Medical devices, systems, and methods utilizing antithrombin-heparin compositions

专利摘要:
Medical devices, systems and methods for the treatment of conditions are described, which use antithrombin-heparin conjugates. For example, medical devices can be coated with antithrombin-heparin (ATH) with the result of a reduction in thrombogenicity. Similarly, various conditions with ATH can be treated. (Machine-translation by Google Translate, not legally binding)
公开号:ES2671844A1
申请号:ES201790052
申请日:2016-06-10
公开日:2018-06-08
发明作者:Leslie Roy Berry；Anthony Kam Chuen Chan
申请人:Attwill Medical Solutions Inc；
IPC主号:

专利说明:

Medical devices, systems and methods that use anti-thrombin heparin compositions.
Background
Coagulation is the process by which blood passes from the liquid state to a gel state. This is part of hemostasis, or the process by which the body 10 stops the loss of blood through damaged blood vessels. Although coagulation is a known function of the human organism, coagulation can lead to arduous problems during medical procedures such as surgery and intravenous or intra-arterial catheterization. A considerable part of the population may also experience coagulation disorders, in which case the presence of unwanted blood clots is a serious
15 health risk.
Invasive procedures, such as cardiopulmonary bypass (BPC), generate huge amounts of fibrin micro-emboli that can lodge in the brain and cause long-term cognitive dysfunctions. The PCB is carried out worldwide for the treatment of cardiovascular diseases. The use of the CPB in the pediatric area
20 represents a specialized and sensitive operation. About 750 pediatric operations are practiced every year in Canada, with 7,500 the corresponding figure for the US. or to Europe. If operations performed on adults are included, more than every year they practice
800,000 The estimated cost of a single CPB operation in Canada is more than $ 10,000. Currently, heparin is the medication used in the PCB and its annual cost is approximately $ 50 / CPB, or what is the same, $ 40,000,000 annually. Heparin cannot prevent thromboembolisms from moving to the brain during CPB, which is associated with both chronic and acute cognitive dysfunction. Thromboembolisms also constitute a risk factor for many other invasive surgeries. Uncontrolled hemorrhage after the PCB is one of the main sources of concern when it comes to
30 attend these high-risk patients.

Venous thromboembolism (VTE) is another problem associated with unwanted blood clotting. Each year, VTE affects 1-2 out of every 1,000 people, usually in the form of deep vein thrombosis (DVT) of the leg, or pulmonary embolism (PE). The incidence rate increases from 1 case per 10,000, in the case of individuals under 40, to 1 in 100 in the case of those over 60. Approximately 1 million people in the US develop DVT every year, and another 500,000 develop PD, resulting in 30% of cases fatal. In general, approximately two thirds of all VTE cases require hospitalization. In a study of 66,000 patients with neutropenic cancer in adults, the mortality rate among hospitalized patients was substantially higher among those who developed VTE (14.85%) compared to those who did not develop it (7.98%) . VTE is also considered responsible for 46.3% of all deaths after surgery for cancer. In the United Kingdom, the Government has estimated that there are more than 25,000 deaths each year caused by VTE, a figure higher than the sum of the number of deaths caused by breast cancer, HIV, and traffic accidents. The number of LV cases is expected to increase, due to the aging of the general population and the increased exposure to risk factors, such as surgery, oral contraceptives, long distance travel and higher levels of obesity.
Plasminogen deficiency is a well-known disorder, which causes reduced levels of plasminogen to cause the development of pseudo-membranes on mucosal surfaces. One of these problems is woody conjunctivitis. Woody conjunctivitis is another example of a disease in which dangerous coagulation occurs. Pediatric patients with a mutation that causes a homozygous plasminogen deficiency have a membrane on their eyes (woody conjunctivitis) caused by coagulation, which causes fibrin clots to form. These pseudo-membranous eye lesions can cause chronic eye problems, which can be serious, to the point of virtually complete loss of vision. In 2003, more than 100 cases of children from around the world who suffered from this condition were described, but many more have been identified in recent years. Current treatment consists of frequent scraping of the cornea. However, these surgical manipulations do not offer lasting relief, and remodeling of the epithelial surface layers can cause permanent damage that affects ocular function. Long-term disease progression, even when this intermittent physical withdrawal is performed, can cause children

affected less physical development and difficulties in the learning process. Thus, apart from the important physical suffering suffered by each individual, it is a very serious problem, which greatly affects the health of the child throughout his life. It would be highly desirable that a non-invasive method that preserves the sight of these patients be conceived. The current number of patients affected by woody conjunctivitis is unknown. However, a 1996 study showed that the prevalence of heterozygous type I plasminogen deficiency was 0.25% (25 of 9611 subjects), which corresponds to a prevalence of the homozygous / heterozygous compound of 1 , 6 per 1,000,000 inhabitants. Topical treatments using corticosteroids, hyaluronidase and antibiotics have had variable success. Normally, surgery is usually performed to remove the woody mass, but it is of little use to prevent recurrence of the problem, so it is necessary to repeat the procedure.
Respiratory Difficulty Syndrome (RDS) is a common disorder that implies the existence of pulmonary fibrosis. Approximately 1% of all children are born weighing less than 1,500 grams, and the vast majority suffer from RDS. Babies weighing less than 1,000 grams at birth have a very high incidence of RDS, and between 50 and 80% of these children shed or develop bronchopulmonary dysplasia (BPD). In fact, RDS remains the leading cause of mortality and morbidity among premature newborns. Although the use of surfactants has reduced the severity and slightly improved the mortality rate caused by RDS in premature infants, there is still a significant incidence of RDS / DBP and severe cases of BPD. In the case of children as well as adults, infections, environmental factors and genetic components can cause acute episodes of RDS that evolve into chronic dysfunctional conditions of the disease, which involve remodeling of tissue and structures. pulmonary
Lung damage is a situation that has a great impact on the quality of life, and that can imply a significant risk of mortality. The underlying underlying causes of lung lesions are a consequence of both chronic and acute factors. Recently, it is increasingly thought that mechanical damage caused by high pulmonary stress contributes significantly to the appearance of lung lesions. The treatment of assisted breathing by mechanical ventilation can cause trauma that encourages the advance of localized lesions in patients with acute RDS. Although a low tidal volume may offer better results, the

mortality remains unacceptably high. In fact, fans can directly cause lung damage on their own, and reports are currently highlighting the biological markers of lung lesions associated with ventilation. Following the basic causes that lead to clinical damage to the lungs, there is growing awareness of associations with new interlocking mechanisms. It has been increasingly proven that coagulation, triggered by an initial lung injury, contributes to the development of pulmonary inflammation and fibrosis in cases of acute SOR. The increase in tissue factor (TF) is a marker associated with the development of SOR. For some time it has been known that alveolar fibrin deposition may predispose infants to complications related to BPD, although fibrin-related damage during RDS / BPD is not always obvious, due to its rapid separation from the lung. However, even in the absence of crosslinked fibrin clots, low levels of fibrin monomer products interfere with surfactant function and contribute to the remodeling of the alveolar surface and fibrosis caused by the accumulation of fibroblasts. Similarly, the relationships between pulmonary lesions induced by ventilation and coagulation are being admitted. Again, it is suggested that the increase in FT expression caused by ventilatory aggression is one of the factors that contribute to the complications caused by thrombi, and that cause an increase in lung damage. In fact, ventilation treatment with a high tidal volume can cause pulmonary and systemic coagulation associated with TF in newborns.
Blood clotting can also cause problems with devices that are in contact with blood, such as catheters and stents. These devices tend to suffer failures caused by surface induced thrombosis. Coagulation is an important clinical problem with central venous access catheters. Catheters are used to draw blood and administer medications to numerous patients affected by various problems that require frequent and long-term venous access. Ideally, it should be possible to leave a catheter placed for months or even years, but catheters are characterized by their proclivity towards clots. In fact, in the case of children, most thrombosis are related to catheters. For example, 89% of venous and arterial thrombosis in newborns involve the presence of an intravascular catheter, and 78% of aortic thrombosis in children are related to catheters. One study concluded that the presence of a catheter was the greatest risk factor for thrombosis in children. In total, of patients of all ages at whom

apply venous catheters, up to 26% suffer thrombotic complications related to the catheter. Although the mechanism of surface-induced thrombosis has not been elucidated completely, it is believed that protein absorption takes place very shortly after the surface comes into contact with blood, and is followed by adhesion and activation of platelets, as well as leukocytes, which ultimately results in the formation of a clot. The thrombi formed inside a catheter's lumen makes the catheter useless for blood collection or the administration of fluids and medications. Clots that form on the outside of the catheter can cause deep vein thrombosis and embolization, and can damage the integrity of the vessel, resulting in pain and inflammation. Both situations cause patient discomfort, alter the patient's medical care and make more resources necessary for their care. Treatment of a clogged catheter requires thrombolytic therapy or catheter replacement. The result is greater discomfort and inconvenience for the patient, as well as a higher cost of treatment.
The replacement of a central venous catheter (CVC) is a very invasive procedure that causes patients to experience pain and have to suffer prolonged hospital stays, and prevents doctors from having enough time to attend to other patients, because of the replacement surgery Patient care is also degraded, as treatments are interrupted or canceled due to the failure of their CVC. Clots that form in CVCs can also break up and travel through the bloodstream to other parts of the body, causing serious complications. Therefore, these clots can lead to prolonged hospital stay, interruption of essential treatment to patients, neurocognitive dysfunctions and even death.
It is expected that the aging of the population will result in an increase in the incidence of coronary artery disease, heart failure and cerebrovascular accidents. Therefore, the problems caused by coagulation-related diseases, unwanted clot formation in medical devices and coagulation during medical interventions are also expected to increase, which will carry a heavy financial burden on the health sector.
Brief description of the figures

Figure 1A is a graph of the values of the second order constant (k2) for the inhibition of Ila by ATH and the AT + H conjugate not covalenle, according to the examples of the present invention.
Figure 1B is a graph of the values of the second order constant (k2) for the inhibition of Xa by ATH and the non-covalent AT + H conjugate, according to the examples of the present invention.
Figure 2 is a graph showing the delay time of the inhibition of fibrin formation induced by Ila caused by ATH and AT + H in the presence of endothelium, according to the examples of the present invention.
Figure 3A is a graph showing the delay time of heparan sulfate-linked fibrin-induced inhibition of the formation of fibrin, according to the examples of the present invention.
Figure 38 is a graph showing the delay time of thrombomodulin (TM) -inflated fibrin-induced inhibition of fibrin formation, according to the examples of the present invention.
Figure 4A is a graph showing the coagulation time of plasma samples from rats treated with an intrapulmonary P8S buffer solution, with and without a high volume of mechanical ventilation for one hour, according to the examples of the present invention.
Figure 48 is a graph showing the coagulation time of plasma samples from rats treated with intrapulmonary ATH, with and without a high volume of mechanical ventilation for one hour, according to the examples of the present invention.
Figure 5 shows immunoblotting of cellular media incubated with a buffer (control) solution or with ATH, according to the examples of the present invention.
It should be noted that the figures are merely mere examples of various embodiments, and are not intended to establish any limitations on the scope of the present technology.
Detailed description
Reference will now be made to a series of embodiments, and a specific language will be used to describe them. However, it should be understood that this is not intended to introduce any limitation in the scope of the invention. Any additional alterations and modifications of the features of the invention described in the

This document, as well as the additional applications of the principles of technology described herein, and which could occur to any person skilled in the art who is in possession of this invention, should be considered to be included within the scope of the invention. Also, before disclosing and describing specific embodiments, it should be understood that such disclosure is not limited to the specific process and materials described herein, as they may vary to some extent. It should also be understood that the terminology used in this document is used only for the purpose of describing specific embodiments, and is not intended to limit the scope of the present invention, which is defined exclusively by the appended claims and others equivalent thereto.
When describing and claiming the present technology, the following terminology will be used.
The singular forms, "un," and "el" include references to the plural, unless the context clearly indicates otherwise. Thus, for example, the reference to "an additive" includes references to one or more of said components, "a solution" includes references to one or more of said materials, and "a mixing phase" refers to one or more of these phases.
As used herein, "substantial" when used in reference to a quantity of a material, or a specific characteristic thereof, refers to a quantity sufficient to achieve the effect that said material or characteristic is intended to achieve. The exact degree of permissible deviation may sometimes depend on the specific context.
As used herein, "approximately" refers to a degree of deviation based on the experimental error characteristic of the specific property identified. The latitude inherent in the term "approximately" will depend on the specific context and the specific property, and can be easily perceived by any person skilled in the art. The term "approximately" is not intended to expand or limit the degree of equivalents that otherwise could be assigned to a specific value. In addition, unless otherwise indicated, the term "approximately" expressly includes "exactly," in line with what is discussed below in relation to ranges and numerical data.
The concentrations, dimensions, quantities and other numerical quantities may adopt in this document a range format. It is to be understood that said range format is used exclusively for convenience and brevity purposes, and that it must be interpreted flexibly, so that it includes not only the numerical values explicitly indicated as

range limits, but also all of the individual numerical values or sub-ranges included in that range, as if each of the numerical values and sub-ranges is explicitly listed. For example, a range ranging from about 1 to about 200 should be interpreted to include not only the explicitly listed limits of 1 and 200, but also individual quantities, such as 2, 3, 4, and sub-ranges, such as 50, from 20 to 100, etc.
As used herein, a plurality of components, structural elements, compositional elements, and / or materials may be presented for convenience in a common list. However, these lists were to be interpreted as if each of the members of the list were individually identified as a unique and differentiated member. In this way, it cannot be interpreted that none of the individual members of said list constitutes a de facto equivalent to any other member of the same list, based exclusively on their presentation within a common group without any indication in any other way.
As used herein, "hexose" refers to a carbohydrate (CSH120 S) with six carbon atoms. Hexoses can be aldohexoses, such as glucose, mannose, galactose, idosa, gulose, talose, alose. and altrosa, whose open chain form contains an aldehyde group. Alternatively, hexoses can be ketoses, such as fructose, sorbose, allulose and tagatose, whose open chain form contains a ketone group.
As used herein, "uronic acid" refers to the carboxylic acid formed by the oxidation of the primary hydroxyl group of a carbohydrate, and they are usually named after the carbohydrate from which they were obtained. Therefore, by oxidation of the C6 hydroxyl of the glucose, glucuronic acid is obtained, with that of the hydroxyl CS of the galactose, galacturonic acid is obtained, and by the oxidation of the hydroxyl CS of the idid, iduronic acid is obtained
As used herein, "hexosamine" refers to a derivative of hexose in which at least one hydroxyl group, usually the C2 hydroxyl group, has been substituted by an amine. The amine may have been optionally alkylated, acylated (for example, with murmic acid), usually by an acetyl, sulphonated (N-sulfated), sulfonylated, phosphorylated, phosphonylated and the like group. Representative examples of hexosamines include glucosamine, galactosamine, tagatosamine, fructosamine, their modified analogues and the like.

As used herein, "glycosaminoglycan" refers to long repetitive linear chains of disaccharide units containing a hexosamine and a uronic acid. The precise identities of hexosamine and uronic acid can undergo great variations, and representative examples of each can be found in the previous definitions. Optionally, the disaccharide can be modified by alkylation, acylation, sulfonation (0- or N-sulphated), sulfonylation, phosphorylation, phosphonylation and the like. The degree of such modification can vary greatly, and can be found in a hydroxyl group or an amino group. Normally, hydroxy e6 and C2 amine are sulfated. The chain length can be variable, and the glycosaminoglycan can have a molecular weight greater than 200,000 daltons, usually up to 100,000 daltons, and more normally, less than 50,000 daltons. Glycosaminoglycans can usually be found as mucopolysaccharides. Among the most representative examples are heparin, dermatan sulfate, heparan sulfate, chondroitin-6-sulfate, chondroitin-4 sulfate, keratan sulfate, chondroitin, hyaluronic acid, polymers containing Nacetyl monosaccharides ( such as N-acetylneuraminic acid, N-acetyl glucosamine, N-acetyl galactosamine, and N-acetyl muramic acid) and the like, as well as gums, such as gum arabic, gum tragacanth and the like.
"Heparin" is a sulfated polysaccharide that largely consists of an alternating sequence of hexauronic acid and 2-amino-2-deoxy-D-glucose. Heparin and a compound related to it, dermatan sulfate, function properly as anticoagulants for clinical use in the prevention of thrombosis and related diseases. They are members of the glycosaminoglycan family, (GAGs), which are linear chains of sulfated repetitive disaccharide units containing a hexosamine and a uronic acid. Anticoagulation using GAGs (such as heparin and dermatan sulfate) is carried out through its catalysis of inhibition of coagulant enzymes (one of the most important being thrombin) by inhibitors of serine protease (serpins) such as antithrombin 111 (called simply "antithrombin" or "AT" in this document) and cofactor 11 of heparin (HCII). The binding of the serpins by the catalysts takes place by action the above, and is produced by specific sequences along the linear carbohydrate chain of glycosaminoglycan (GAG). Heparin acts by binding to Al through a sequence of pentasaccharides, thereby enhancing the inhibition of various coagulant enzymes (in the case of thrombin, heparin also binds to the enzyme). Heparin can also enhance thrombin inhibition by linking with the

HCI serpina !. The dermatan sulfate acts by specifically binding to HCII through a sequence of hexasaccharides, thereby enhancing only thrombin inhibition. Given that glycosaminoglycans (especially heparin) can bind to other molecules in vivo or lose the site of action due to various mechanisms, it would be advantageous to keep GAGs permanently associated with serpine by a covalent bond. In more detail, it would be desirable to provide covalent conjugates of heparin and similar glycosaminoglycans that retain high biological activity (eg, their anticoagulant activity) and better pharmacokinetic properties, as well as simple methods of preparation.
As used herein, "protein" includes, among others, albumin, globulins (eg, immunoglobulins), histones, lectins, protamines, prolamines, glutelinasphospholipases, antibiotic proteins and scleroproteins, as well as conjugated proteins, such as phosphoproteins, chromoproteins, lipoproteins, glycoproteins and nucleoproteins.
As used herein, "serpine" refers to a serine protease inhibitor, and among its examples are species such as antithrombin and cofactor I1 of heparin.
As used herein, "amine" refers to primary amines, RNH2, secondary amines, RNH (RI, and tertiary amines, RN (R ') (R ").
As used herein, "amino" refers to the group NH or NH2 •
As used herein, "imine" refers to the group C = N and its salts.
As used herein, the terms "treatment" of a condition and / or a disease in a mammal, means: the prevention of said condition or disease, that is, avoiding any type of clinical symptoms of the illness; inhibition of the condition or disease, that is, stop the development or progression of clinical symptoms; and / or alleviate the condition or disease, that is, cause the retraction of clinical symptoms. Treatment also includes the use of the compositions according to the present invention associated with a medical procedure, being administered before, during or after said medical procedure.
In accordance with the preliminary information described above, the present invention relates to methods and compositions for the treatment of conditions involving coagulation. In one example, a method of coating a polymeric surface with a

Antithrombin-heparin conjugate may include contacting the polymeric surface with a solution of the antithrombin-heparin conjugate. The antithrombinheparin conjugate can directly coat the polyurethane surface without any binding group between the antithrombin-heparin conjugate and the polymeric surface.
In another example, a medical device with reduced thrombogenicity may include a polymeric surface coated with an antithrombin-heparin conjugate without binding groups between the antithrombin-heparin conjugate and the polymeric surface.
In a further example, a method of coating a polymeric surface of a medical device by lyophilization may include contacting the polymeric surface of the medical device with an antithrombin-heparin solution comprising an antithrombin-heparin conjugate and a solvent , in the absence of binding groups. In this way, the excess antithrombin-heparin solution can be removed from the polymer surface allowing it to dry. The solvent can then be evaporated from the polymeric surface, in an at least partial vacuum.
An additional example involves a method of treating a medical condition by inhibiting thrombogenesis in a mammal. The method may include administration to the mammal of a dose of an antithrombin-heparin conjugate, in which at least 98% of the heparin chains of the antithrombin-heparin conjugate have a molecular weight greater than 3,000 daltons.
In another example, a method of treating woody conjunctivitis in a mammal may include the administration of a dose of an antithrombin-heparin conjugate in one of the mammal's eyes.
In another additional example, a method of treating a lesion caused to a mammal by mechanical ventilation may include the administration of a dose of an antithrombin-heparin conjugate in the injured lung of the mammal.
In a further example, a method of treating woody gingivitis in a mammal may include the administration of a dose of an antithrombinaheparin conjugate in the gums of the mammal.
In an independent example, a wash and seal solution of an intravenous or intra-arterial catheter may include an antithrombin-heparin conjugate.
Another example relates to a method for maintaining the permeability of a catheter. The method may include the insertion of a catheter into a vein or artery of a patient, so that an inner opening of the catheter is opened inside the vein or artery,

opening an outer opening of the catheter on the outside of the patient. A solution containing an antithrombin-heparin conjugate can be injected into the catheter through the outer opening of the catheter. Then, the outer opening of the catheter can be sealed, so that at least a part of the solution comprising the antithrombinaheparin conjugate remains inside the catheter.
In another additional example, a composition for the treatment of blood clots may include antithrombin, heparin, and fibrin. At least 50% by weight of heparin can be conjugated with antithrombin to obtain an antithrombin-heparin conjugate. At least a portion of the fibrin can adhere to the antithrombin-heparin conjugate.
In another example, a method for the treatment of a condition or disease may include the administration, to a mammal that requires it, of an antithrombinaheparin conjugate prepared in accordance with the examples of current technology. In more detail, these treatments can be carried out by administering the heparin and antithrombin conjugates of the present invention to a patient, such as a human being, in need of such treatment. Conditions and diseases that can be treated by using the conjugate compositions described herein include myocardial infarctions and a wide variety of thrombotic conditions. These include fibrin deposits in the case of neonatal respiratory distress syndrome, adult respiratory distress syndrome, primary lung carcinoma, non-Hodgkins lymphomas, fibrosing alveolitis, and lung transplants, to name just a few . Likewise, by means of the present invention, states of acquired AT deficiency can be treated, such as neonatal respiratory distress syndrome, L-asparaginase-induced deficiency, cardiopulmonary bypass-induced deficiency, sepsis or congenital deficiency states of AT. In the case of congenital AT deficiency, thrombotic complications that pose a threat to life may occur, with AT levels below 0.25 Units / mi in heterozygotes that require AT plus heparin, in up to 1 or 2 children at year in the US Conditions and diseases treated by the present invention include those that are characterized by an excess generation or thrombin activity. These conditions often occur when the patient has been exposed to trauma, for example in the case of surgical patients. Traumas caused by wounds or surgery cause vascular damage and secondary activation of blood coagulation. These undesirable effects may occur after a general or orthopedic surgery intervention, surgery

gynecological, cardiac or vascular surgery, or other surgical procedures. An excess of thrombin can also complicate the evolution of natural diseases, such as atherosclerosis, which can be the cause of heart attacks, cerebrovascular accidents
or gangrene of the members. Therefore, the methods and compositions of the present invention can be used for the treatment, prevention or inhibition of various cardiovascular complications, including unstable angina, acute myocardial infarction (heart attack), cerebrovascular accidents (stroke), pulmonary embolisms, deep vein thrombosis, arterial thrombosis, etc. The compositions and methods of the invention can be used to reduce or prevent coagulation during dialysis and reduce or prevent intravascular coagulation during open heart surgical procedures. In more detail, in some aspects of the invention, methods and compositions are provided to prevent or inhibit thrombin generation or its activity in patients with an increased risk of thrombus development, because of medical conditions that disturb hemostasis (by example, coronary artery disease, arteriosclerosis, etc.). In another aspect, methods and compositions are provided for patients suffering from an increased risk of developing a thrombus after a medical procedure, such as cardiac surgery, vascular surgery or percutaneous coronary interventions. In one embodiment, the methods and compositions of the present invention are used for surgical procedures of cardiopulmonary bypass. The compositions can be administered before, during and after said procedure.
Turning now to the antithrombin-heparin conjugate used in the compositions and treatments described in the present invention, the antithrombin-heparin conjugate provides various advantages as an anti-thrombotic against heparin.
The antithrombin-heparin conjugate (ATH) can be prepared by covalently binding heparin chains to the antithrombin (AT). Heparin contains terminal aldose bonds that coexist in equilibrium between the hemiacetal and aldehyde forms. Heparin can be conjugated with antithrombin by reducing the simple Schiff base formed spontaneously between the aldehyde of the terminal aldose of heparin and an N-terminal amino lysyl O of the antithrombin. Heparin remains unmodified (without reducing its activities) prior to conjugation, and binds at a specific location at one end of the molecule without any unblocked activation group or exchange of antithrombin bonds.

Normally, the reaction is carried out at a pH of between about 4.5 and about 9, or between about 5 and about 8, or even between about 7 and about 8. The reaction generally takes place in aqueous medium. . However, organic media, and especially polar hydrophilic organic solvents, such as alcohols, ethers, formamides and the like can be used in proportions of up to about 40% to increase the solubility or reactivity of the reagents, if necessary. Non-nucleophilic buffers, such as phosphate, acetate, bicarbonate and the like can also be used.
The imines formed by the condensation of the amines of the AT with the heparin residues of the terminal aldose can be reduced and the corresponding amines can be obtained. This reduction can be carried out simultaneously with or after the formation of the imine. A wide range of reducing agents can be used, with hydride reducing agents, with specific useful examples being sodium borohydride or sodium cyanoborohydride. In general, any reducing agent that does not reduce disulfide bonds can be used.
Alternatively, if reduction of the intermediate imine is not desired, the imine may be incubated for a sufficient period of time, generally from one day to one month, more generally between 3 days and 2 weeks, in order to allow Amadori transposition in the intermediate amine. The terminal aldose residues of the conjugated heparin by the methods described in this invention may possess C2 hydroxyl groups in the terminal aldose residue, that is, a 2-hydroxy carbonyl fraction that is converted into a 2-hydroxy imine by condensation with the AT amine conjugated to heparin. In Amadori transposition, the a-hydroxy imine (imine in C1, hydroxy in C2) formed by the initial condensation can be transposed to form an a-ketoamine by enolization and reprotonation (keto in C2, amine in C1). The resulting a-carbonyl amine is thermodynamically favored with respect to the a-hydroxy precursor imine, thereby facilitating a stable additive with minimal disruption of the heparin chain. Thus, heparin can be covalently conjugated in C1 of the terminal aldose residue of heparin to obtain an AT chain containing amines via an amine bond. Covalent complexes can be formed simply by mixing heparin and AT in a buffer solution and allowing a keto-amine to spontaneously form through an Amadori transposition between the aldose terminal of heparin and a lysyl-AT or N-terminal amino group. Thus, Amadori transposition can be used to make heparin conjugates with AT. This

Conjugation method is especially useful and simple, and minimizes the modification of glycosaminoglycan, thereby maximizing the maintenance of its biological activity.
In some cases, the antithrombin-heparin conjugate can be prepared using unfractionated heparin. In other cases, the antithrombin-heparin conjugate can be prepared using heparin from which the low molecular weight heparin chains have been removed. As is known, heparin can be easily obtained in an unfractionated form, which contains molecules with a wide range of molecular weights. By eliminating most or all of the heparin molecules with molecular weights below 3,000 daltons prior to the conjugation of heparin with antithrombin, the activity of the antithrombin-heparin conjugate can be improved. In a further embodiment, heparin molecules with a molecular weight of less than 5,000 daltons can be removed mostly or completely prior to their conjugation with antithrombin.
Antithrombin-heparin conjugates formed using heparin from which the low molecular weight heparin molecules have been removed differ in their composition from other antithrombin-heparin conjugates. Low molecular weight heparin chains can be removed from heparin prior to their reaction with AT to synthesize the antithrombin-heparin conjugate (ATH). Therefore, low molecular weight heparin chains conjugated to AT are removed from ATH.
The low molecular weight heparin chains can be removed from the commercially available heparin before reacting the heparin with the AT to obtain the ATH. In this way, an ATH is obtained whose composition differs from that of the ATH formed from unfractionated heparin without eliminating low molecular weight heparins before its reaction with AT. Additionally, the formation of ATH from unfractionated heparin and the subsequent elimination of low molecular weight ATH conjugates does not allow obtaining the same product as the ATH of the present invention. Without having to hold on to any specific theory, it is believed that low molecular weight heparin chains (such as those of less than 3,000 daltons or less than 5,000 daltons) compete with the longer chain heparins for conjugation with AT. Very low molecular weight heparin chains have a high proportion of aldose terminals that react with AT. Therefore, very low molecular weight heparin chains tend to conjugate with AT more rapidly, surpassing heparin chains with a higher molecular weight. However, once the very low molecular weight heparin chains bind to the AT, the chains no longer have enough gaps or length for the thrombin and Factor Xa bonds, a

enzyme that participates in the coagulation cascade. The inhibitory activity against factor Xa and thrombin drops dramatically in the range of lower molecular weight heparin molecules. Thus, the ATH formed from these very low molecular weight heparin chains has essentially zero activity for the prevention of thrombogenesis. Although commercial grade heparin contains a relatively small percentage of heparin chains of less than 5,000 Daltons, these very low molecular weight heparin chains have such high reactivity with AT that a very large amount of the ATH formed contains heparin chains. of very low molecular weight.
If heparin of very low molecular weight is not eliminated prior to conjugation, there will be a higher proportion of reactive terminals in this population compared to that of heparin with a higher molecular weight, which will exceed the rest of the heparin molecules in a variable proportion throughout the entire molecular weight spectrum (since the proportion of aldose terminals varies continuously throughout the entire molecular weight range of heparin). This can have an adverse effect on the final ATH. First, ATH will contain a significant population of ATH molecules that contain very small heparin chains without activity. Secondly, the rest of ATH molecules (apart from this range of very low molecular weight ATH molecules) will contain a heparin population with a reduced proportion of heparin chains located in discrete ranges of molecular weights and that have less Aldose terminals to compete with inactive low molecular weight heparin chains. This type of low-aldose heparin usually has much longer chains, but is not entirely defined by a direct relationship between the length of the heparin chains and the aldose terminals necessary to bind to the AT.
In addition, heparin with at least 18 units of monosaccharides may be more effective in inhibiting thrombin. At least 18 units of monosaccharides are used to bind antithrombin and thrombin. The mechanism by which heparin binds to antithrombin and thrombin is called the template or bridge mechanism. Heparin can exert its effect through its conformational activation by binding to the AT and converting the Al allosterically into a structural form that is much more reactive against coagulation proteases. Alternatively, heparin can act as a template, linking with both the inhibitor and the enzyme, thereby locating the molecules for the reaction. In this mechanism, conformational activation of Al is produced by heparin, but the further improvement in the reaction rate is obtained by linking

simultaneous heparin with the enzyme, thus contributing to the approach of the coagulation factor to the activated inhibitor. The minimum specific chain length of 18 monosaccharides can explain why there is a very sharp drop in activity against thrombin within the heparin fraction with a lower molecular weight. Starting from the
5 structure corresponding to a monosulfated-disaccharide uronic acid of bisulfated glucosamine heparin, that is, without sodium or other ions that can be found in a salt format, the molecular weight of a chain of 18 saccharides (9 disaccharides) would be located around to 4,500 daltons.
Heparin chains with a slightly lower molecular weight may be useful for
10 inhibition of factor Xa. A specific sequence of heparin pentasaccharide can bind to AT and activate AT for inhibition of factor Xa. The specific pentasaccharide sequence is available on its own as the drug "Fondaparinux," but the sequence can also occur in heparin chains. The monosaccharide sequence is shown in formula 1:
or
COO
HN, _
OH SO, O
~ o
HN,. BEAR;
SW,
(one)
Thus, heparin chains that contain less than 18 monosaccharides that in turn contain this pentasaccharide sequence may be able to activate AT for
20 inhibit factor Xa even when the chains are not long enough to bind to AT and thrombin.
In some cases, longer heparin chains may have the highest inhibitory activity. However, some heparin chains of medium and low molecular weights may have significantly less undesirable rates of binding with 25 other plasma proteins and platelets. Therefore, these typical type heparin chains may be more selective for thrombin and factor Xa inhibition without causing effects.
'"

unwanted collaterals, such as platelet dysfunctions, by ceasing to bind platelets and bind to other materials.
Isolation of the higher molecular weight ATH after conjugation, to obtain a very long chain ATH provides a different and less desirable product compared to the present invention, which separates (substantially or entirely) heparin prior to conjugation . For example, the proportion of 2 high activity pentasaccharide molecules in this sub-population can be altered because of a differential ability of these high activity chains to compete for conjugation with very low molecular weight heparins. Additionally, the isolation of high molecular weight ATH after conjugation eliminates ATH molecules with medium and small heparin chains, which are also active and have other desirable characteristics, such as reduced non-selective bonds with plasma proteins and platelets .
Alternatively, attempts to react all heparin chains with aldose terminals with the AT by increasing the proportion of AT versus heparin in the reaction mixture do not have a great chance of success, since many experiments have shown that only a maximum rate of conversion of AT into ATH of 60% by weight, even when the aldose content of heparin exceeds several times the highest practical concentrations. If the proportion of heparin versus AT is reduced further, the performance of ATH will be further reduced, with no promise that all of the longer active chains will be incorporated into the product.
In some embodiments, a thrombogenesis prevention composition may contain ATH obtained from commercial grade heparin, from which almost all heparin chains with a molecular weight of less than
3,000 daltons (for example, at least 98% by weight of the rest of the heparin chains may have a molecular weight greater than 3,000 daltons). In other embodiments, heparin chains with a molecular weight of less than 5,000 daltons can be substantially or totally eliminated. Thus, the ATH product can contain heparin chains whose molecular weight ranges between 3,000 daltons (or 5,000 daltons) and the highest molecular weights that commercial heparin contains. In some examples, this range of molecular weights can range between 3,000 daltans and 50,000 daltans, to between 5,000 daltans and 50,000 daltans. In the additional examples, at least a portion of the heparin chains can be found in a medium range of molecular weights. For example, at least a portion of ATH's heparin chains may have a molecular weight ranging from 3,000 daltons to 30,000

dallans, between 3,000 dallans and 20,000 dallans, between 3,000 dallans and 15,000 dallans, between 3,000 dallans and 10,000 dallans, between 5,000 dallans and 30,000 dallans, between 5,000 dallans and 20,000 daltons, between 5,000 aaltons and 15,000 daltons, or between 5,000 and 10,000 daltons daltons. In this way, all or practically all heparin chains whose molecular weight is less than 3,000 Daltons or 5,000 Daltons can be removed from ATH.
Normally, commercial heparin may contain a series of heparin chains whose molecular weights range from 1,000 daltons or less and 50,000 daltons or more. The fraction of lower molecular weight, such as chains whose molecular weight is less than 3,000 or
5,000 Daltons can be eliminated by any suitable method. Among some of the methods of elimination of low molecular weight chains are dialysis, diafiltration, gel filtration and electrophoresis. Dialysis and diafiltration can be carried out under conditions of high salinity. For example, among the high salinity conditions for dialysis or diafiltration are saline concentrations between about 1 M NaCl and about 4 M NaCI. Other salts other than NaCI can also be used. The high saline concentration can help small chains move through membranes with adequate pore sizes. Gel filtration can be carried out using suitable means for the separation of molecules according to their size. In a specific example, gel filtration can be carried out in SephadeX® G-200, which is a gel-shaped medium for the separation of molecules with molecular weights ranging from 1,000 to 200,000 daltons. Commercial heparin can be gel filtered on a gel column, and a series of fractions can be eluted with the first fractions containing the chains of higher molecular weight and the subsequent fractions containing gradually lower molecular weights. The heparin molecular weights of each fraction can be determined, and fractions having the desired molecular weights can be grouped. Using this method, fractions containing heparin with molecular weights below the threshold of 3,000 or 5,000 daltons can be excluded. If desired, heparin chains that exceed a certain threshold can also be excluded. For example, fractions containing heparin with a molecular weight of more than 50,000 daltons, 30,000 daltons, 20,000 daltons, 15,000 daltons, or 10,000 daltons can be excluded, if desired. Clustered fractions possessing the desired range of molecular weights can then be used to synthesize ATH.

It should be noted that the methods of elimination of the very low molecular weight heparin chains described above are just examples, and should not be considered exhaustive. Any commercial heparin treatment method can be used in the present invention for the elimination of heparin chains that are below a certain molecular weight threshold.
In various embodiments of the present invention, the treatments and methods described herein can be carried out using ATH to which the low molecular weight heparin has been removed, or alternatively, using ATH formed from unfractionated heparin.
ATH can be formed by conjugating AT with the heparin from which the low molecular weight chains have been removed. Examples of methods of conjugation of heparin with AT can be found in US Pat. No. 7.045.585, which is incorporated herein by reference. These methods can be applied to the formation of ATH using heparin from which the very low molecular weight chains have been removed, as described herein. Heparin can be conjugated to AT by a simple process consisting of a single stage, and which allows direct covalent bonding of the amine of a fraction containing amines (such as, for example, oligo (poly) saccharides containing amines , lipids, proteins, and nucleic acids containing amines, as well as any xenobiotic containing amines) with a terminal aldose residue of a heparin chain. For the formation of ATH, the fraction containing amines is present in the TA, although other proteins can be conjugated using the same methods. The minimum processing and non-destructive methods described herein allow maximum preservation of the biological activity of the protein and allow direct protein binding without the need for intermediate separator groups.
In one embodiment, the heparin is incubated with AT at a pH suitable for imine formation between the amine and the terminal aldose or keto residue of heparin. Terminal aldose and ketose residues usually exist in equilibrium between the cyclic form of the hemiacetal or hemicetal closed ring and the corresponding equivalents of open ring aldehyde or ketone. Usually, amines are able to react with the open ring shape to produce an imine (Schiff base). Normally, the aldoses are more reactive because the corresponding aldehydes of the open ring form are more reactive against the amines. Therefore, the formation of the covalent conjugate between the amines and the residues of

Terminal aldose of heparin allows a method of binding with the heparin of the AT containing an amine.
Normally, the reaction is carried out at a pH between about 4.5 and about 9, and more usually between about 5 to about 8, and even more normally, between about 7 to about 8. The reaction is usually carried out at out in an aqueous medium. However, organic media, and especially polar hydrophilic organic solvents, such as alcohols, ethers and fomamides and the like, can be used in proportions of up to about 40% to increase the solubility or reactivity of the reagents, if necessary. Non-nucleophilic buffer solutions, such as phosphate, acetate, bicarbonate and the like, can also be used.
In some cases, the imines formed by condensation of the amines of the AT with the terminal aldose residues of heparin are reduced to the corresponding amines. This reduction can be carried out simultaneously with or after the formation of imines. Various reducing agents can be used, such as hydride reducing agents, including sodium borohydride or sodium cyanoborohydride. In one example, any reducing agent that does not reduce disulfide bonds can be used.
Alternatively, if the reduction of the intermediate imine is not desired, the imine can be incubated for a sufficient period of time, usually between one day and one month, more usually between 3 days and 2 weeks, to allow the Amadori transposition in the intermediate imine. The terminal aldose residues of the conjugated heparins by the methods described in this invention usually possess C2 hydroxyl groups in the terminal aldose residue, that is, a 2-hydroxycarbonyl fraction that is converted to a 2-hydroximin by condensation with the AT amine that is conjugated with heparin. In the transposition of Amadori, which is especially common in carbohydrates, ahydroximin (imine in C1, hydroxy in C2) formed by initial condensation can be transposed to form an a-ketoamine by enolization and reprotonation (keto in C2, amine in C1 )). The resulting a-carbonylamine is thermodynamically favored against the precursor a-hydroximin, thereby facilitating a stable additive with minimal disruption of the heparin chain. Thus, in this embodiment, the invention allows to obtain a covalently conjugated heparin chain in C1 of the terminal aldose residue of heparin with an amine containing AT, by means of an amine bond. If desired, the resulting conjugate can be reduced or labeled by group reduction.

C2 carbonyl with a labeling reagent, such as a radiolabel (for example, NaB3H4), or conjugated with a second species, such as a fluorescent label.
Although the above description focuses on heparin and AT, there are several species that contain amines and that can be conjugated to obtain various glycosaminoglycans by the methods described herein. The primary amine can be found in a small molecule, such as a fluorescent or chromophore drug or label or a macromolecule, such as a protein (antibodies, enzymes, receptors, growth factors and the like), a polynucleotide (DNA, RNA and mixtures of its polymers), a lipid or a polysaccharide. In general, when the proteins are conjugated to form glycosaminoglycans, the bonds will be made through the r.-amino groups of the lysine residues. Alternatively, the linkages can also be carried out by the a-amino group of the acid residue of the terminal amyl N. In addition, many other methods known to any person skilled in the art can be used for the introduction of an amine function into a macromolecule.
Specifically, the present technology can be applied to a variety of other therapeutically useful proteins when considerations of the longer average life span type and blood clotting may be useful. Among these are blood enzymes, antibodies, hormones and the like, as well as the plasminogen activators related to them, streptokinase and its derivatives. Specifically, this technology provides heparin or dermatan sulfate conjugates with antithrombin, heparin cofactor 11 (HCII) or heparin coolant II analogs.
The methods of the present invention provide glycosaminoglycan conjugates with maximum retention of biological activity. Specifically, heparin or dermatan sulfate conjugates are provided with AT or HCII, which have> 60% by weight, usually> 90% by weight, more normally> 95% by weight, and especially> 98% by weight of antithrombin activity intact unconjugated heparin. The methods of the present invention provide intact heparin molecules conjugated to heparin antithrombin or cofactor 1I. In this way, the loss of the biological activity associated with fragmentation or any other modification of heparin prior to conjugation is avoided. The heparin conjugates of the invention retain their anticoagulant activity due to their preparation from intact heparin. Therefore, the methods described here can be used to prepare active heparin conjugates by first adding binding and separator groups to the species that are to be conjugated with heparin (or with the

glycosaminoglycan being used) and then adding them to heparin. Numerous methods of incorporating reactive amino groups into other molecules and solid supports are described in the ImmunoTechnology Catalog and Handbook, Pierce Chemical Company (1990), which is incorporated by reference. Therefore, any species that possesses reactive amino groups or that is capable of being modified in order to contain said amino groups, by any method known at present or that may be known in the future, can be covalently conjugated with glycosaminoglycans, such as heparin, by the methods described herein, and all of said conjugates is contemplated by the present invention.
As described above, the present technology takes advantage of the fact that native heparin (isolated from the intestinal mucosa), as well as dermatan sulfate, already contain molecules with terminal aldoses, which would exist in equilibrium between the hemiacetal and aldehyde forms. Thus, heparin or dermatan sulfate can be conjugated to form antithrombin serpins by spontaneously reducing the Schiff base between the terminal aldehyde of the aldose in heparin or dermatan sulfate and an amino acid of the serpine. Heparin or dermatan sulfate remain unmodified (without reducing its activities) before conjugation, and it is bound at a specific point at one end of the molecule, without any unblocked activation groups or cross-linking of the serpine.
In another aspect of the present invention, covalent complexes can be produced by simply mixing heparin and AT in a buffer solution and allowing a keto-amine to spontaneously form by transposing Amadori between the terminal aldose of heparin and an amino group of the AT . Thus, this technology provides methods of using Amadori transposition to produce glycosaminoglycan conjugates with species containing amines, and especially with proteins. This is a particularly simple method of conjugation, which minimizes the modification of glycosaminoglycan, thereby maximizing the retention of its biological activity.
Another aspect of this technology provides covalent conjugates of glycosaminoglycans, and especially of heparin, labeled at its end with a species containing amines in the terminal aldose residue of glycosaminoglycan. For example, heparin and AT can be linked directly to each other so that the active sequence of pentasaccharide for AT in heparin is very close to its link. This is one of the fundamental reasons for the realization of a covalent heparin-AT complex, since heparin accelerates inhibition through AT only if the AT can bind

The active sequence. It is notable that ATH has the exclusive property that the H (heparin) of the conjugate stoichiometrically activates endogenous AT while catalytically activating exogenous AT. Normally, a species containing amines will bind to each glycosaminoglycan. However, it is clear that the proportion between the species containing amines and the glycosaminoglycan below the unit can be reduced by adjusting the molar proportions of the reagents or the reaction time.
Glycosaminoglycans are available in various forms and molecular weights. For example, heparin is a mucopolysaccharide, isolated from pig intestines or bovine lungs, and is heterogeneous with respect to molecular size and chemical structure. It basically consists of linked residues (1-4) of 2-amino-2-deoxy-a-Dgluopyranosyl, and α-L-idopyranosyluronic acid with a relatively reduced amount of j3-D-glucopyranosyluronic acid residues. The hydroxyl and amine groups are derived to varying degrees by sulfation and acetylation.
Heparin molecules can also be classified according to their pentasaccharide content. About one third of the heparin contains chains with a copy of the single pentasaccharide with a high affinity for AT, while a much smaller proportion (calculated around 1% of total heparin) consists of chains containing more than a copy of the high affinity pentasaccharide. The rest (approximately 66%) of heparin does not contain pentasaccharide. Thus, the so-called "standard heparin" constitutes a mixture of the three species, the "low affinity" heparin that lacks a copy of the pentasaccharide, the "high affinity" heparin, which is enriched for the species that contain the minus one copy of the pentasaccharide, and the "very high affinity" heparin which refers to approximately 1% of molecules that contain more than one copy of the pentasaccharide. These three species can be separated from each other using routine chromatographic methods, such as chromatography on an antithrombin affinity column.
An advantage of forming a conjugate between heparin and a species containing at least one primary amino group (for example, AT) using the slow glycation process described herein is the apparent selection of heparin chains with two pentasaccharides. Thus, for example, the ATH prepared according to the method of this invention appears to be enriched with respect to the heparin species containing two pentasaccharides. When standard heparin (containing approximately 1% heparin formed by two pentasaccharides) is used as a starting material, therefore

In general, more than 10% of the resulting ATH comprises two pentasaccharide heparin, often more than about 20%, often more than 35%, and more frequently about 50% of the ATH comprises two pentasaccharide heparin.
This enrichment may be responsible for a number of useful properties of ATH. The ATH of the present invention activates the TA with which it is conjugated, stoichiometrically, but activates the exogenous AT catalytically. Thus, the heparin found in the ATH complex acts catalytically, both when ATH is administered as a systemic coagulant and when ATH is used to coat the surfaces and make them non-thrombogenic. With the method of the invention, an ATH complex with a very high specific anti-factor Ila activity is obtained. In addition, the second pentasaccharide chain of the ATH complex can interact with exogenous AT molecules, thus allowing conjugated heparin to have a catalytic activity. In addition, the heparin of the ATH complex can be oriented such that the pentasaccharide is available to bind and activate the circulation of AT molecules when the ATH complex joins a prosthetic surface.
It will be noted that a heparin conjugate of interest (for example, ATH) can also be obtained by incubating a species containing at least one primary amino group (for example, Al) with purified heparin of very high affinity (i.e., containing two pentasaccharide groups) or an enriched fraction for a very high activity heparin.
Although this technology has been basically illustrated with respect to heparin, it is clear that all glycosaminoglycans, regardless of their molecular weight and derivatization, can be conjugated by the methods described in this invention, provided they possess a terminal aldose residue. The conjugates of all these glycosaminoglycans and their preparation through the methods indicated herein are within the scope of these invention. For example, heparin conjugates derivatized with phosphates, sulfones and the like, as well as glycosaminoglycans with lower molecular weights
or greater than the molecular weights of heparin are within the scope of the present invention.
In a further aspect of the present invention, a method of manufacturing a composition for the prevention of thrombogenesis can include conjugation of AT with heparin outside the body of a subject to obtain an antithrombinheparin conjugate, where the amount of antithrombin obtained in the antithrombin-heparin conjugate is greater than 60% by weight, greater than 65% by weight, greater than 75% by weight, greater than 85

% by weight, greater than 90% by weight, greater than 95% by weight, or greater than 99% by weight depending on the initial antithrombin used in the synthesis. The yield can be increased by various methods. In one example, AT can be conjugated with heparin by the methods described above. After conjugation, any unbound Al may be recycled and used in another conjugation reaction with heparin. After each stage of incubation of Al with heparin, unbound Al can be recycled and used to obtain additional AlH.
In another example, the AlH yield can be increased using as a catalyst a Amadori transposition. Some of the examples of catalysts that can increase the proportion of Amadori transposition include 2-hydroxypyridine, tertiary amine salts, ethyl malonate, phenylacetone and acetic acid, as well as other acids. In a specific example, Al and heparin can be reacted in the presence of 2-hydroxypyridine while they are heated in water or in very amphiphilic solvents, such as formamide. In additional examples, the AT and heparin can be reacted in the presence of trimethylamine or trimethylamine salts.
The speed of Amadori transposition can also be increased by solvent accelerator systems of Amadori transposition. Examples of such solvents include mixtures of water with formamide, dimethylformamide, dioxane, ethanol, dimethylsulfoside, pyridine, acetic acid, trimethylamine, triethylamine, acetonitrile and combinations thereof. Heparin and Al can be reacted in these solvent systems to accelerate Amadori transposition to obtain ATH.
An additional method of increasing the rate of conjugation of the heparin aldose with a molecule containing an amine involves the use of a binding agent. The binding agent can be a heterobifunctional agent, with a group that reacts to the heparin aldose at one end and a different group at another end that can be used to bind to the AT or a second binding agent which in turn can bind to Al. In a specific example, the binding agent may contain hydrazine at one end and an amino group at the other end, such as 2-aminoethylhydrazine. This binding agent can be reacted with heparin to form a hydrazone with the aldehyde of the heparin aldose. The product can be dialyzed or diafiltered with membranes that allow the removal of heparin chains with a molecular weight of less than 3,000 or 5,000 Daltons together with any unreacted binding agent. The heparin-hydrazone product can then be reacted with a large amount of a secondary binding agent. The secondary binding agent

it can be a homobifunctional reagent that has activated carboxyl groups at each of its ends, such as the succinic acid di (N-hydroxysuccinimide) ester (prepared by esterification of succinic acid with N-hydroxysuccinimide using condensing agents such as carbonyldiimidazole or a carbodiimide) so that the amino group of the hydrazine-containing binding agent reacts with only one of the activated carboxyls of the secondary binding agent. The reaction mixture can be dialyzed or diafiltered to remove the secondary binding agent that has remained unreacted. At this point, the product will be heparin modified by the amino-hydrazine-containing binding agent, as well as the secondary binding agent. This product can be incubated with AT in an H20 buffer solution so that the AT amino group reacts with the second activated carboxyl group of the secondary binding agent to form an AT-Heparin conjugate, where the AT and heparin are linked to through the binding agent and the secondary binding agent.
After the formation of ATH, ATH can be lyophilized (freeze dried) for storage. In one embodiment, the ATH may be prepared in a solution containing exclusively water and subsequently lyophilized. In another embodiment, ATH may be prepared in a solution with water and alanine at a concentration ranging from 0.01-0.09 molar, subsequently lyophilized. In another additional embodiment, the ATH can be prepared in a solution containing water and mannitol, and then lyophilized. Each of these methods can be used independently, and each of them has its own advantages. After lyophilization using any of these methods, ATH can be reconstituted and preserve an important level of thrombin inhibition activity, compared to its level of activity prior to lyophilization. In some cases, ATH may retain at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of its thrombin inhibitory activity. It has been shown that by using other ATH lyophilization methods, such as the preparation of ATH in a solution containing salt in a concentration of more than 1 molar prior to lyophilization, the activity of ATH can be destroyed.
Whether or not ATH has been lyophilized, ATH can be prepared in an aqueous solution containing between 9 and t t mg / mL of ATH with respect to the entire volume of the solution. It has been shown that if solutions with an concentration of ATH greater than 11 mg / mL are prepared, an aggregation of ATH can occur that is difficult or impossible to reverse. However, stable aqueous solutions with ATH concentrations of 9-11 mg / ml can be prepared. This solution can be formulated for administration.

to a patient for the treatment of any of the conditions described herein. The solution may also include various additives that are suitable for administration to a patient.
In clinical practice, the heparin conjugates of the present invention can generally be used in the same manner and in the same pharmaceutical preparation format as commercially available heparin for clinical use. Thus, the heparin conjugates provided by the present invention can be incorporated into aqueous solutions for injection, (intravenous, subcutaneous and the like) or intravenous infusion, or in ointment preparations for administration through the skin and mucous membranes. . Any type of therapy, both prophylactic and curative, known today or available in the future, can be performed using the heparin conjugates provided by this technology.
The heparin conjugates of the present invention are especially useful for the treatment of respiratory distress syndrome (SOR) in neonates and adults. Contrary to what happens with the use of non-covalent heparin-AT complexes, the use of heparin covalent conjugates of the present invention prevents the loss of heparin in the lung space caused by its dissociation of AT. In this case, a solution formed by a covalent complex can be offered in a regulatory solution, in the form of atomized spray in the lung through the airways by means of a catheter or inhaler. Due to its large size, ATH will remain in the alveoli for a prolonged period of time. ATH is also useful for the treatment of idiopathic pulmonary fibrosis.
Its long-term use in the bloodstream can be carried out by intravenous or subcutaneous injection of complex in a physiological regulatory solution. The covalent conjugates of this technology can also be used in the treatment of acquired states of AT deficit characterized by thrombotic complications, such as cardiopulmonary bypass, extracorporeal molecular oxygenation, etc. since the longer half-life of the covalent complex allows reducing the number of treatments and control. Additionally, this invention allows prophylactic treatment of adult patients at risk of deep venous thrombosis.
The ATH conjugate of the present invention has numerous advantages over non-complex AT and standard heparin. Since AT is bound to heparin by a covalent bond, the non-specific binding of ATH to plasma proteins will be less than in the case of standard heparin, with the result of less variation.

interindividual in the dose response in the case of ATH than that given with standard heparin. The longer half-life of ATH after its intravenous injection in humans means that a sustained anticoagulant effect can be achieved with less frequent administration of ATH than would be required in the case of non-complex AT and standard heparin. ATH is a thrombin and factor Xa deactivator much more efficient than AT, and can be effective when used in much lower concentrations than AT in patients with AT deficit. In addition, ATH can access fibrin-bound thrombin and inhibit it. Finally, when it is attached (for example, by covalent bonds) to prosthetic surfaces (for example, endovascular grafts), ATH has shown to have an in vivo antithrombitic activity far superior to that of TA bound by covalent bond, heparin bound by covalent bond, or hirudin bound by covalent bond.
Premature babies have a higher incidence of respiratory distress syndrome (SOR), a serious lung disease that requires treatment by assisted ventilation. Long-term assisted ventilation leads to the appearance of bronchopulmonary dysplasia (oBP) as a result of lung injury, which allows plasma coagulation proteins to travel through the alveolar spaces of the lung. The result is the generation of thrombin and subsequently fibrin. A widespread presence of fibrin in the lung tissue and air spaces of babies who died due to SOR has been constantly observed. This fibrin gel located inside the air spaces makes it difficult to transport fluid from the air spaces of the lung, with the result of persistent and aggravated edema. The present invention allows the treatment of said fibrin-mediated diseases in lung tissue, by preventing the formation of intra-alveolar fibrin, by maintaining an "anti-thrombotic environment" and / or improving fibrinolysis inside the lung tissue, thereby reducing the fibrin load in the air spaces of the lung.
Heparin conjugates can be administered directly into the air spaces of the lung through the airways, prophylactically (before the baby breathes for the first time). This ensures that the antithrombotic agent is available directly at the point where fibrin deposition can occur, and that the risk of bleeding associated with systemic antithrombotic therapies is avoided. In addition, the antithrombotic agent will already be present in the lung prior to the beginning of the ventilatory support associated with the initial lesion, that is, unlike the systemic administration of antithrombin in which the passage of the drug administered through the airspace of the

Lungs do not occur until after lung injury occurs. Since heparin is covalently bound to AT, it will remain in the air spaces of the lung. It can also consist of a complementary therapy of the surfactants that are being administered to prevent RDS and BPD. "Pulmonary surfactant" means the soapy substance that is usually present in the airspaces of the lung, whose main function is to prevent the collapse of airspace, and assist in the transfer of gases. Conjugates can also be administered repeatedly through the endotracheal tube or as an inhaled aerosol. Complementary therapy can also be practiced with asthma medications administered by an inhaler (for example, anti-inflammatory steroids such as beclomethasone dipropionate), other asthma medications, such as sodium chromolin (1,3-bis (2-carboxychromon disodium salt) -5-yloxy) -2-hydroxypropane, ("INTAL") and bronchodilators such as albuterol sulfate.
Various other diseases associated with high thrombin activity and / or fibrin deposits can be treated by administration of the conjugates of the present invention. The inflammatory processes typical of respiratory distress syndrome in adults are basically similar to neo natal SDR, and can be treated by the described antithrombotic therapy. It has also been shown that spontaneous pulmonary fibrosis activates coagulation / fibrinolytic cascades in the air spaces of the lung. Fibrotic lung disease is often a side effect associated with cancer chemotherapy and antithrombotic administration in case of RDS of the covalent heparin conjugates of the present invention can be performed prophylactically prior to cancer chemotherapy. , to prevent pulmonary fibrosis. Administration is repeated after chemotherapy to ensure that fibrin has not formed. A decrease in antithrombin activity and an increase in thrombin activity in the case of sepsis have also been well documented. Sepsis is the most common risk factor for the development of RDS in adults. Thus, the heparin conjugates of the present invention can be used to reduce mortality associated with septic shock.
The conjugates of the present invention can be administered at a therapeutically effective dose, that is, the amount that, when administered to a mammal that requires it, is sufficient to effect the treatment, as described above (for example, to reduce or otherwise treat thrombosis in the mammal, or to deactivate coagulation-related thrombin, or to inhibit the accumulation of thrombi). The administration of active components and salts described in this document

it can be carried out by any of the accepted modalities of administration of agents that serve similar purposes.
In general, an acceptable daily dose is approximately between 0.001 and 50 mg per kilogram of body weight of the recipient and per day, approximately between 0.05 and 25 mg per kilogram of body weight and per day, or approximately between 0.01 and 1 mg per kilogram of body weight and per day. Thus, for administration to a 70 kg person, the dose can range from approximately 0.07 mg to 3.5 g per day, approximately 3.5 mg to 1.75 g per day, or approximately between 0.7 mg and 0.7 g per day, depending on the individuals and the situation of the disease being treated. In the case of ATH, its long half-life allows the compound to be administered less frequently than standard heparin (for example, once or twice a week).
Administration may be carried out by any accepted local or systemic route, for example, parenterally, intravenously, nasally, bronchially (ie, aerosol formulation), transdermally or topically, in the form of solid, semi-solid or liquid, such as tablets, suppositories, pills, capsules, powder, solutions, suspensions, aerosols, suspensions or the like, as suitable unit doses to easily administer a precise dose. Generally, aqueous formulations can be used. The conjugate can be formulated in a non-toxic, inert and pharmaceutically acceptable excipient medium, with a pH of around 3-8 or with a pH of around 6-8. Generally, the aqueous formulation may be compatible with the culture or perfusion medium. The compositions will include a conventional pharmaceutical carrier or excipient, and a glycosaminoglycan conjugate, and may also include other medicinal agents, pharmaceutical agents, excipients, adjuvants, etc. The vehicles can be selected from the various oils, including those derived from petroleum or animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Suitable liquid vehicles include water, saline, dextrose, aqueous or mannitol solutions, and glycols, especially for injectable solutions. Among the most suitable pharmaceutical excipients are starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, chloride sodium, concentrated skim milk, glycerol, propylene glycol, water, ethanol and the like. Other suitable pharmaceutical excipients and their formulations are described in Remington's Pharmaceutical Sciences, by E. W. Martin (1985).

If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH regulating agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc. .
The compounds of the invention can be administered as a pharmaceutical composition comprising a pharmaceutical excipient combined with ATH. The level of ATH in a formulation can vary within the entire range used by those skilled in the art, for example, between about 0.01 percent by weight (% w / w) and about 99.99% w / w of the drug, based on the total formulation, between about 0.01% w / w and 99.99% w / w excipient. In one example, the formulation may contain between about 3.5 and 60% by weight of the pharmaceutically active compound, the remainder being suitable pharmaceutical excipients.
The present invention also extends to the treatment of various conditions by ATH. In some examples, this includes treatment methods, compositions containing ATH, and medical devices comprising ATH. As described in this document, ATH has several advantages over heparin. Normally heparinoid anticoagulants are used for the treatment and prevention of thrombotic diseases. Heparinoids work by catalyzing the anticoagulant activity of the plasma protease inhibitor, antithrombin. Unfractionated heparin (UFH) and its low molecular weight derivatives (LMWH) have several deficiencies, including their reduced average life, their variable anticoagulant response, their limited efficacy in inhibiting thrombin (especially thrombin linked to coagulation), the induction of hemorrhage and the induction of thrombocytopenia.
ATH can solve many of these problems when used as a therapeutic agent. Compared to traditional heparinoids, AlH has a longer average life span, lower linkage with plasma proteins and endothelial cells and greater antithrombotic efficacy in animal models without an increased risk of bleeding. In vitro, ATH directly inhibits various coagulation factors, with significantly higher rates compared to the non-covalent mixtures of Al and UFH (Al + H). AlH is also more effective in inhibiting coagulation-related thrombin, compared to Al + H.
In some embodiments of the present invention, AlH can be used to make coatings for medical devices. In one embodiment, a coating method of

a polymeric surface with an antithrombin-heparin conjugate may include contacting the polymeric surface with a solution of the antithrombin-heparin conjugate, such that the antithrombin-heparin conjugate directly coats the polyurethane surface, without the presence of groups binders between the antithrombin-heparin conjugate and the polymeric surface.
The known methods of covalent fixation of the molecules to the polymeric surfaces can be used to fix the ATH to a polymeric surface of a medical device. For example, the polymeric surface can be activated first by treatment with an oxidant or reducer, and then a binding group can be attached to the activated surface. Next, ATH can bind to the monomer. In one example, a polyurethane surface is activated by reacting it with sodium hypochlorite or with lithium aluminum hydride. Next, allyl glycidyl ether is grafted onto the surface, to act as a binding group. Next, ATH joins the binding group.
Although ATH can thus be fixed to surfaces using binding groups, it has been shown that, surprisingly, ATH can also be directly attached to a polymeric surface, without any linker groups. Thus, the present invention provides very simple methods for coating polymeric surfaces with ATH without activating the polymeric surface or attaching a binding group to the surface. In some examples, the polymeric surface can simply be contacted with an ATH solution by immersion or by any other method. ATH can be attached directly to the surface and remain attached to it even after washing with detergents. This method may be useful for coating medical devices that come into contact with blood, in order to reduce the thrombogenicity of medical devices.
The polymeric surface may be composed of any polymer used for the manufacture of medical devices. In some embodiments, the polymeric surface may be a polyurethane surface, a polyethylene surface, a polypropylene surface, a polyethylene tetrafluoride surface, a polydimethylsiloxane surface, an ethylene-acrylic acid copolymer surface, a Dacron surface , a polyester polyurethane surface, a polyurethane polycarbonate surface, a polyvinyl chloride surface, a silicone surface, a latex rubber surface, a nitinol surface, a Nylon surface, a polyethylene Terephthalate surface, a surface of polystyrene, or combinations of said products. In other embodiments, the polymeric surface may include materials of loplex and other hydrogels, such as those based on 2-hydroxyethyl methacrylate.

or polyether polyurethane acrylamide and ureas (PEUU), including Biomer (Ethicon Corp.) and Avcothane (Avco-Everretl Laboratories).
The polymeric surface may be part of a medical device that comes into contact with blood. In some embodiments, the medical device may be an intravenous catheter, an intra-arterial catheter, a central peripheral insert catheter, a central catheter, a Swan-Ganz catheter, a coronary stent, an arteriovenous shunt, a vein filter lower cava, a dialysis catheter, a dialysis blood circuit line, a dialysis membrane, an extracorporeal membrane oxygenation line, an extracorporeal membrane oxygenation membrane, an in vivo prosthesis, a pacemaker electrode, a suture, a blood filter, a mechanical valve, an artificial organ, or a blood storage container. Any internal or external medical device that comes into contact with blood and because of which it would be desirable to reduce blood clotting could be coated with ATH.
In a specific embodiment, the medical device can be a catheter with an ATH coating. Catheters often fail due to surface-induced thrombosis. The coating of these devices with conventional anticoagulants, such as heparin, may provide limited improvements, but occlusion of the device due to clots remains a serious problem. ATH coated devices exhibit clearly superior antithrombotic properties, and can be used without any occlusion caused by thrombi.
In one example, a catheter can be coated with ATH, soaking the catheter in an ATH solution. The inside of the catheter can be contacted with the ATH solution by passing the solution to the lumen of the catheter using a syringe. Next, the catheter can be left incubating for a period of time, to allow ATH to adhere to the polymeric surfaces of the catheter. This period of time can range, for example, between 0.1 and 48 hours, 1 and 48 hours, 1 and 24 hours, 2 and 8 hours, or any other period of time that is sufficient for the ATH to adhere to the polymeric surface. Then, the catheter can be removed from the ATH solution, and the excess ATH solution can be emptied from the catheter, allowing the remaining coating of the ATH solution to dry. Drying can last for a sufficient period of time for the solvent to evaporate from the ATH solution with which the catheter is coated. In one embodiment, the catheter can be air dried, at room temperature. The drying time can range, for example, between 1 and 48 hours, 1 and 24 hours, 1 and 8 hours, or between 1 and 2 hours. In other embodiments, the catheter

it can be allowed to dry to the jet of air coming from a blowing device, to the ambient air
or by exposure to a jet of hot air (which can be air heated to approximately 60 "C), or to ambient air or by exposure to a jet of dehumidified air, under a nitrogen atmosphere or by exposure to a stream of nitrogen, under an atmosphere of noble gas or by exposure to a jet of noble gas, or vacuum, partial or total.The drying time can be reduced when using a jet, air or other gas, dehumidified or hot. it can reduce substantially when carried out under vacuum, partial or total, for example, the drying time can range between 1 minute and 48 hours, 1 minute and 24 hours, 1 minute and 1 hour, from 1 to 30 minutes, or any other Sufficient drying time Once dried, the catheter can be used in vitro or sterilized for use in medical applications.A sterilization method may include placing the catheter in an airtight container containing a gas permeable membrane and sterilizing the catheter and container by exposure to ethylene oxide.
Various methods of coating a catheter or other medical device with ATH can be used. For example, a medical device can be immersed in an ATH solution and incubated for a sufficient period of time to allow the ATH to adhere to the surfaces of the medical device. In another example, a circulation method may be used, in which the medical device is contacted with a circulating ATH solution. In a specific example, the medical device can be a catheter, and the ATH solution can circulate around the catheter so that the ATH solution circulates through the outer and inner surfaces of the catheter. The ATH solution can be continuously recycled and circulate through the catheter for a period of time sufficient for the ATH to adhere to the catheter surfaces.
Multiple medical devices can be simultaneously coated in a single batch of ATH solution. For example, a sufficient volume of ATH solution can be prepared to submerge any number of sanitary products for incubation in the solution. Additionally, the ATH solution can be used to coat multiple batches of method products sequentially. Taking into account that the coating of ATH formed on the surface of the medical device may have a thickness of only one molecule, most of the ATH of the solution will continue to remain in the solution after the coating of a medical device. For example, an ATH layer with a thickness of one molecule may contain about 2 pmoles of ATH / cm2 of surface. However, 100 mL of a 1 mg solution ATH / mL contain a total of 1.69 x 106 pmoles of ATH. For the

Thus, the solution contains enough ATH to form a coating with a thickness of one molecule in many medical devices. In one embodiment, a batch can be prepared with an ATH solution, and then a plurality of medical devices can be dipped and incubated in the ATH solution. After incubation, the medical devices can be removed, and the remaining ATH solution can be poured back into the batch of ATH solution. Next, this same batch of ATH solution can be reused to coat another plurality of medical devices. This process can be repeated multiple times to coat a large number of medical devices with a single batch of ATH solution. In some cases, the process can be repeated up to five times, up to 10 times, up to 20 times, or even up to 50 times before preparing a new batch of ATH solution.
In a continuous circulation method, the ATH solution could potentially be recycled indefinitely so that the entire ATH of the solution becomes used. For example, the ATH solution can be recycled as a series of sanitary products are coated with the solution. When the ATH concentration of the ATH solution falls below a threshold value, such as 0.1 mg ATH / mL, 0.5 mg ATH / mL, 0.8 mg ATH / mL, or 0.9 mg ATH / mL, more ATH can be added to the solution of this example.
In a specific example, a group consisting of 50 catheters can be coated with ATH. This operation can be carried out by immersing the catheters in 200 mL of a buffer solution of 1 mg ATH / mL to simultaneously submerge the 50 catheters. Alternatively, 5 or 10 catheters can be coated by immersion simultaneously using a single batch of ATH solution. In this way, the batch of required ATH solution can be reduced from 200 mL to 40 mL or 20 mL.
Catheters and other ATH-coated medical devices can remain operative for a considerably longer period of time than heparin-coated devices. For example, catheters coated with ATH can remain permeable in a vein or artera for up to a year without using any type of anticoagulant. In certain cases, an ATH-coated catheter can remain permeable in a vein or artery for a period of between 1 week and 1 year, between 1 month and 6 months, between 2 and 6 months or any other prolonged period of time. In some cases, a medical device coated with ATH can be kept indefinitely free of clots, or at least for as long as the product is used. Thus, it is not necessary to remove and replace the implanted device to a patient due to the formation of clots. Is older

ATH's capacity may be due, at least partially, to the fact that ATH may be a substantially 100% active anticoagulant, while heparin may contain a high percentage of inactive heparin chains.
In a specific embodiment, lyophilization coating can be used to coat a polymeric surface of a medical device with ATH. The polymeric surface can be contacted with the ATH solution in the absence of binding groups. The excess ATH solution can be allowed to drain from the polymeric surface. Then, the solvent can be allowed to evaporate from the polymer surface under vacuum, at least partially. This can form a dry coating of ATH on the polymeric surface. In some cases, the lyophilized coating may have greater uniformity compared to the coatings obtained by other coating methods.
In addition to coating catheters and other medical devices with ATH, the present invention also extends to catheter washing and sealing solutions. Many institutions have a habit of washing and sealing the catheters with a diluted heparin solution to prevent the appearance of clots, although there is no basic agreement in the scientific literature about the effectiveness of heparin washing to keep the catheters permeable. As an anticoagulant, heparin has several limitations, including its dependence on adequate levels of antithrombin in the plasma, and the fact that only one third of commercial heparin preparations have anticoagulant activity. The dependence of antithrombin on the functioning of heparin is a matter of particular concern in very young children, when neonates, and especially premature infants, have plasma antithrombin concentrations markedly lower than those of adults.
Coatings with heparin are frequently used to make surfaces more antithrombogenic. However, these surfaces are not ideal, due to surface heparin seepage, non-uniform replacement and different anticoagulant activities of the product. Taking into account that two thirds of the unfractionated standard heparin do not contain the sequence of pentasaccharides required for anticoagulation, this circumstance limits the level of anticoagulant activity that can be achieved on the modified surface. In addition, non-anticoagulant heparin chains may continue to favor the deposition of proteins on the surface, which may favor the appearance of thrombi. These problems can be avoided using ATH.

In one embodiment, a solution for washing and sealing an intravenous or intra-arterial catheter may include an antithrombin-heparin conjugate. In another embodiment, a washing and sealing system may include: an intravenous catheter
or intra-arterial; a cleaning and sealing solution configured for introduction through the catheter, said cleaning and sealing solution comprising an antithrombinheparin conjugate; and a syringe configured to inject the cleaning and sealing solution into the catheter.
In another additional embodiment, a method of maintaining the permeability of a catheter may include: inserting a catheter into a vein or artery of a patient, such that an inner opening of the catheter opens inside the vein or artery, and an outer opening of the catheter opens towards the outside of the patient; injecting a solution comprising an antithrombin-heparin conjugate into the catheter through the outer opening of the catheter; and sealing the outer opening of the catheter so that at least a part of the solution comprising the antithrombin-heparin conjugate remains inside the catheter.
When a cleaning and sealing solution with a catheter is used in this way, the catheter remains in contact with the ATH solution after preparation and closure. While the catheter remains in contact with the ATH solution, the ATH can be attached to the catheter surfaces as described above. Thus, each time the catheter is washed and prepared with a solution of ATH, the catheter can safely be coated by ATH. When the cleaning and sealing solution is injected through the catheter, the ATH can be attached to both the inner surfaces and the mouthpiece of the catheter. In this way thrombus formation can be prevented inside the catheter's lumen, as well as at the tip of the catheter.
ATH may be present in the cleaning and sealing solution in a variable amount between 0.01-10 mg ATH / mL, 0.1-1 mg ATHJmL, or another effective amount. Other components of the solution may include water, sodium chloride and regulators.
Taking into account that the heparin fraction present in ATH already has a permanently bound AT molecule, the part of the heparin chain that is fully available for interaction with plasma proteins and cell surfaces is smaller than the total heparin ATH binds less to plasma proteins and endothelial surfaces (single-layer HUVEC) compared to heparin. If the ATH coating binds to platelets, unlike heparin coatings, ATH does not activate platelets. Therefore, catheters that have the lining of ATH remain thrombus free when the catheters with heparin lining will begin to form tops and become stuck. Even when platelets are activated at

Because of stasis or other biophysical phenomena, ATH is superior to heparin (UFH) in terms of inhibition of the prothrombinase complex and concomitant thrombin generation on activated platelet surfaces. Given the great superiority of ATH as an anticoagulant against heparin, ATH can be much more effective in preventing the onset of coagulation. This is especially underlined by the fact that, although heparin will not be able to inhibit activated clotting factors inside the catheter nozzle, unless AT is also present in the plasma, ATH already has a Extremely reactive AT molecule that can directly inhibit clotting factors.
Additionally, ATH has anti-Xa or anti-Ila activities, and a catalysis of the inhibition by the AT of factor Xa or thrombin much higher. In fact, it has been shown that the power of the ATH complex is more than four times higher, in terms of the thrombin inhibition rate, than that of AT + H (heparin). It has also been shown that the catalytic activity of anti-factor Xa is similar for ATH versus heparin. ATH can also directly inhibit clotting factors on its own without adding AT, something that heparin cannot do. ATH can directly inhibit all coagulation factors of the coagulation cascade. On the contrary, the need for AT in the heparin anticoagulant mechanism has been perfectly documented. Unlike heparin, ATH can easily inhibit thrombin attached to fibrin clots and inhibit factor Xa attached to phospholipid surface complexes, such as prothrombinase. In fact, once ATH inhibits thrombin on the surface of the clots, the ATH complex remains attached, turning the surface of the clot into an anticoagulant due to the active heparin chain of ATH. These and other advantages make ATH a very useful product for use in a cleaning and sealing solution. Taking into account the superior power of ATH, as well as other advantages, much lower concentrations of ATH will be required in the sealing solution compared to heparin solutions. For example, ATH may have at least 5 times more catalytic anti-Xa inhibitory activity compared to unfractionated heparin.
Citrate is sometimes used for cleaning and sealing solutions. Compared to citrate as a cleaning and sealing solution, ATH can maintain the permeability of the catheter for a longer period of time at lower concentrations. Citrate prevents coagulation from occurring by chelating calcium from the blood. However, once the citrate has bound to a calcium ion, it can no longer prevent clotting.

joining more calcium. Thus, once the citrate is saturated, there will be more calcium from the blood flow that starts the coagulation cascade. On the other hand, ATH can continue to catalyze inhibition by the presence of AT in the plasma. In this way, ATH is never saturated or consumed, and will continue to operate.
ATH can inhibit thrombin directly (without added TA) 3.2 times faster than AT + heparin. ATH can inhibit Vlla factor complexes with tissue factor 28 times faster than AT + heparin. Depending on these differences, a concentration of ATH 3.2 x 28 = 89.6 lower may give an equivalent reaction rate when inhibiting the coagulation cascade, compared to heparin. Thus, the concentration of ATH can be at least 89.6 times less than the concentration of heparin used in cleaning and sealing solutions, even without taking into account the non-covalent coating of the catheter surface with ATH that will occur because of the presence of the sealing solution of ATH.
Current cleaning and sealing solutions that contain heparin result in 20% of central venous catheters becoming clogged and needing to be replaced. If ATH is used in the cleaning and sealing solution, this 20% of CVCs may maintain their permeability without having to be replaced.
Catheter replacement rises to an average of 10,000 units per day in the US alone. Because of the complications. The use of ATH cleaning and sealing solutions can reduce the number of catheters to be replaced. This would reduce replacement costs, increase the patient's quality of life (shorter hospitalization time), prevent days of treatment from being lost (as in the case of cancer therapies to be stopped while waiting for it to be scheduled an intelVención for a change of CVC), would reduce the surgical costs and increase the time that the doctor would have to take care of other patients.
In one example, ATH was used to permanently coat the surface of stents and CVCs, which were then tested in animal models. ATH coated stents and CVCs exhibited lower clot formation compared to heparin coated stents and CVCs and uncoated stents and CVCs.
Since it is not feasible to coat the different types and brands of CVCs with an anticoagulant, many institutions choose to rinse the catheters with an anticoagulant solution to prevent the appearance of clots related to the catheter. The utilization

A cleaning and sealing solution containing ATH may be more effective than using a heparin solution.
The present invention also extends to methods of treating conditions using ATH. ATH can be used to treat various conditions that require thrombogenesis inhibition. In some cases, ATH formed from unfractionated heparin may be used. In other cases, ATH can be manufactured from heparin from which the low molecular weight heparin chains have been removed. In one embodiment, a method of treating a medical condition by inhibiting thrombogenesis in a mammal may include administering to the mammal a dose of an antithrombin-heparin conjugate, where at least 98% of the heparin chains of the Antithrombin-heparin conjugate having a molecular weight greater than 3,000 Daltons.
In some embodiments, ATH may be administered during an invasive procedure to reduce the risk of thromboembolic complications. Invasive procedures, such as cardiopulmonary bypass (BPC), generate massive amounts of fibrin microemboli that can lodge in the brain, causing long-term cognitive dysfunctions. It has been shown that ATH significantly reduces transient High Intensity Signals (H ITS) in the carotid arteries of pigs undergoing cardiopulmonary bypass (PCB). The reduction in HITS may indicate a lower risk of thromboembolic complications during CPB and a reduction in related post-surgical neurological dysfunctions. The decrease in HITS with ATH, compared with treatment using UFH + AT, was achieved without a significant increase in bleeding (during or after the BPC). The superior ability of ATH to inhibit thrombin related to fibrin clots and its longer half-life compared to heparin may be responsible for this improved performance during PCB.
For prophylactic purposes (in prothrombotic patients not undergoing coagulation treatment), ATH can be administered intravenously at a variable dose between 1 unit (in terms of activity of anti-factor Xa) per kilogram (unit / kg) and 1000 unitslkg, the typical dose being around 100 unitslkg. If ATH shows an anti-factor Xa activity of 130 units per milligram (units / mg), in terms of the ATH component of ATH, the doses of ATH can range from about 0.008 mg to about 8 mg, being a typical dose of about 0.8 mg Doses can also be determined in terms of mg of the heparin component of ATH knowing that the proportion of AT versus heparin in ATH is 59 mg versus 18 mg. Having in

It has the longer intravenous half-life of ATH than that of heparin, ATH does not need to be administered as frequently. In this way, ATH could be administered between once a day and once a week, being the most common once a day.
For resolution of a clot, ATH can be administered intravenously with a variable dose between 50 units (in terms of anti-factor Xa activity) per kilogram (units / kg) and 2000 units / kg, the typical dose being of approximately 300 units / kg. If ATH has an anti-factor Xa activity of 130 units / mg (in terms of the ATH component of ATH), then the doses of ATH may then range between approximately 0.4 mg and approximately 15 mg, the typical dose being approximately 2 , 3 mg. Given that the intravenous half-life of ATH is longer than that of heparin, it will not have to be administered as frequently. Thus, ATH may be administered three times a day and twice a week, being the most common twice a day. Once the clot has shrunk sufficiently, the dose and frequency of treatment with ATH can be reduced, or such treatment discontinued.
In the case of prophylactic administration during surgery, the dose may depend on the type of surgery, with the most invasive procedures requiring higher doses of ATH. One of the most invasive and most damaging surgeries is bypass surgery, which can be treated with doses of ATH (in terms of the ATH component of ATH), varying between 1 and 6 mg / kg body weight. In some cases, a dose of ATH of 3 mglkg may be used during bypass surgery.
ATH can be administered intravenously, in the form of a solution containing simple iso-osmotic salts (such as 0.15 M NaCI) and physiologically acceptable regulators (such as HEPES at a pH of 7.4). No other protein, solvent, adjuvant or other type of additives is required, but may optionally be included without producing any significant detrimental effect on the function of ATH.
ATH can provide several advantages over heparin as a systemic anticoagulant. One of the problems associated with anticoagulants is bleeding. Bleeding may occur when you have too much blood thinner. ATH has a high anticoagulant activity, but causes less bleeding, compared to heparin. ATH also has other advantages over heparin. ATH AT is always activated and the heparin binding step to AT has been eliminated, which determines the frequency. Additionally, ATH directly inhibits activated coagulation factors, such as thrombin, with faster rates than heparin. Heparin can bind to

non-selective form to proteins of plasma and cell supersite in vivo, and yet, said non-selective binding is reduced with ATH. In addition, ATH has a longer intravenous half-life than heparin. Finally, since covalently bound AT encompasses a significant portion of ATH's heparin chains, adverse interactions with platelets are reduced, so that normal platelet function is maintained.
In another embodiment of the present invention, ATH can be used for the treatment of woody conjunctivitis. A method of treating woody conjunctivitis in a mammal may include the administration of a dose of the antithrombin-heparin conjugate in a mammalian eye.
Retinal venous occlusion caused by ocular thrombosis is only overcome by diabetic retinopathy as a cause of deficiencies or loss of vision caused by retinal vascular diseases. In the event that there is a plasminogen deficiency, woody conjunctivitis caused by a fibrin membrane that covers the eye and obscures vision. The treatment of this thrombotic problem with plasminogen-based eye drops has modest proportions due to the inhibitors, and heparin-based eye drops are only partially effective due to the variable presence of plasma antithrombin that is activated by heparin. ATH, unlike heparin, can remain isolated outside vascular spaces, due to its large dimensions. Also, ATH contains antithrombin, so it does not depend on the patient's system to provide this agent so that heparin operates as an anticoagulant. In this way, ATH can treat or prevent ocular thrombosis more effectively. In one example, woody conjunctivitis can be treated by daily administration of an eye drops containing ATH. ATH may be present in eye drops with a concentration of 0.01 to 10 mg of ATH / mL, 0.1 to 2 mg of ATH / mL, or about 1 mg of ATH / mL.
In another embodiment, a contact lens can be coated with ATH. The patient may wear the coated lens in the eye to prevent ocular thrombosis.
Woody gingivitis is another condition related to plasminogen deficiency. ATH can be used for the treatment of woody gingivitis. In one embodiment, a solution of ATH may be periodically applied topically to the patient's gums, to prevent fibrin formation in the gums.
In a further embodiment, ATH may be used for the treatment of coagulation disorders in the lungs. These may include respiratory distress syndrome.

(SOR) And acute lung injuries, caused, for example, by mechanical ventilation. Plasma experiments on fetal distal pulmonary epithelial cells (FOLE) have confirmed that ATH inhibits thrombin generation to a greater extent than an equivalent dose of AT + UFH. This result demonstrates that ATH can be used for the prevention or treatment of intrapulmonary coagulation. The intratracheal instillation of ATH achieves a high anticoagulant activity in the washing liquid over 48 hours, without measurable systemic activity. In addition, ATH shows a tendency for selective proliferation of epithelium in relation to fibroblasts. Thus, ATH has many of the characteristics necessary to alleviate the main factors that contribute to SOR, OBP and pulmonary diseases induced by ventilation.
Neonatal and adult SOR is characterized by the leakage of plasma proteins of various sizes into the airspace, which results in the generation of interstitial and intra-alveolar thrombin, with the consequent deposition of fibrin. There is serious and convincing evidence that clearly shows that coagulation, which causes fibrin formation, remains one of the effectors, key in lung lesions. Absent in a normal lung, the presence of fibrin in the alveolar and interstitial compartments, throughout the evolution of diffuse alveolar lesions, remains one of the most prominent features of SOR. Since the introduction of surfactant therapies, the amount of fibrin directly observed in SOR patients has been reduced. However, taking into account that in the SOR an important activity of the pulmonary thrombin is present, a lack of crosslinked fibrin polymer can occur due to the fact that the fibrin can quickly leave the lung, as is the case with various solid tumors. In fact, it has been observed that improvement in intrapulmonary fibrinolysis decreases fibrosis, which is another demonstration of fibrin activity in lung lesions. Fibrin rotation in the lungs of SOR / OBP patients is altered locally, similar to what happens with acute SOR. In addition, fibrin deposits in the vasculature and pulmonary artery indicate the existence of vasoconstrictor mechanisms when greater pulmonary vascular resistance occurs in the SOR. Intra-alveolar fibrin deposits can have significant negative effects, both in the short and long term. Fibrin has been shown to significantly impair surfactant function, while products related to fibrin degradation are linked to increased permeability of the alveolar-capillary membrane, thereby increasing leakage into the plasma protein airspace. Another test of the

Activation of the coagulant and fibrinolytic systems in the intra-alveolar space is the presence of an excess of procoagulant and a deficit of fibrinolytic activities in the broncho-alveolar lavage fluid (BAL) from patients of SOR. Protein leaks into the lungs have been associated with systemic activation of polymorphonuclear lymphocytes, complement lymphocytes and coagulation lymphocytes. Fibroblasts are activated by fibrin and proliferate in areas that contain it, which leads to improper remodeling of lung tissues, as in the case of fibrosis.
Fibrin is produced by the division of fibrinogen by thrombin, subsequently intersecting. It has been shown that the onset of coagulation in vivo is basically the result of the appearance of active tissue factor (TF). Experiments in which thrombin generation from plasma in a mixture of FDLE and fetal lung cells has been investigated has shown that procoagulant activity in the pulmonary epithelium or fibroblasts was due to the activation of the Vlla factor by the FT attached to the cell surface In fact, FT has been identified as the only coagulation activator in lung cancer cell cultures. Additional data have shown that premature infants have a thrombotic activity in the airspace similar to that of TF. As a conjunctive feature, the less developed lungs in the premature state
or fetal are more permeable than those of adults to molecules that access the airspace from systemic circulation. Thus, any pulmonary substrate of the TF or other coagulant activity has greater opportunities to contact the clotting cascade factors in newborns, producing fibrin deposits and complications detected in the SOR. In this way, the state of development can influence the susceptibility of the individual to prothrombotic aggressions. Other acute lesions may occur as a result of biological or mechanical factors, which may be associated with coagulant pathologies. For example, in the case of an infection, there is important evidence of pulmonary thrombin generation with harmful results. More recently, the existence of certain physical mechanisms that cause thrombin generation and fibrin deposits has been demonstrated. It has been known for some time that patients (especially younger ones) who require prolonged mechanical ventilation show evidence of damage to the tissue structures of the lung. In addition, the existence of pulmonary and circulatory coagulation has been indicated as a result of ventilation, which can be counteracted by the administration of systemic AT. Also, the ventilatory volutrauma has produced lung lesions, which in turn have caused a deterioration in the

gas exchange, which has been improved by heparin. Once again it can be inferred that the ventilation-induced damage associated with thrombosis is especially important in young patients. Experiments have shown that ventilation with a high tidal volume in rats causes the release of functional TF, both in the circulation and in the airways of the lung. The generation of TF and thrombin was only induced in neonates, but not in adult animals. This is in line with other results, in which the coagulant activity related to TF was only expressed in adult humans with very high tidal volumes, and without positive final expiratory pressure. The appearance of thrombotic activity peaks in newborn rats occurred after less than 15 min and persisted over periods of one hour. Taking into account the relationship between fibrin deposits and pulmonary viability, control of the procoagulant activity of TF during an acute (ventilatory) or chronic lesion (BPD) can benefit resistance to pulmonary dysfunction and adverse alteration of the tissues. Interestingly, damage control is likely to also attenuate the permeability of lung tissue, which leads to the presence of fibrin and other plasma proteins that inhibit tenside action. In fact, an agent that could neutralize the action of TF, block the pathways that lead to lung cell dysfunction and moderate tissue remodeling during lung injury would greatly improve the outcome in patients, especially among the pediatric population.
ATH can address many of the limitations found in the treatment of pulmonary fibrin deposits by UFH. ATH has a direct non-catalytic inhibitory activity against thrombin that is 4 to 10 times faster than that of non-covalent mixtures of AT + UFH. The rapid rate of direct thrombin inhibition by ATH would guarantee neutralization of the low thrombin levels involved in the activation of thrombin generation, and would be present in the edema fluid, in which the AT concentrations of the Plasma may be insufficient to inhibit thrombin. In addition, the great capacity of ATH to catalyze the reaction of AT with thrombin would also allow it to use any AT that accesses the alveolar spaces. Apart from the rapid inhibition of thrombin in the liquid phase, ATH can inhibit fibrin-bound thrombin that is resistant to reaction with non-covalent AT · UFH. Inhibition of thrombin attached to clots helps reduce venous clots by ATH without significantly increasing hemorrhagic side effects. In the case of FDLE surfaces, ATH has proven to be far superior when it comes to inhibiting

Plasma thrombin generation compared to similar doses of AT + UFH. In addition, the comparison of ATH with unfractionated and low molecular weight heparins has shown that ATH can more effectively inhibit thrombin generation in plasma of adults, children or neonates. Intra-tracheal administration of ATH in rabbits and newborn rats showed that high levels of anticoagulant activity can be detected in the washing liquid at least 2 to 4 days later, without any presence of antigens or systemic activity. In this way, ATH can be used as a rapid and potent anticoagulant, which can inhibit pulmonary fibrin deposits over prolonged periods of time, without systemic side effects. After a single administration in the lungs of premature babies at risk of acute lung injury, ATH exerts an interesting differential effect on in vitro epithelial growth against fibroblasts, as opposed to the impact exerted by Al. The extensive research conducted on ATH immobilized in Polyurethane catheters and endoluminal grafts have shown that the conjugate exhibits significantly greater inhibitory activity against thrombin in vitro and anticoagulant activity in vivo. ATH adhered to alveolar matrices can also help control excess thrombin generation locally and fibroblast accumulation compared to other cells.
Specifically, ATH can be administered by providing mechanical ventilation. Mechanical ventilation can damage the lungs, especially in the case of preterm infants. Therefore, by administering ATH to the lungs of newborns, before or during mechanical ventilation, complications caused by fibrin formation in the lungs can be avoided. A single dose of ATH may be sufficient to provide anticoagulant effects in the lungs for a prolonged period of time. For example, a single dose can last between 6 hours and 1 week, in some cases.
The present invention also extends to a specific composition that includes ATH together with fibrin. ATH can be incubated with fibrin outside the body, so that fibrin adheres to ATH. This composition can then be used for the treatment of blood clots. The composition can be injected or otherwise administered to attack a clot. The ATH: Fibrin complex of the composition tends to bind to the other fibrin present in the clot. When the ATH: Fibrin complex adheres to the surface of the clot, the clot's surface acquires a net anticoagulant property. This stops clot growth and allows it to break.

In one embodiment, a composition for the treatment of blood clots may contain antithrombin, heparin, and fibrin. At least 50% by weight of heparin can be conjugated with antithrombin to obtain an antithrombin-heparin conjugate. At least a portion of the fibrin can bind to the antithrombin-heparin conjugate. In
In some cases, at least 50% by weight of the fibrin may bind to the antithrombin-heparin conjugate. The percentage may be higher, joining in the order of 90-100% by weight of the fibrin of the composition to the antithrombin-heparin conjugate.
The composition of the ATH: Fibrin complex can be prepared by mixing an ATH solution with a fibrin solution. The fibrin may be sufficiently diluted or contain a
10 fibrin polymerization inhibitor, such as glycine-proline-arginine-proline amide, so that fibrin does not polymerize in the solution. When the fibrin binds to ATH, if necessary, the ATH: Fibrin complex can be separated from any unbound fibrin, so that the composition does not introduce additional active fibrin into the patient's organism.
15 Examples
The following examples show the embodiments of the invention that are best known today. However, it should be understood that the following are but illustrative examples of the application of the principles of the present invention. Any
A person skilled in the art could conceive of numerous modifications and alternative compositions, methods and systems without departing from the spirit and scope of the present invention. The attached claims try to cover said modifications and configurations. Thus, although the present invention has been described above in particular, the following examples provide additional details regarding what is currently being
25 consider practical embodiments of the invention.
Example 1
Direct incubation of ATH with a polyurethane catheter was carried out in the manner
30 next. An ATH solution, labeled with C251-ATH radiolabelled ATH, ATH radiolabelled using Na1251 (from Perkin Elmer, Woodbridge, ON) and iodobeads markers (from Thermo Fisher, Ottawa, ON) according to the manufacturer's instructions) was prepared, used to perform incubations with non-activated catheters. Two of the catheters (catheters of

15 cm long polyurethane. and caliber FR 7 manufactured by Saloman) were immersed in a cylinder containing 53 mL of a solution of 1 mg / mL ATH¡'25 I-ATH in PBS diluted tltO. The liquid inside each catheter was absorbed by a syringe attached thereto, held in place by the fixed syringe. Next, the catheters were incubated for 24 hours at room temperature by stirring with a rod. After 24 hours of incubation, the catheters were removed from the incubation solution and transferred to another container for washing. The catheters were washed sequentially, in 60 mL volumes of: a) 0.15 M sodium phosphate, pH 8.0, b) 2 M NaCl 0.15 M sodium phosphate, pH 8.0, e) 0.1% SDS sodium phosphate 0 , 15 M, pH 8.0 (performed 3 times in this solution), d) 0.15 M NaCl, 0.02 M sodium phosphate pH 7.4 Y e) H20. During each wash, the wash solution was stirred with a rod and made up and down the inside of the catheter 50 times with a syringe. After washing, 0.5 cm sample segments (3 per catheter) were cut and subjected to gamma-radioactivity count to determine the amount of radiolabelled ATH that remained attached to the catheter. Each segment had a total area of 1 cm2 (interior + exterior). The
15 results are shown in table 1: Table 1
Catheter sample number Counts per minuteATH pmoles calculated
one t75830.265
2 136420.206
3 180700.273
4 157460.237
5 147270.222
6 145670.220
Average 157230.237
The calculated "pmoles of ATH" values were calculated according to a conversion factor that 66,300 counts per minute equaled 1 pmol of ATH. The ELlSA trials of
20 0.5 cm segments corresponding to similar catheter segments to calculate the amount of surface ATH (in terms of the TA detected by the test) verified that the total ATH mass of the catheter was also of the order of one tenth of 1 pmol . This experiment demonstrates that, surprisingly, the solution-free ATH can adhere strongly to the surface of the unmodified catheter. This also shows that

A cleaning and sealing solution based on ATH can help anticoagulate the catheter on the surface of the catheter, since ATH will adhere to the surface. The opposite occurs with heparin, which does not adhere to the surface of the catheters. The ATH remained adhered after a copious wash with a buffer solution, detergent and a high salt content. On the contrary, it was not observed that heparin adheres to polyurethane surfaces.
Example 2
Direct incubation of ATH with a polyurethane catheter, including drying, can be performed as follows. The following example shows in detail how an unmodified covalent antithrombin-heparin (ATH) coating can be applied to non-reactive and passive polymer surfaces without previously modifying or activating ATH or the polymer surface. An ATH solution is prepared with a suitable regulating liquid (with a variable pH range between 4.0 and 10.0, and in some cases approximately 7.4) with an ATH concentration (in terms of the AT fraction ) variable between 0.01 mg / mL and 10 mg / mL and in some cases around 1 mg / mL. Up to 20 catheters (such as 15 cm long polyurethane catheters) were immersed in a cylinder containing 53 mL of the ATH solution. The liquid was aspirated from the inside of each catheter through a syringe attached thereto and retained by the syringe. Next, the catheters were incubated for 24 hours at room temperature by stirring with a rod. After 24 hours of incubation, the syringes were removed from the incubation solution, the catheters were removed from the incubation solution, each catheter was held vertically with the lower end in contact with a cellulose paper filter, and left that the liquid should come out by gravity from the inside and outside of the catheter over a period of time (usually around 1 minute). Next, the catheters were suspended vertically to dry (evaporation of the solvent) without the bottom tip touching any solid surface. Drying, for a maximum of 48 hours (in some cases, around 18 hours) can be carried out: in the air, at room temperature, in a nitrogen atmosphere at room temperature, in a atmosphere of noble gases at room temperature, in an open atmosphere of gas heated to 60 degrees Celsius or in vacuum (for lyophilization). Once dry, the catheters are used in vitro or placed in sealed bags (with gas permeable membranes) and sterilized by exposure to ethylene oxide, for future use in patients or other medical applications.

Example 3
Effectiveness of the inhibition by the ATH of the Coagulation factors in the endothelial cells that cover the blood vessels (surface of artificial blood vessels). In vitro investigations of coagulation inhibition by ATH have been carried out basically in plasma or buffer solutions containing purified proteins. In vivo, the endothelial surface can modulate coagulation in various ways, such as facilitating sites for the binding of thrombin protein receptors that alter its activity (e.g., thrombomodulin (TM)), and expressing anticoagulant glycosaminoglycan (GAG) molecules. (for example, heparan sulfate (HS)). The objective of this study was to compare the anticoagulant activities of ATH and AT + H in the presence of endothelium.
Venous endothelial cells from human umbilical cords (HUVEC), EBM-2 media and EGM-2MV Bullet Kits were purchased from Lonza (Walkersville, MD, USA). An Essential Minimum Medium (MEM) was acquired from Invitrogen. Ila and Xa were from Enzima Research Laboratories (50uth Bend, IN, USA). The batches of normal human plasma and purified human Al came from Affinity Biologicals (Ancaster, ON, CA). Heparin, hexadimethrin bromide (polybrene), heparan sulfate and gelatin were purchased from Sigma (Mississauga, ON, CA). Fibrinogen (plasminogen, fibronectin, without FXIII) and human recombinant thrombomodulin came from American Diagnostica Inc. (Stamford, CT, USA). 5-2238 and 5-2222 came from Diapharma (West Chester, OH, USA). AlH was produced in the manner described above. The remaining reagents consisted of commercial reagents.
HUVEC cells were cultured on plastic treated with a tissue culture (Primary, BO, Mississauga, ON, CA) coated with 2% gelatin. For the experiments, the cells were seeded in 96 well plates at a rate of 20000-40000 cells / mL and grown to confluence in an EBM-2 medium supplemented with the EGM-2MV Bullet kit, in a humidified air atmosphere - 5% CO2 Cells were used between passes 2 and 5.
The values of the second order constant (kú for the inhibition of the ATH conjugate and the inhibition of non-covalent AT + H of Ilay Xa at 37 "C were measured by a discontinuous test under pseudo-first order conditions (inhibitor ratio: enzyme = 10: 1) Ila, Xa, AT, UFH and ATH were diluted in a Minimum Essential Medium (MEM) containing

10mM of HEPES at pH 7.4 and 0.1% (w / v) of PEG3000 (MEMPH). Ellla or Xa were incubated with AT + H or ATH in wells of a 96-well plate containing unique confluent HUVEC plates. The single layers were washed with MEMPH before adding the reaction components. The molar proportions of H: AT in the non-covalent mixtures of AT + H were
23: 1 and 10: 1 for reactions with Ila and Xa respectively. It was previously observed that these H: AT ratios produced maximum values of k2 for inhibition in the absence of HUVEC. After incubation over various time intervals, the reactions were stopped by adding 1.25 mg / mL of polybenzene in a TSP regulatory solution (20mM Tris-CI, 150mM NaCI, 0.6% (p / v) PEG8000 pH 7.4) containing 0.4 mg / mL of the appropriate chromogenic substrate (8-2238 for Ila or 8-2222 for Xa). Residual enzyme activities in each well were measured as the change in A405 over time, using a SpectraMax Plus 384 spectrometer (Molecular Oevices, Sunnyvale, CA, USA). The values of k2 were calculated by taking the negative value of the slope of the traces of the residual enzyme activity against the time of inhibition prior to the addition of the polybrene / substrate and dividing this negative value of the slope by the inhibitor concentration (ie say, ATH or AT). Identical tests were also performed on a plastic surface (not HUVEC) for comparison, using flat-bottomed Falcon ProBind 96-well plates (BO, Franklin Lakes, NJ, USA).
In separate experiments, wells containing HUVEC monolayers were washed with MEMPH, and 20 µL of 2nM Ila (dissolved in MEMPH) was added to multiple wells. After 3 min incubation at 37 "C, 80 IlL of MEMPH containing 1 mg / mL of human fibrinogen and various concentrations of AT + H or ATH (01.25 ATH ATH or AT with an H ratio) were added simultaneously to all wells : 23: 1 AT.) The formation of fibrin clots at 37 "C was monitored by turbidimetry, measuring the value of 00350 by an 8pectraMax Plus 384 spectrometer. The delay time in clot formation was defined as the time that It took 00350 to reach a value of 0.005. If 0 0 350 did not reach this value of 0.005 after 90 min, a delay time of 90 min was assigned. Tests were also performed on plastic (not HUVEC) for comparison. In some plastic tests, 2nM of Ila was mixed with 10nM of thrombomodulin (TM) or 5 ~ M of heparan sulfate (H8) prior to the 3 min incubation stage
The results are expressed as the mean of ± 8EM. The analysis of statistical importance using Student's {-test, where p <0.05 was considered significant. The {-test of 8tudent was carried out using Minitab 13 for Windows.

To assess the effect of the endothelium on the inhibition of Ila and Xa catalyzed by heparin by non-covalent AT + H or ATH, the k2 values were determined for the inhibition rates of these proteases in the absence or presence of a HUVEC monolayer . FIGs. 1 A and 1 B show the rate of inhibition of coagulation factors by AT + H and ATH in the presence of endothelium. K2 values were determined for the inhibition of Ila (A) and Xa (8) by ATH and AT + H in wells with plastic surface, or in wells covered by a single layer of HUVEC. K2 values were measured under pseudo first order conditions, using a discontinuous method. The molar ratios of H: AT in the AT + H mixture were 23: 1 (A) and
10: 1 (B), which has been previously shown to offer the maximum values of k2 for the inhibition of Ila and Xa respectively. Data represent the mean of ± SEM (n2 :: S). In the absence of HUVEC, the k2 values for the inhibition of Ila and Xa by ATH were higher than the k2 values corresponding to the inhibition by non-covalent AT + H (FIGs. 1A and 18). The degree of increase was greater in the case of dellla, standing at a factor of 2.3. When endothelium was present, the inhibition of Ila and Xa by ATH was again faster than with AT + H (FIGs. 1A and 1B). In comparison with plastic, the absolute values of k2 for ATH and AT + H in endothelial cells decreased significantly (p <O, OS) in the case of lIa (FIG. TAl, Y were reduced slightly, but not significantly for Xa ( p> O, t) (FIG. 1 B).
Ila inhibition was further investigated by a fibrin formation assay, in which Ila was incubated in wells with or without a HUVEC monolayer, followed by the addition of fibrinogen and anticoagulants to the wells. Figure 2 shows the inhibition of fibrin formation induced by Ila by AT + H and ATH in the presence of endothelium. She was incubated in plastic or HUVEC coated wells, before adding a mixture of purified fibrinogen, CaCI2 and AT + H or ATH. The final concentration of Ila was 0.4nM. The final inhibitor concentrations (AT or ATH) are indicated on the abscissa axis, and the molar ratio of H: AT in the AT + H mixture was 23: 1. Fibrin formation was monitored by turbidimetry, and the delay time represents the time required to achieve a value of 00350 = 0.005. The data represent the mean ± SEM (n2 :: 5). Figure 2 shows that, unlike in the rate experiments, the presence of endothelium did not exert effects on the inhibition of Ila by the fibrinogen cleavage activity by ATH or AT + H. As in the case of rate experiments. ATH was more effective than AT + H at the time of preventing fibrin formation by it.

The two molecules that constitute the main binding points of factor lI on the endothelial surface are HS and TM. To determine whether Ila binding to any of these molecules has affected anticoagulant activity, soluble HS or TM were pre-incubated with lIa and fibrin formation was evaluated in the presence or absence of ATH or AT + H. HS did not affect Ila-induced fibrin formation (onset time = 97.3 ± 6.6 s versus 95.4 ± 6.4 s in the presence and absence of HS respectively, n = 9). The presence of TM significantly delayed fibrin formation with an onset time of 122.4 ± 1.7 s, compared with 97.9 ± 6.4 s in the absence of TM (p = O, 003, n <!: 8). Figures 3A and 38 show the inhibition of fibrin formation induced by Ila adhered in HS or TM. She was mixed with an excess of HS (A) or TM (B) before carrying out the fibrin formation tests in plastic wells, as described in Figure 2. The final concentrations of Ila, HS and TM were of O, 4nM, 1 ~ M and 4nM respectively. The final concentrations of the inhibitor (AT or ATH) are indicated on the X axis and the molar ratio of H: AT in the AT + H mixture was 23: 1. The data represent the mean ± SEM (n <!: 4). ATH maintained its superior anticoagulant function compared to AT + H in the presence of HS (FIG. 3A) and TM (FIG. 38).
Example 4
Washing and sealing catheters using an ATH solution. A solution of ATH in 0.15 M NaCI was prepared, with or without a buffer solution (such as HEPESO, 05 M) by setting the pH to 7.4. The concentration of ATH in this solution is around 1.5 U / mL in terms of the activity of anti-factor Xa, but in some cases it can range between 0.01 U / mL and 100 U / mL of anti activity -factor Xa. The ATH solution is sterilized by filtration through a sterile filter with 0.2 micrometer pores inside a laminar flow hood with sterile air flow. When still inside the sterile environment, the sterile filtered solution of ATH is sealed inside sterile bottles containing a partition for subsequent removal by sterile syringes immediately before use, or measured amounts are taken in sterile syringes which are then plugged.
After inserting a catheter into a patient's vein or artery, a syringe containing about 2 mL (in some cases, between 0.1 mL and 50 mL total volume) of the sterile ATH solution is connected, as described above, to the outer end of the catheter, and all the ATH solution of the syringe is injected into the catheter, followed by sealing the catheter with a sterile cap.

After a variable period of 1 hour to 7 days has elapsed, the cap can be removed and replaced with a syringe, so that a blood sample can be drawn or medication can be injected. After blood collection or medication injection, the syringe used to draw blood samples or administer the medication is replaced by
5 another syringe containing an ATH solution similar to the previous one, injecting the ATH solution and covering the catheter as in the previous case. This process is repeated whenever necessary, until the catheter is removed.
Example 5
10 Treatment of woody conjunctivitis. A solution of ATH in 0.15 M NaCI is prepared, with or without a regulator (such as 0.05 M HEPES) with a pH of 7.4. The concentration of ATH in this solution would normally be around 1 milligram / mL (in terms of AT content) but it could range between 0.001 milligrams / mL and 11 milligrams / mL. ATH's solution is sterilized
15 by filtering through a sterile filter with 0.2 micrometer pores inside a laminar flow hood with sterile air flow.
A patient requiring treatment to prevent the appearance of woody conjunctivitis on tissue surfaces outside the vascular system regularly receives aliquots of a solution containing ATH. In the case of woody conjunctivitis of the
20 eye, 1 or more drops (approximate volume of the drop between 10 microliters and 100 microliters) of the ATH solution described above are applied one or more times a day, depending on the severity of the specific patient's disease. This process is repeated continuously throughout the patient's life.
25 Example 6
The most recent preliminary data suggest that ATH exerts an interesting differential effect on epithelial growth versus that of in vitro fibroblast that is opposite to the impact exerted by AT (see Table 2 below). Extensive research
30 performed on the immobilized ATH in polyurethane catheters and endoluminal grafts have shown that the ATH conjugate exhibited significantly greater inhibitory activity against thrombin in vitro and anticoagulant activity in vivo. This results

suggest that ATH adhered to alveolar matrices may help control the generation of excess thrombin and the accumulation of fibroblasts in relation to other cells.
Table 2. Effect of AT and ATH on lung cell growth in MEM media
Percentage change in the number of cells with respect to the Control solution
Treatment Fibroblast epithelial cells lung lung cells
500 nM AT -19.3 ± 3.3 -0.141 ± 1.76 500 nM ATH 29.1 ± 13.6 "13.9 ± 4.16"
Primary lung cell cultures of newborn rats were prepared in 24 wells of a well plate for 1 day in a humid atmosphere with 5% 10 CO2 / 95% air at 37 ° C in a MEM medium supplemented with antithrombin ( AT) or covalent ATheparin (ATH). The monolayer cells were then released using trypsin, and the total number of cells was determined using a hemocytometer. The results are expressed as the mean value ± SEM (n = 5) of the percentage change in the number of cells with respect to control cultures in a non-supplemented MEM medium. Some are indicated
15 important differences in the results with ATH versus those obtained with AT in the same group of r cells, and ••• mean P = 0.002, P = 0.026 and P = 0.027, respectively). Although AT alone significantly inhibited the proliferation of epithelial cells, but not fibroblasts, ATH preferentially improved epithelial growth against fibroblasts.
20 Example 7
Investigation of treatment with ATH of lungs suffering from stress / acute injuries. Preliminary experiments were performed using ATH intratracheally in newborn animals that randomly breathed normally or were subjected to a high volume
25 (1 h) of mechanical ventilation. Figure 4 shows the coagulation times of recalcified plasma samples from citrated blood from rats 2 to 6 days old treated intrapulmonally with ATH or a PBS regulatory solution, with or without a high

mechanical ventilation volume for 1 hour. Coagulation tests reliably demonstrated that with respect to control solutions instilled with a regulator, ATH eliminated the reduced plasma coagulation time derived from the systemic activity of tissue factor (TF) associated with ventilation (see Figure 4). . Given the obvious generation of pulmonary thrombin and the formation of fibrin related to FT, direct neutralization of the Vlla-FT key complex by the ATH conjugate would block the activation of coagulation in the lung, where plasma inhibitors will be scarce.
Example 8
Experiments were carried out to determine the effect of ATH on the production of surfactants in lung cells to measure the potential for instillation of ATH in the lung (to prevent SOR or lung damage caused by mechanical ventilation) in order to increase surfactant levels and prevent airspace collapse and improve gas transfer. The following preliminary data were collected (see Figure 5). Fetal distal pulmonary epithelium (FOLE) was isolated from 20-day fetal rats and cultured in 24 wells of a plate of culture wells. The culture medium was supplemented with 100 g / mL of ATH or with a control buffer solution. Figure 5 shows immunoblotting of cellular media produced by fetal distal pulmonary epithelial cells of rats (FDLE) incubated with a buffer solution or with ATH (100 µg / mL culture medium), in which the surfactant protein C was detected. Immunoblotting of cell media showed an increase in surfactant protein C produced by FOLE cells incubated with ATH (100 µg / mL of culture medium).
Example 9
ATH: Anticoagulant Fibrin for the treatment of a clot. The following is the proposed methodology for the preparation of ATH linked to a fibrin handle that can be injected to treat a clot in a patient, and make the surface of the clot acquire net anticoagulant properties. The use of ATH: Fibrin can be a very effective coagulation neutralizing agent, which can be used at low concentrations compared to the injection of ATH only, since the ATH: Fibrin complex will tend to bind to other fibrins present in an important concentration , as

in the case of a clot containing polymerized fibrin. Unlike the ATH already adhered to the fibrin, the free ATH would end up reaching other goals, in addition to the fibrin clot and therefore, may need to be administered at much higher concentrations. A method for the preparation of ATH adhered to the Fibrin monomer and its administration to a patient who has a clot is indicated below.
A stock solution of fibrin monomer in acetic acid is prepared, as follows. Commercially available fibrinogen is used (Enzyme Research Laboratories, South Bend, IN, USA). In the previous experiments, the following protocol was followed to prepare soluble fibrin solutions. Initially, any remaining fibronectin contaminant from commercial fibrinogen was removed by 2 incubations of 15 mL of 130 11M fibrinogen (molecular molecular mass 340000 Da) with 5 mL gelatin agarose (Sigma, Mississauga, Ontario, Canada) for 30 min, followed by centrifugation and collection of the supernatant containing the fibrinogen. Fibrinogen concentration was determined by absorbance at 280 nm using an absorbency of 10 mg / mL = 15.1 (after correction by light scattering at 320 nm using the corrected equation A280 = A280 -1.7 x ~ 2 {l ). The soluble fibrin monomer was prepared by the following method. The purified fibrinogen (60 -100 ~ M) was incubated with 2 nM thrombin (Enzyme Research Laboratories, South Bend, IN, USA) at 37 ° C for 4-6 hours, followed by centrifugation at 2000 g for 5 min. The fibrin polymer pellet was placed in a dialysis bag (12,000-14,000 molecular weight cut), dialyzed against H20 (4 "C) to remove fibrinopeptides A and BY was dialyzed against acetic acid 0, 02 M until fibrin dissolved (-8 hours) The concentration of soluble fibrin in the solution was obtained by absorbency at 280 nm and using a molecular weight of 340000 and absorbency of 10 mg / mL = 14.0. 100 11M of soluble fibrin were obtained and stored at -80 ° C.
For the preparation of fibrin solutions for binding with ATH, 6 volumes of the soluble fibrin were neutralized in 0.02 M acetic acid with 4 volumes of 1 M Tris-HCI with pH 7.5 containing 10 mM GPRP- NH2 (Sigma, Mississauga, Ontario, Canada) to block the polymerization of fibrin under neutral conditions. The resulting neutralized soluble fibrin monomer of 40,000 nM could be further diluted or maintained at that concentration before combining it with ATH. Although solutions with various molar proportions of fibrin can be prepared: ATH, ATH can be added to the fibrin (at a pH around 7.5 in the presence of GPRP-NH2) at approximately equal molar concentrations to a slight molar excess (in around 10%) compared to

fibrin. In a typical experiment, 1 volume of 40,000 nM of ATH was mixed rapidly in 0.02 M Tris-HCI 0.15 M Glical of 0.6% palietilena 8000 with a pH of 7.4 (TSP) containing GPRP - 0.01 M NH2 with an equivalent volume of neutralized fibrin solution of 40,000 nM. The resulting mixture, designated as solution 1 of ATH: Fibrin contains 20,000
5 nM ATH + 20,000 nM soluble fibrin monomer.
The solutions of ATH + fibrin can be injected intravenously in patients with clots, to neutralize the procoagulant activity of the clot, so that the organism's own fibrinolytic system itself can successfully digest the clot to eliminate it. The dose of ATH product: Fibrin to be delivered to the patient can vary widely
Depending on the degree of thrombosis or coagulatory activity or size of the patient's clot. Using the ATH: Fibrin solution described above, any amount between 0.01 mUkg of body weight and 10 mLlkg of body weight can be administered by intravenous injection, the most common being 0.5 mUkg of body weight.
Other options for preparing ATH + fibrin monomer solutions include the
15 rapid dissolution of 100 ~ M fibrin in 0.02 M acetic acid with 0.02 M Tris-Hel 0.15 M NaCl 0.6% polyethylene glycol 8000 at a pH of 7.4 (TSP) up to concentration of <100 nM, where the fibrin will not polymerize too quickly. An equivalent volume of ATH at the same concentration will then be rapidly mixed in the diluted fibrin solution and the resulting mixture will be injected at varying doses between 0.1 mLlkg of weight
20 body up to 10 mLlkg of body weight, the most common being 2 mUkg of body weight. It is to be understood that the above configurations are intended to illustrate the application of the principles of the present invention. Thus, although the present invention has been described above in relation to the embodiments, it will be apparent to any person skilled in the art that numerous modifications and configurations can be made.
25 alternatives without departing from the principles and concepts of the invention, as set forth in the claims.

权利要求:
Claims (19)
[1]
1. A method of coating a polymeric surface with an antithrombin-heparin conjugate, which comprises bringing the polymeric surface into contact with a
5 antithrombin-heparin conjugate solution so that the antithrombinheparin conjugate directly coats the polymeric surface without binding groups between the antithrombin-heparin conjugate and the polymeric surface.
[2]
2. The method according to claim 1, wherein the polymeric surface is selected
10 between the members of the group formed by a polyurethane surface, a polyethylene surface, a polypropylene surface, a polytetrafluoride surface, a polydimethylsiloxane surface, an ethylene-acrylic acid copolymer surface, a Dacron surface, a surface polyester-polyurethane, a polyurethane-polycarbonate surface, a polyvinyl chloride surface, a silicone surface, a surface of
15 polydimethylsiloxane, a stainless steel surface, a titanium surface, a latex rubber surface, a nitinol surface, a Nylon surface, a polyethylene terephthalate surface, a polystyrene surface, and combinations of such products.
[3]
3. The method according to claim 1, wherein the polymeric surface is a polyurethane surface 20.
[4]
4. The method according to claim 1, wherein the polymeric surface is a surface of a medical device selected from among the members of the group consisting of an intravenous catheter, an intra-arterial catheter, a peripherally inserted central catheter, a catheter
25 central, a Swan-Ganz catheter, a coronary stent, an arteriovenous shunt, a mechanical valve, an artificial organ, a dialysis catheter, a dialysis blood circuit line, a dialysis membrane, an extracorporeal oxygenation line of the membrane, an extracorporeal membrane oxygenation membrane, and a blood storage container.
[5]
5. The method according to claim 1, wherein the polymeric surface is an inner surface of a lumen of a catheter, and wherein the contacting of the polymeric surface with

The antithrombin-heparin conjugate solution comprises inserting the solution into the lumen of the catheter and incubating the catheter with the solution for a period of time.
[6]
6. The method according to claim 5, wherein the period of time varies between 5 minutes and 48 hours.
[7]
7. The method according to claim 5, further comprising emptying the solution after incubation and evaporating the solvent from the residual solution contained in the lumen.
[8]
8. The method according to claim 1, wherein the polymeric surface is an outer surface of a catheter, and wherein the contacting of the polymeric surface with the antithrombin-heparin conjugate solution comprises immersion of the catheter in the solution and incubation of the catheter with the solution for a period of time.
[9]
9. The method according to claim 8, wherein the period of time ranges from 5 minutes to 48 hours.
[10]
10. The method according to claim 8, further comprising emptying the solution after incubation and evaporating the solvent from the residual solution contained in the catheter.
[11]
eleven. A medical device of reduced thrombogenicity, said medical product comprising a polymeric surface coated with an antithrombin-heparin conjugate without binding groups between the antithrombin-heparin conjugate and the polymeric surface.
[12]
12. The medical device according to claim 11, wherein the polymeric surface is a polyurethane surface.
[13]
13. The medical device according to claim 11, wherein the medical device is selected from the members of the group consisting of an intravenous catheter, an intraarterial catheter, a peripherally inserted central catheter, a central catheter, a SwanGanz catheter, a coronary stent, a shunt arteriovenous, a mechanical valve, an artificial organ, a

dialysis catheter, a dialysis blood circuit line, a dialysis membrane, an extracorporeal membrane oxygenation line, an extracorporeal membrane oxygenation membrane, an in vivo prosthesis and a blood storage container.
5 14. A method of coating by lyophilization of a polymeric surface of amedical device, which includes:
The contact of the polymeric surface of the medical device with an antithrombin-heparin solution comprising an antithrombin-heparin conjugate and a solvent in the absence of binding groups;
10 Allow the excess of antithrombin-heparin solution to be emptied from the polymer surface; and Evaporate the polymer surface solvent at least under partial vacuum.
[15]
15. A method of treating a medical condition by inhibiting the
15 thrombogenesis in a mammal, said method comprising: administration of a dose of an antithrombin-heparin conjugate to the mammal, where at least 98% of the heparin chains of the antithrombin-heparin conjugate have a molecular weight of more than 3,000 daltons.
The method according to claim 15, wherein the medical condition is respiratory distress syndrome and wherein the administration of the dose of the antithrombinheparin conjugate comprises the administration of the antithrombin-heparin conjugate in a mammalian lung.
[17]
17. A method for the treatment of woody conjunctivitis in a mammal, which
25 comprises the administration of a dose of an antithrombin-heparin conjugate in an eye of the mammal.
[18]
18. A method to prevent or treat injuries caused by mechanical ventilation in
mammals, which comprises the administration of a dose of a heparin antithrombin30 conjugate in a mammalian lung.

[19]
19. A method for the treatment of woody gingivitis in a mammal, comprising the administration of a dose of an antithrombin-heparin conjugate in the gums of the mammal.
5 20. A wash and seal solution of an intravenous or intra-arterial catheter, whichIt comprises an antithrombin-heparin conjugate.
[21]
twenty-one. A washing and sealing system, comprising: An intravenous or intra-arterial catheter; 10 A cleaning and sealing solution configured to be introduced through the catheter,
where the cleaning and sealing solution comprises an antithrombin-heparin conjugate; and A syringe configured to inject the cleaning and sealing solution into the catheter.
[22]
22 A method for maintaining the permeability of a catheter, which comprises:
The insertion of a catheter into a vein or artery of a patient so that an inner opening of the catheter opens inside the vein or artery and an outer opening of the catheter opens outside the patient; The injection of a solution comprising an antithrombin-heparin conjugate into
20 the catheter through the outer opening of the catheter;
The sealing of the outer opening of the catheter so that at least a portion of the solution comprising the antithrombin-heparin conjugate remains inside the catheter.
23. A composition for the treatment of blood clots, comprising antithrombin, heparin, and fibrin, where at least 50% by weight of heparin is conjugated with antithrombin to obtain an antithrombin-heparin conjugate, and where at At least a portion of the fibrin adheres to the antithrombin-heparin conjugate.
24. The composition according to claim 23, wherein at least 50% by weight of the fibrin adheres to the antithrombin-heparin conjugate.

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同族专利:

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公开号 | 公开日

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AU2016274868B2|2021-02-18|

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GB201800471D0|2018-02-28|

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ES2671844B1|2019-03-22|

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WO2016201202A1|2016-12-15|

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AU2016274868A1|2018-02-01|

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CA2989110A1|2016-12-15|

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GB2556531A|2018-05-30|

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JP2018524067A|2018-08-30|

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US20180177924A1|2018-06-28|

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优先权:

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申请号 | 申请日 | 专利标题

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US201562174308P| true| 2015-06-11|2015-06-11|

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US62/174,308|2015-06-11|

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PCT/US2016/036855|WO2016201202A1|2015-06-11|2016-06-10|Medical devices, systems, and methods utilizing antithrombin-heparin compositions|

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