats

Rats were intravenously injected with compound 1 (0.1 μιτιοΐ/kg; a 60/40 mixture of diastereoisomers was used in this experiment) either alone, or after complexation of compound 1 with avidin using different avidin/compound 1 molar ratios (1 .5/1 and 5/1 ) . Blood was sampled on citrate at various time points and plasma was prepared. The circulating anti-factor Xa activity was assessed in rat plasma using a commercially available chromogenic assay (Stachrom HP kit, Diagnostica Stago) and a STAR instrument.

The results exposed in figure 1 show that the elimination half-life of the compound 1 -avidin complex (white diamonds and triangles, respectively for a ratio of 1 .5/1 and 5/1 ) is identical (parallel curves) to that of compound 1 alone (black diamonds). Thus, the half-life of the compound 1 -avidin complex (2-3 hours) is higher than that of avidin (2 minutes; J. Drug Target. , 1995, 3, 159-165), which was unexpected since it was believed that the complex was eliminated as rapidly as avidin. The results also show that avidin/compound 1 ratio influences the intensity of the residual activity of the complex. Example 2 - ACT assay

The ACT (Activated Clotting Time) test is a point of care test used during ECC to monitor the level of anticoagulation of the patient. The test was performed using commercially available ACT cartridge and a Hemochron Signature Elite instrument. A typical ACT value for a non anticoagulated patient is in the range 80-120 seconds. Values between 400 and 500 s in the ACT test are typically recommended for UFH during extracorporeal blood circulation for cardiac surgery (Bull BS, "Heparin Therapy During Extracorporeal Circulation", J Thorac Cardiovasc Surg, 69, 1975, 123-133).

Compound 1 was injected into healthy human subjects ( n= 36) as an intravenous bolus, and after 1 .5 h avidin was infused over 30 min. Three doses for compound 1 were tested: 4 mg, 8 mg and 12 mg. Avidin was administered with avidin/compound 1 molar ratios of 1 /1 , 2/1 and 3/1 (weight ratios of 6/1 , 12/1 and 18/1 ).

As can be seen from figure 2a to 2c, after injection of compound 1 the ACT dose dependently increases to reach values in the range 200-350, according to the dose, and then slowly decreases, reflecting the pharmacokinetics of Compound 1 (the peak being reached at different time points is due to interindividual variability). Infusion of avidin between 1 .5 and 2 hours results in an immediate decrease in the ACT values which finally go back to normal ACT values, as measured in the absence of any anticoagulant treatment, and stay at this value along time. Avidin triggered a rapid and irreversible neutralisation of compound 1 without rebound effect (the sometimes observed higher values of ACT are explained by the known variability of the test).

It is noticeable that whatever the dose of compound 1 , the ACT hardly reaches a value above 300 s, whereas clinicians typically require a high value over 400 s for considering that the patient is effectively anticoagulated during ECC.

Therefore, the results of this assay do not give any incentive for using compound 1 and avidin during ECC.

After the end of the infusion of avidin, the ACT comes back to baseline (normal values are in the 80-120 seconds range i n the absence of any anticoagulant treatment). Higher values (e.g. around 200 seconds) would indicate a residual antithrombotic effect.

Example 3 - Anti-factor Xa assay

The circulating anti-factor Xa activity was assessed in plasma using a commercially available chromogenic assay (Biophen Heparin (LRT)) and a STAR- R instrument. It was expressed as fondaparinux equivalent (fondaparinux is a well acknowledged antithrombotic agent).

Compound 1 was injected into healthy human subjects (n=36) as an intravenous bolus, and after 1 .5 h avidin is infused over 30 min. Three doses for compound 1 were tested: 4 mg, 8 mg and 12 mg. Avidin was administered with avidin/compoundl molar ratios of 1 /1 , 2/1 and 3/1 (weight ratios of 6/1 , 12/1 and 18/1 ). Citrated blood was used to prepare plasmas and the plasma were store frozen until anti-factor Xa activity determination.

As can be seen from figure 3a to 3c, after injection of compound 1 , the anti- factor Xa activity increases dose dependently, and then slowly decreases, reflecting the pharmacokinetics of Compound 1 . Infusion of avidin between 1 .5 and 2 hours triggers an immediate and large decrease in the anti-factor Xa activity.

However, the results show that, in contrast with what is observed for the ACT test, that the anti-factor Xa activity remains at a level of 0.2-0.6 Mg/mL fondaparinux equivalent. Also in contrast to the ACT the anti-Xa activity level at 2 hours slightly continues to decrease to go back to the baseline in 12 to 24 hours.

Claims

Products containing:

compound (I) of formula

or a salt -thereof; and

avidin,

as a combination product for the separate and successive administration in prevention of blood clotting disorders during and after a surgical intervention or a clinical procedure, wherein compound (I) or a salt -thereof is administered just before the surgical intervention or clinical procedure and avidin is administered after the surgical intervention or clinical procedure.

2. The products as defined in claim 1 , as a combination product for the separate and successive administration in prevention of blood clotting disorders during and after extracorporeal circulation, including ECMO wherein compound (I) or a salt thereof is administered at the beginning of the extracorporeal circulation— and avidin is administered just at the end of extracorporeal circulation.

3. The products according to any of claim 1 or 2, wherein the administration of antiplatelet and/or anticoagulant drugs after the administration of avidin is postponed in particular in case of major bleeding for example by 24 hours.

4. The products according to any of claim 3, wherein the administration of antiplatelet and/or anticoagulant drugs after the administration of avidin is postponed by at least 12 hours, preferably by at least 24 hours, more preferably by at least 48 hours.

5. The products according to any of claims 1 to 4, wherein the blood clotting disorders are thrombosis and embolism or bleeding.

6. The products according to any of claims 1 to 5, wherein compound (I) or the salt thereof, is administered at a dose of from 1 to 24 mg as bolus potentially followed by a continuous infusion of 4 to 20 mg/h and avidin from 0.5 to 5 molar ratio over compound (I).

7. The products according to claim 6, wherein the continuous infusion of avidin is from 1 to 3 molar ration over compound (I).

8. The products according to claim 6 or 7, wherein compound (I) or the salt, thereof, is administered at a bolus dose of from 8 to 16 mg potentially followed by a continuous infusion of 6 to 16 mg/h / surgical intervention or a clinical procedure.

9. The products according to any claim 1 to 8, wherein they are in a form suitable for parenteral administration.

10. The products according to any claim 2 to 9, wherein the extracorporeal circulation is chosen among: cardiopulmonary bypass, ECMO, haemodialysis and haemofiltration.

11 . The products according to claim 10, wherein the extracorporeal blood circulation is cardiopulmonary bypass.

12. The products according to any claim 1 to 11 , wherein the patient has a high risk of bleeding and/or has heparin-induced thrombocytopenia.

13. A kit containing the products as defined in claim 1 and instructions for use during and after surgical intervention or clinical procedure.

14. The kit containing the products as defined in claim 1 and instructions for use in ECC during cardiac surgery.