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    • 专利摘要:
    Compositions and methods for preventing thrombogenesis can include antithrombin and heparin. In one example, a conjugate of antithrombin and heparin where at least 50 wt% of the heparin is conjugated to antithrombin can be present. Furthermore, in one example, at least 98 wt% of the heparin in the composition has a molecular weight greater than 3,000 Daltons.
    公开号:ES2710313A2
    申请号:ES201790037
    申请日:2016-04-15
    公开日:2019-04-24
    发明作者:Leslie Roy Berry；Attilio Difiore；Anthony Kam Chuen Chan
    申请人:Attwill Medical Solutions Inc；
    IPC主号:
    专利说明:

    [0001]
    [0002] Composition for the prevention of thrombogenesis and procedure for its manufacture.
    [0003]
    [0004] Background
    [0005]
    [0006] Heparin is a polysulphated saccharide consisting largely of an alternating sequence of hexonic uronic acid and 2-amino-2-deoxy-D-glucose. Heparin and a related compound, dermatan sulfate, are of great importance as anticoagulants for clinical use, for the prevention of thrombosis and related diseases. Both are members of the glycosiaminoglycan family, (GAGs), which consist of linear chains of repeating units of sulfated disaccharides containing a hexosamine and a uronic acid. Anticoagulation through GAGs (such as heparin and dermatan sulfate) takes place through its catalysis of inhibition of coagulant enzymes (the most important being thrombin) by serine protease inhibitors (serpins) such as antithrombin III (to which in the present document it is denominated simply "antithrombin" or "AT") and the cofactor II of the heparin (HCII). The union of the serpins by the catalysts is fundamental for its action, and takes place by means of sequences The heparin acts by binding to the AT through a sequence of pentasaccharides, thus potentiating the inhibition of various coagulant enzymes (in the case of thrombin, the heparin also binds to the enzyme.) Heparin can also potentiate thrombin inhibition by binding to the HCII serpin.The dermatan sulfate acts by specifically joining to the HCII through of a hexasaccharide sequence, thus potentiating only thrombin inhibition. Since the glycosiaminoglycans (and specifically, heparin) can bind to other molecules in vivo or be lost from their site of action because of various mechanisms, it would be advantageous to keep the GAG permanently associated with the serpin by a covalent bond. In a more detailed manner, it would be desirable to provide covalent conjugates of heparin or other related glycosiaminoglycans that maintain a high biological activity (for example, their anticoagulant activity) and better pharmacokinetic properties, and whose preparation procedure is simple.
    [0007] 30
    [0008] Brief description of the figures
    [0009]
    [0010] Figure 1 is a graph showing the absorption (A215 vs. H2O) of each heparin fraction eluted from a Sephadex® G-200 chromatography column,
    [0011] Figure 2 is a graph showing the absorption of the fractions of AT conjugated with heparin and the fractions of AT alone eluted from a column of chromatography Sephadex® G-200,
    [0012] Figure 3 is a graph showing the absorption (A405 vs. H2O) of reaction mixtures of four different reactions with which a covalent antithrombin-heparin complex was investigated.
    [0013] Figure 4 is a graph showing the absorption (A405 vs. H2O) of reaction mixtures of three different reactions with which a covalent antithrombin-heparin complex was investigated and made lyophilized from a solution of high salinity (concentrated ).
    [0014] It should be emphasized that the figures are mere examples of various embodiments, and that they do not intend to introduce any type of limitation within the scope of the present invention.
    [0015]
    [0016] DETAILED DESCRIPTION
    [0017]
    [0018] Next, reference will be made to a series of realization examples, and a specific language will be used for the description thereof. However, it should be understood that this does not intend to introduce any limitation to the scope of what is disclosed in this document. The alterations and subsequent modifications of the characteristics of the invention described in this document, as well as the additional applications of the principles of the technology described therein, that would occur to any expert in the subject that was in possession of what is disclosed herein, must be considered included in the scope of the disclosed. Also, before disclosing and describing specific embodiments, it is to be understood that what is disclosed herein is not limited to the process and specific materials disclosed herein, as they may vary to some extent. It should also be understood that the terminology used herein is used for the exclusive purpose of the specific embodiments, and is not intended to be limiting, since the scope of what is disclosed herein will be defined exclusively by the appended claims and their equivalents. .
    [0019] When describing and claiming the present technology, the following terminology will be used.
    [0020] 35 The singular forms "a,""an," and "the" include references to the plural, unless the context clearly indicates otherwise, in this way, for example, the reference to "a additive "includes reference to one or more of said components," a solution "includes reference to one or more of said materials, and a" mixing step "refers to one or more of said steps.
    [0021] As used herein, "substantial", when used in reference to an amount of a material, or a specific characteristic thereof, refers to an amount that is sufficient to provide the effect that said material or characteristic The exact degree of permissible deviation may depend in certain cases on the specific context.
    [0022] As used in this document, "around" refers to a degree of deviation based on the experimental error of the identified specific property.The degree of amplitude of the term "around" will depend on the context and of specific property, and may be easily identified by any expert in the field. The term "around" does not intend to extend or limit the degree of equivalents that could be attributed to a specific value, and, unless otherwise indicated, the term "in 15 around" will expressly include "exactly," in line with the observations made later in relation to the ranges and numerical data.
    [0023] The concentrations, dimensions, quantities and other numerical data can be presented in this document in rank format. It is to be understood that said range format is used exclusively for convenience and should be interpreted flexibly, so as to include not only the numerical values explicitly indicated as range limits, but all the individual numerical values or sub-ranges included within the range. range, as if each of said numerical values and sub-ranges was explicitly mentioned. For example, a range between 1 and around 200 should be interpreted so that it not only includes the explicitly stated limits of 1 and 200, 25 but also includes individual sizes such as 2, 3, 4, and sub-ranges as 10 to 50, from 20 to 100, etc.
    [0024] As used herein, a plurality of elements, structural elements, compositional elements and / or materials may be presented for convenience in a common list. However, such lists should be interpreted as if each item on the list was individually identified as an independent and unique number. Thus, none of the individual elements of said list should be interpreted as a de facto equivalent to any other element of the same list, based exclusively on its presentation in a common group, unless otherwise indicated.
    [0025] As used herein, "hexose" refers to a carbohydrate (C6H12O6) containing six carbon atoms.The hexoses may be aldohexoses such as, for example, glucose, mannose, galactose, idosa, gulose, talose, alose and altrose, whose open chain form contains an aldehyde group. Alternatively, hexoses can be ketoses such as fructose, sorbose, alulose and tagatose, whose open chain form contains a ketone group.
    [0026] As used herein, "uronic acid" refers to the carboxylic acid formed by the oxidation of the primary hydroxyl group of a carbohydrate, and they usually take their name from the carbohydrate from which they are obtained. Therefore, the oxidation of the C6 hydroxyl group of the glucose gives rise to the glucuronic acid, the oxidation of the C6 hydroxyl group of the galactose gives rise to the galacturonic acid and the oxidation of the C6 hydroxyl group of the idose gives rise to the iduronic acid.
    [0027] As used herein, "hexosamine" refers to a hexose derivative in which at least one hydroxyl group, typically the C2 hydroxyl group, has been replaced by an amine. The amine may be optionally alkylated, acylated (for example, with muriate acid), typically through an acetyl, sulphonated, sulphonated , sulfonylated, phosphorylated, or phosphorylated (O or N- 15 ) acetyl group and the like. Representative examples of the hexosamines include glucosamine, galactosamine, tagatosamine, fructosamine, their modified analogs and the like.
    [0028] As used herein, "glycosiaminoglycan" refers to linear chains of long-repeating disaccharide units that contain a hexosamine and a uronic acid. The precise identity of hexosamine and uronic acid can vary widely, and representative examples of each are given in the above definitions. The disaccharide can optionally be modified by alkylation, acylation, sulfonation (O- or N-sulfated), sulfonylation, phosphorylation, phosphonylation and the like. The degree of said modification may vary, and may be carried out in a hydroxyl group or in an amino group. In general, the C6 hydroxyl group and the C2 amine are sulfated. The length of the chain may vary and the glycosaminoglycan may have a molecular weight greater than 200,000 Daltons, typically up to 100,000 Daltons, and more usually, less than 50,000 Daltons. Glycosiaminoglycans are usually found as mucopolysaccharides. Among its most representative examples are heparin, dermatan sulfate, heparan sulfate, chondroitin-6-sulfate, chondroitin-4-sulfate, keratan sulfate, chondroitin, hyaluronic acid, polymers containing N-acetyl monosaccharides. (such as N-acetylneuramic 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.
    [0029] As used herein, "protein" includes, among others, albumins, globulins (such as immunoglobulins), histones, lectins, protamines, prolamines, glutelins, phospholipases, antibiotic and scleroprotein proteins, as well as conjugated proteins such as phosphoproteins, chromoprotemes, lipoproteins, glycoproteins and nucleoprotemes.
    [0030] As used herein, "serpin" refers to an inhibitor of serine protease and among its examples are species such as antithrombin and cofactor II of heparin.
    [0031] As used herein, "amine" refers to primary amines, RNH2, secondary amines, RNH (R '), and tertiary amines, RN (R') (R ").
    [0032] As used herein, "amino" refers to the group NH or NH2.
    [0033] As used herein, "imine" refers to the group C = N and the salts thereof.
    [0034] As used herein, the terms "treat" or "treatment" of a disease or a condition in a mammal means: preventing the disease or disease, that is, avoiding all types of clinical symptoms of the disease ; inhibit the ailment or disease, that is, stop the development or advancement of the classical symptoms; and / or alleviate the ailment or disease, that is, cause the regression of the classical symptoms. Treatment also includes the use of the compositions disclosed in this document associated with a medical procedure, administered before, during or after the medical procedure.
    [0035] Accordingly, the present document relates to compositions and methods for the preparation of heparin and antithrombin conjugates, as well as to the treatment of patients with the compositions herein. In one example, a composition for preventing thrombogenesis can comprise antithrombin and heparin, where at least 50% by weight of the heparin is conjugated to antithrombin, and where at least 98% by weight of the heparin of the composition has a molecular weight greater than 3,000 Daltons.
    [0036] In another example, a method for the manufacture of a composition for preventing thrombogenesis may comprise the steps of conjugating antithrombin with heparin outside the body of the patient to form an antithrombin-heparin conjugate;
    [0037] the preparation of the antithrombin-heparin conjugate in a solution; and lyophilization of the antithrombin-heparin conjugate. In this example, the antithrombinheparin conjugate can be found in a solution formed solely by water, by water and alanine 0.01-0.09 molar, or by water and mannitol, for example.
    [0038] In another example, a composition for preventing thrombogenesis can include an aqueous solution of antithrombin-heparin conjugate, wherein the conjugate of Antithrombin-heparin is present at a concentration of 9-11 mg / mL with respect to the total volume of the solution. The antithrombin-heparin conjugate can be formed by conjugating the antithrombin with heparin outside the patient's body.
    [0039] In a further example, a method for the manufacture of a composition for preventing thrombogenesis can include antithrombin with heparin outside the body of a patient, to form an antithrombin-heparin conjugate, where the performance of the antithrombin-heparin conjugate is defined such that at least 60% by weight of the initial antithrombin is reacted and conjugated with heparin (ie, it is used to obtain the antithrombin-heparin conjugate).
    [0040] In yet another example, a composition for preventing thrombogenesis can include antithrombin, heparin and antithrombin-heparin conjugate, where the antithrombin-heparin conjugate is present with a yield of at least 60% by weight of the initial antithrombin used. to obtain the antithrombin-heparin conjugate.
    [0041] In another example, a method for the treatment of a disease or disease may include the administration of an antithrombin-heparin conjugate prepared according to the examples of the technology of the invention to a mammal in need thereof. In more detail, these treatments can be carried out by administering the heparin and antithrombin conjugates of the present invention to a patient, for example, human, who requires said treatment. Among the ailments and diseases that can be treated using the conjugate compositions described herein are myocardial infarction and a wide variety of thrombotic conditions. These include the formation of fibrin deposits associated with the syndrome of neonatal respiratory distress syndrome, respiratory distress syndrome in adults, primary lung carcinoma, non-Hodgkin's lymphoma, fibrosing alveolitis and lung transplants, to mention only a few. Also, situations of acquired deficiency of AT, such as neonatal respiratory distress syndrome, L-asparaginase-induced deficiency, deficiency induced by cardiopulmonary bypass, sepsis or situations of congenital deficiency of AT can be treated with the compositions of the present invention. In the case of congenital AT deficiency, potentially fatal thrombotic complications can occur , with AT levels below 0.25 units / ml in heterozygotes requiring AT plus heparin in up to 1 or 2 children each year in the USA. . The ailments and diseases treated by the present invention include those that are characterized by an excess of thrombin generation or activity. These situations usually occur when a patient has been exposed to trauma, for example in the case of 35 surgical patients. The trauma caused by the injuries or the surgery causes vascular damages and the secondary activation of the coagulation of the blood. These effects do not The desired results may occur after a general or orthopedic surgery, gynecological, cardiac or vascular surgery, or other surgical interventions. An excess of thrombin can also complicate the evolution of natural diseases, such as arteriosclerosis, which can lead to heart attacks, strokes or gangrene of the limbs. Therefore, the procedures and compositions of current therapy can be used to treat, prevent or inhibit a series of important cardiovascular complications, including unstable angina, acute myocardial infarction (heart attack), cerebrovascular accidents (effusion). cerebral), pulmonary embolism, deep vein thrombosis, arterial thrombosis, etc. The compositions and methods of the technology can be used to reduce or prevent coagulation during dialysis and reduce or prevent intravascular coagulation during an open heart surgery. Additionally, in some aspects of the invention, methods and compositions are provided to prevent or inhibit the generation of thrombin or its activity in patients with an increased risk of developing thrombi due to clinical situations that alter hemostasis (eg, coronary artery disease). , arteriosclerosis, etc.). In another aspect, procedures and compositions are provided for those patients who have a higher risk of developing a thrombus after medical treatment, such as cardiac surgery, vascular surgery or percutaneous coronary interventions. In one embodiment, the methods and compositions of the present invention are used in cardiopulmonary bypass operations. The compositions can be administered before, during or after the intervention.
    [0042] Turning now to the various embodiments and details related to the present invention, it is known that heparin is readily available in a non-fractionated 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, the heparin molecules with a molecular weight of less than 5,000 Daltons can be eliminated in the majority or in their entirety.
    [0043] The antithrombin-heparin conjugates formed using heparin from which the low molecular weight heparin molecules have been removed are different in their composition from other antithrombin-heparin conjugates. The low molecular weight heparin chains can be removed from heparin prior to their reaction with AT to synthesize the antithrombin-heparin conjugate (ATH). Therefore, ATH remains devoid of low molecular weight heparin chains conjugated with AT.
    [0044] The low molecular weight heparin chains can be removed from commercially available heparin before reacting heparin with AT to obtain ATH. This produces an ATH whose composition differs from that of the ATH formed from unfractionated heparin without removing the low molecular weight heparin before reacting with the AT. Additionally, the formation of ATH from unfractionated heparin and the subsequent elimination of the ATH of low molecular weight does not produce the same product as the ATH of the present invention. Without adhering to any particular 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 to be conjugated with 10 AT. Very low molecular weight heparin chains have a high proportion of terminal aldose that can react with AT. Therefore, the very low molecular weight heparin chains tend to conjugate with the AT faster, displacing the heparin chains with a higher molecular weight. However, once the very low molecular weight heparin chains bind to the AT, the chains do not contain sufficient positions or length for the thrombin and factor Xa linkages, an enzyme that participates in the cascade of the coagulation. The inhibitory activity against factor Xa and thrombin dramatically decreases in the lag of the heparin molecules of lower molecular weights. Thus, ATH formed from these very low molecular weight heparin chains have essentially zero activity to prevent thrombogenesis. Although commercial heparin contains a relatively small percentage of heparin chains below 5,000 Daltons, these very low molecular weight heparin chains have such high reactivity with AT that a significant amount of the ATH formed contains very low heparin chains. molecular weight.
    [0045] 25 If heparin of very low molecular weight is not removed first prior to conjugation, a higher proportion of reactive terminals of this population, in comparison with that of higher molecular weight heparin, will tend to displace the others Heparin molecules in various measures throughout the molecular weight spectrum (since the proportion of aldose terminals varies continuously throughout the molecular weight range of heparin). This can have adverse effects on the final ATH. First, the ATH will contain a large population of ATH molecules that contain very small heparin chains with no activity. Second, the remaining ATH molecules (apart from this range of very low molecular weight ATH) will contain a heparin population with a reduced proportion of heparin chains with discrete molecular weight ranges with fewer terminal aldoses to compete with inactive low molecular weight heparin chains.
    [0046] This low heparin in aldose tends to be found in much longer chains, but is not entirely defined by a direct relationship between the length of the heparin chain and the aldose terminals required for its binding to the AT.
    [0047] In addition, heparin with at least 18 units of monosaccharides may also be more effective at inhibiting thrombin. At least 18 monosaccharide units are used to bind antithrombin and thrombin. The mechanism by which heparin binds to antithrombin and thrombin is called a template or bridge mechanism. Heparin can exert its effect through conformational activation by binding to the AT and the alosteric conversion of the AT in a structural form that is much more reactive to the coagulation proteases. Alternatively, heparin can act as a template through its linkage to the inhibitor and to the enzyme, thereby locating the molecules for the reaction. In this mechanism, the activation of conformation of the AT is produced by heparin, but the reaction rate is improved by means of simultaneous heparin bonds to the enzyme, thus collaborating in the approximation of the coagulation factor towards the activated inhibitor. The specific minimum chain length of 18 monosaccharides may explain why there is a very abrupt fall in activity against thrombin within the fraction of low molecular weight heparin. Starting from the structure corresponding to a monosulphated urine acid-bisulfated disaccharide of glucosamine heparin, that is, without sodium 20 or other of the ions found in a salt, the molecular weight of a chain of 18 saccharides (9 disaccharides) would be around 4500 Daltons.
    [0048] Heparin chains with a somewhat lower molecular weight may be useful in the inhibition of factor Xa. A specific pentasaccharide sequence of heparin can bind with AT and activate AT for the inhibition of factor Xa. This specific sequence of pentasaccharide is available on its own as the drug
    [0049] "Fondaparinux," but the sequence can also occur in heparin chains. The sequence of monosaccharides is shown in formula I:
    [0050]
    [0051]
    [0052] Thus, heparin chains with less than 18 monosaccharides containing this pentasaccharide sequence may be able to activate AT to inhibit factor Xa even when the chains are not long enough to bind AT and thrombin.
    [0053] The longer heparin chains may in some cases have the highest inhibitory activity. However, some heparin chains of 10 medium and lower molecular weight can bind significantly less undesirably to other plasma and platelet proteins. Therefore, these mid-range heparin chains can be more selective in inhibiting thrombin and factor Xa without causing unwanted side effects, such as the dysfunction of platelets that stop binding to platelets and bind to other materials.
    [0054] The isolation of ATH with higher molecular weight after conjugation, to obtain a very long chain ATH provides a different and less desirable product compared to the technology of the present invention, which separates (substantially or in its entirety) heparin with before the conjugation. For example, the proportion of molecules of high activity with 2-pentasaccharides in this subpopulation can be altered due to a different capacity of these high activity chains to compete for conjugation with the very low molecular weight heparins. In addition, the isolation of ATH of high molecular weight after conjugation eliminates ATH molecules with heparin chains of medium range and smaller size, which are also active and have other desirable characteristics, such as the reduction of non-selective bonds with plasma proteins and platelets.
    [0055] Alternatively, it is likely that attempts to react all heparin chains with terminal aldoses with AT by increasing the ratio of AT to heparin in the reaction mixture will not be successful, because many experiments have demonstrated that only a maximum conversion of AT in ATH of up to 60% by weight is obtained even when the aldose contains heparin in the form of multiples and at the highest practical concentrations. The additional reduction in the proportion of heparin against AT will only further reduce the performance of the ATH without any guarantee 5 that all of the longer active chains will be incorporated into the product.
    [0056] In some embodiments, a composition for the prevention of thrombogenesis may contain ATH formed from commercial heparin from which substantially all heparin chains with a molecular weight of less than 3,000 Daltons (eg, at least 98%) have been removed. by weight of the rest of the heparin chains 10 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 eliminated or substantially eliminated. Thus, the ATH product may contain heparin chains whose molecular weight varies from 3,000 Daltons (or 5,000 Daltons) to the highest molecular weights that commercial heparin contains. In some examples, this range of molecular weights may range between 3,000 Daltons and 50,000 Daltons, or between 5,000 Daltons and 50,000 Daltons. In additional examples, at least a portion of the heparin chains may be within the average range of molecular weights. For example, at least a portion of the heparin chains of the ATH can have a molecular weight ranging from 3,000 Daltons to 30,000 Daltons, between 3,000 Daltons and 20,000 20 Daltons, between 3,000 Daltons and 15,000 Daltons, between 3,000 Daltons and 10,000 Daltons, between 5,000 Daltons and 30,000 Daltons, between 5,000 Daltons and 20,000 Daltons, between 5,000 Daltons and 15,000 Daltons, or between 5,000 Daltons and 10,000 Daltons. Thus, ATH may be devoid or substantially devoid of heparin chains with a molecular weight below 3,000 Daltons or 5,000 Daltons.
    [0057] Commercial heparin may typically contain a range of heparin chains with molecular weights ranging from 1,000 Daltons or less to 50,000 Daltons or more. The lower molecular weight fraction, such as chains whose molecular weights are below 3,000 or 5,000 Daltons, can be eliminated by any suitable method. Among the non-limiting examples of elimination of 30 low molecular weight chains is dialysis, diafiltration, filtration by gel and electrophoresis. The dialysis or diafiltration can be carried out under conditions of high salinity. For example, conditions of high salinity for dialysis or diafiltration may include salt concentrations varying between about 1 M NaCl and about 4 M NaCl. Other salts other than NaCl may also be used.
    [0058] The high salt concentration can assist in the displacement of the small chains through membranes with the appropriate pore size. Filtering by gel can be carried out using a suitable medium for the separation of molecules according to their size. In a specific example, the gel filtration can be carried out on Sephadex® G-200, which is a gel medium for the separation of molecules with variable molecular weights between 1,000 and 200,000 Daltons. Commercial heparin 5 can be filtered by gel on a gel column, a series of fractions can be eluted with the first fractions containing the chains of higher molecular weight and with the following fractions with progressively lower molecular weights. The molecular weights of the heparin of each fraction can be determined, the fractions having the desired molecular weights can be grouped. By means of this method, those fractions containing heparin with molecular weights below the threshold of 3,000 or 5,000 Daltons can be excluded. If desired, heparin chains that are above a certain threshold can also be excluded. For example, fractions containing heparin above 50,000 Daltons, 30,000 Daltons, 20,000 Daltons, 15,000 Daltons, or 10,000 Daltons can be excluded, if desired . The pooled fractions having the desired range of molecular weights can then be used for the synthesis of the ATH.
    [0059] It should be noted that the methods of elimination of the very low molecular weight heparin chains described above are only examples and should not be considered limiting. Any commercial heparin treatment method for the removal of heparin chains located below a certain molecular weight threshold can be used in the present invention .
    [0060] The ATH can be formed by the conjugation of the AT with heparin, which has been stripped of the chains with very low molecular weight. Examples of heparin conjugation procedures with AT are described in the US Pat . No. 7,045,585, which is incorporated herein by reference. These procedures can be applied to the formation of ATH using heparin from which the chains with very low molecular weight have been removed, as described herein. Heparin can be conjugated to the AT by a simple one-step process, which provides the covalent attachment of the amine of an amine containing 30 fractions (including, among others, amines containing oligo (poly) saccharides, amines containing lipids, proteins, nucleic acids and any amine containing xenobiotics) with a terminal aldose residue of a heparin chain. For the formation of the ATH, the amine containing fractions is present in the AT, although other proteins can be conjugated using the same procedures. The non-destructive soft means provided in this document allows maximum retention of the biological activity of Protein and allow the direct link of the protein without the need for intermediate separating groups.
    [0061] In one embodiment, heparin is incubated with AT at a pH suitable for the formation of imines between the terminal amine and aldose or the heparin ketose residue . The terminal aldose and the ketose residues generally exist as an equilibrium between the closed ring-like semiacetal or semicetal form and the corresponding aldehyde or open-ring ketone equivalents. In general, amines are able to react with the open ring form to obtain an imine (Schiff base). Normally, aldoses are more reactive because the corresponding 10 aldehydes of the ring-open form are more reactive towards amines. Therefore, the formation of the covalent conjugate between amines and terminal aldose residues of the heparin provides a method for linking the AT containing an amine to the heparin.
    [0062] The reaction is usually carried out at a pH of between approx. 4.5 to approx. 9, and more usually 15 , between approx. 5 and approx. 8, to even more typically between approx. 7 and approx. 8. The reaction usually takes place in aqueous medium. However, the organic media, and especially the polar organic hydrophilic solvents, such as alcohols, ethers and 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.
    [0063] In certain cases, the imines formed by condensation of the amines of the AT with the terminal aldose residues of the heparin are reduced to the corresponding amines. This reduction can be carried out simultaneously with the formation of the imine or later. A wide variety of 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 the disulfide bonds can be used.
    [0064] Alternatively, if the reduction of the intermediate imine is not desired, the imine can be incubated for a sufficient period of time, normally from about 1 day to 1 month, more usually from 3 days to 2 weeks, to allow it to have place Amadori's transposition of the intermediate imine. The terminal aldose residues of the conjugated heparins by the methods provided in this invention usually possess C2 hydroxyl groups in the terminal aldose residue, ie, a 2-hydroxy carbonyl fraction that has been converted to a 2-hydroximine by condensation with the amine of the AT that is conjugated with the heparin. In the transposition of Amadori, which it is very frequent in carbohydrates, the a-hydroximine (imine in C1, hydroxy in C2) formed by the initial condensation can be transposed to form one (a-ketoamine by enolization and reprotonation (keto in C2, amine in C1)). The resulting a-carbonylamine is thermodynamically favored with respect to the a-hydroxymine precursor, thus facilitating a stable adduct, with a minimum perturbation of the heparin chain. Thus, in this embodiment, the technology facilitates a heparin chain covalently conjugated to the C1 of the terminal aldose residue of the heparin with an amine containing AT via an amine bond. If desired, the resulting conjugate can be reduced or labeled by reduction of the C2 carbonyl group with a labeling reagent (eg, NaB3H4), or conjugated with a second species-containing amine, such as a fluorescent label.
    [0065] Although the above description focuses on heparin and AT, various amine-containing species can be conjugated to a series of glycosiaminoglycans by the procedures described herein. The primary amine can be found in a small molecule, such as, for example, a fluorescent or chromophoric 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 polymers of said acids) or a polysaccharide. In general, when the proteins are conjugated with glycosiaminoglycans, the linkages will take place through the £ -amino groups of the 20 lysine residues. Alternatively, the linkages can also be carried out by the a-amino group of the N-terminal amino acid residue. In addition, many other methods known to any person skilled in the art can be used to introduce the amine functionality into a macromolecule.
    [0066] Specifically, the technology of the present invention can be applied to a diversity of therapeutically useful proteins, when considerations regarding the half-life and coagulation of blood are important. Said proteins include blood enzymes, antibodies, hormones and the like, as well as related plasminogen activators, such as streptokinase and its derivatives. Specifically, this technology facilitates conjugates of heparin or dermatan sulfate with antithrombin, with heparin cofactor II (HCII) or with analogs of heparin cofactor II.
    [0067] The methods described in the present invention facilitate conjugates of glycosiaminoglycan with a maximum retention of biological activity. Specifically, conjugates of heparin or dermatan sulfate are provided with AT or with HCII, which are> 60% by weight, usually> 90% by weight, more frequently> 95% by weight, and more typically> 98% by weight of activity intact of unconjugated heparin and antithrombin.
    [0068] The methods of the present technology facilitate intact heparin molecules conjugated with antithrombin or with cofactor II of heparin. In this way, the loss of biological activity associated with fragmentation or other type of heparin modification prior to conjugation is avoided. Heparin conjugates according to this technology maintain their anticoagulant activity because they have been prepared from intact heparin. Therefore, the methods described in this document can be used to prepare active heparin conjugates by first linking groups and separators to the species that it is desired to conjugate with heparin (or the glycosaminoglycan in question that is used) by joining it subsequently to heparin. In the 10 Inmunotechnology Catalog and Handbook, by Pierce Chemical Company (1990), which is incorporated herein by reference, numerous methods of incorporating amino groups reactive to other molecules and solid supports are described. Therefore, any species that possesses reactive amino groups or that is capable of being modified to contain said amino groups by any method known at present or that may be known in the future can be covalently conjugated to glycosiaminoglycans such as heparin, by the methods described in this document, and all of said conjugates is contemplated in the present invention.
    [0069] As described above, the technology of the present invention takes advantage of the fact that the native heparin (isolated from the intestinal mucosa), as well as the dermatan sulfate, already contains molecules with terminal aldoses that would exist in equilibrium between the hemiacetal and the aldehyde forms. Thus, heparin or dermatan sulfate can be conjugated to antithrombin serpenins by reducing the unique Schiff base formed spontaneously between the terminal aldose aldehyde of the heparin or dermatan sulfate and an amino of the serpin. Heparin or dermatan sulfate remains unchanged (without reduction of activity) prior to conjugation, and binds at a specific site at one end of the molecule, without giving unblocked activation groups or seric reticulation.
    [0070] 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 ketoamine to spontaneously form by an Amadori transposition between the terminal aldose of heparin and an amino group of the AT. In this way, this technology facilitates procedures for using the Amadori transposition to prepare glycosaminoglycan conjugates with amine-containing species, especially proteins. This method of conjugation is especially simple and simple, and minimizes the modification of the glycosiaminoglycan, thereby maximizing the conservation of its biological activity.
    [0071] Another aspect of the present technology facilitates covalent conjugates of glycosiaminoglycans, and in particular heparin, labeled at their end with an amine containing species in the terminal aldose residue of the glycosaminoglycan. For example, heparin and AT can be directly linked together, so that the active pentasaccharide sequence corresponding to the heparin AT is very close for its binding. This is one of the fundamental reasons for the realization of a covalent heparin-AT complex, since heparin accelerates the inhibition through AT 10 only if the AT can bind the active sequence. It is remarkable that the ATH has the exclusive property that the H (heparin) of the conjugate activates the endogenous TA stoichiometrically, while activating the exogenous TA by catalysis. Normally, an amine containing species will bind to each glycosiaminoglycan. However, it will be apparent that the ratio of amine containing species to glycosaminoglycan can be reduced below one, by adjusting the molar ratios of the reactants or the reaction time.
    [0072] Glycosiaminoglycans are available in various molecular weights and forms. For example, heparin is a mucopolysaccharide, isolated from pig intestines or beef lungs, and is heterogeneous in relation to its molecular size and chemical structure.
    [0073] It consists basically of residues of (1-4) 2-amino-2-deoxy-α-D-glucopyranosyl, and α-L-idopyranosyluronic acid linked with a relatively small amount of p-D-glucopyranosyluronic acid residues. The hydroxyl and amino groups are derived to various degrees by sulfation and acetylation.
    [0074] Heparin molecules can also be classified based on their pentasaccharide content. About a third of the heparin contains chains with a single copy of the single pentasaccharide with a high affinity for AT, while a much smaller proportion (calculated in approximately 1% of the total heparin) consists of chains that contain more of a copy of the pentasaccharide with high affinity. The rest (approximately 66%) of heparin does not contain 30 pentasaccharide. In this way, the so-called "standard heparin" constitutes a mixture of the three species, the "low affinity" heparin, which lacks a copy of the pentasaccharide, the "high affinity" heparin that 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.
    [0075] 35 These three species can be separated from each other using routine chromatography procedures, such as chromatography on an antithrombin affinity column.
    [0076] One advantage of the formation of a conjugate between heparin and a species containing at least one primary amino group (eg, AT) using the slow glycation process described herein, is the apparent selection of heparin chains that contain two pentasaccharides. Thus, for example, the ATH prepared by the method of the present invention appears enriched for the heparin species containing two pentasaccharides. When standard heparin (containing approximately 1% heparin with two pentasaccharides) is used as a starting matral, generally more than 10% of the resulting ATH comprises two pentasaccharide heparin, more often more than 20%, often more than 35%. %, and 10 more frequently more than 50% of the ATH, approximately, comprises heparin with two pentasaccharides.
    [0077] This enrichment may be responsible for a series of useful properties of ATH. The ATH of the present technology stoichiometrically activates the TA with which it has been conjugated, but activates the exogenous TA by catalysis. Thus, the heparin located inside the ATH complex acts catalytically when the ATH is administered as a systemic anticoagulant and when the ATH is used for the coating of surfaces to turn them into non-thrombogenic. The technology procedure produces an ATH complex with a very high anti-factor IIa specific activity. In addition, the second chain of pentasaccharides of the ATH complex can interact with the exogenous molecules of AT 20 thereby allowing the conjugated heparin to exhibit catalytic activity. In addition, the ATH complex heparin can be oriented in such a way that the pentasaccharide is available to bind and activate the circulation of AT molecules when the ATH complex binds to the prosthetic surface.
    [0078] It will be noted that a heparin conjugate of interest (eg, ATH) can also be obtained by incubating a species containing at least one primary amino group (eg, AT) with purified heparin of very high activity (ie, containing two pentasaccharide groups) or a fraction enriched for a very high affinity heparin.
    [0079] Although this technology has been exemplified basically in relation to heparin, it is evident that all glycosaminoglycans, irrespective of their molecular weight and their derivatization, can be conjugated by the procedures described herein, provided they possess a terminal aldose residue. The conjugates of all these glycosaminoglycans and their preparation by the methods described herein are within the scope of what is disclosed by this invention. For example, heparin conjugates derived with phosphates, sulfonates and the like, as well as glycosaminoglycans with molecular weights lower or higher than the molecular weights of heparin are within the scope of the present invention.
    [0080] In a further aspect of the present invention, a method of making a composition for preventing thrombogenesis can include conjugating the AT with heparin outside the body of a patient to obtain an antithrombinheparin conjugate, wherein the amount of antithrombin obtained in the conjugate of antithrombinheparin 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, weight, or greater than 99% by weight as a function of the antithrombin initially used in the synthesis.
    [0081] 10 Performance can be increased through various procedures. In one example, TA can be conjugated with heparin by the methods described above. After conjugation, any unbound AT can be recycled and used in another heparin conjugation reaction. After each stage of AT incubation with heparin, unbound TA can be recycled and used to obtain additional ATH.
    [0082] In another example, the ATH performance can be increased using a catalyst in the Amadori transposition. Among some of the examples of catalysts that can increase the rhythm of the Amadori transposition are 2-hydroxypyridine, tertiary amine salts, ethyl malonate, phenylacetone and acetic acid, as well as other acids. In a specific example, AT and heparin can react with each other in the presence of 2- 20 hydroxypyridine while heating in water or in very amphiphilic solvents, such as formamide. In other examples, AT and heparin can react with each other in the presence of trimethylamine or trimethylamine salts.
    [0083] The speed of the Amadori transposition can also be increased by solvent systems that accelerate the Amadori transposition. Some of the examples of these solvents include mixtures of water with formamide, dimethylformamide, dioxane, ethanol, dimethylsulfoxide, pyridine, acetic acid, trimethylamine, triethylamine, and combinations thereof. Heparin and AT can be reacted in these solvent systems to accelerate the transposition of Amadori to form ATH.
    [0084] A further method for increasing the conjugate speed of aldose 30 heparin with a molecule containing amines involves the use of a binding agent. The binding agent can be a heterobifunctional agent, with a group that reacts against the aldose of heparin at one end and against a different group at the other end, which can be used to bind to an AT or a secondary binding agent who can then join an AT. In a specific example, the binding agent can contain hydrazine at one end and an amino group at the other, such as 2-aminoethylhydrazine. This binding agent can react with heparin to form hydrazine with the aldehyde aldose of heparin. The product can be dialyzed or diafiltered by membranes that eliminate the heparin chains with less than 3,000 or 5,000 Daltons molecular weight, along with any unreacted binding agent. Thereafter, the heparin hydrazone product can be reacted with a large amount of the excess secondary binding agent. The secondary binding agent can be a homobifunctional reagent having activated carboxyl groups at each end, such as succidic acid di (N-hydroxysuccinimide) an ester (obtained by esterification of succid acid with N-hydroxysuccinimide using condensation agents such as carbonyldiimidazole or a carbodiimide ) so that the amino group of the hydrazine binding agent reacts precisely with one of the activated carboxyl in the secondary binding agent. The reaction mixture can be dialyzed or diafiltered to remove the unreacted secondary binding agent. At this point, the product will be heparin modified by the amino-hydrazine binding agent as well as the secondary binding agent. This product can be incubated with AT in an H2O buffer solution such that the amino group of the AT reacts with the activated second carboxyl group in the secondary binding agent to form an AT-Heparin conjugate, where the AT and heparin are present. linked through the link agent and the secondary link agent.
    [0085] Once the ATH is formed, the ATH can be lyophilized (desiccation by freezing) for storage. In one embodiment, the ATH can be prepared in a solution containing only water, then lyophilized. In another embodiment, the ATH can be prepared in a solution with water and alanine at a variable concentration between 0.01-0.09 molar, and then lyophilized. In another further embodiment, the ATH can be prepared in a solution containing water and mannitol, then freeze-dried. Each of these 25 procedures can be used independently, and each of the procedures has its own advantages. Following lyophilization by any of these methods, the ATH can be reconstituted and maintain a significant amount of its thrombin inhibition activity, compared to its activity prior to lyophilization. In some cases, the ATH can 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 inhibition activity. It has been demonstrated that the use of other lyophilization procedures of the ATH, such as the preparation of the ATH in a solution containing a salt more than 1 molar before lyophilization can destroy the ATH activity.
    [0086] 35 Whether the ATH has been freeze-dried or not, the ATH can be prepared in an aqueous solution containing between 9 and 11 mg / ml of ATH with respect to the total volume of the solution. It has been shown that the manufacture of solutions with an ATH concentration greater than 11 mg / ml can cause an aggregation of ATH that is difficult or impossible to reverse. However, stable aqueous solutions can be prepared with ATH concentrations of between 9 and 11 mg / ml. This solution can be formulated for its administration to a patient for the treatment of any of the ailments described in this document. The solution may also include various additives, as long as they are suitable for administration to a patient.
    [0087] In clinical practice, the heparin conjugates of the present invention can be used in general in the same manner and under the same pharmaceutical preparation modality as commercially available heparin for clinical use. Thus, the heparin conjugates of the present invention can be incorporated into aqueous solutions for injection (intravenous, subcutaneous or the like) or intravenous infusion, or in balm preparations for administration through the skin and mucous membranes. Any type of therapy, both prophylactic and curative, currently known or available in the future, for which heparin therapy is indicated, with the heparin conjugates indicated in the present invention can be practiced.
    [0088] The heparin conjugates according to the present invention are especially useful for the treatment of respiratory distress syndrome (RDS), neonatal syndrome and in adults. In contrast to the use of non-covalent heparin-AT complexes, the use of 20 covalent conjugates of heparin of the present invention prevents the loss of heparin in the pulmonary space when the TA is dissociated. In this case, a solution of covalent complex in a physiological buffer solution can be facilitated as an atomized spray in the respiratory tract, by means of a catheter or inhaler. Due to its large size, the ATH will remain in the alveoli for a longer period. ATH 25 is also useful for the treatment of idiopathic pulmonary fibrosis.
    [0089] Long-term use in the circulation can be carried out by intravenous or subcutaneous injection of the complex in a physiological buffer solution. The covalent conjugates of this technology can also be used for the treatment of acquired TA deficiency states characterized by thrombotic complications such as cardiopulmonary bypass 30, extracorporeal molecular oxygenation, etc. Since the longer half-life of the covalent complex allows fewer treatments with less monitoring. Additionally, the present invention allows the prophylactic treatment of adult patients at risk for venous thrombosis.
    [0090] The ATH conjugate according to this technology has numerous advantages compared to the AT 35 and the standard heparin that have not formed complexes. Since AT binds by covalent binding to heparin, the non-specific binding of ATH to plasma proteins it will occur less frequently than in the case of standard heparin, which results in fewer inter-individual variations in the dose response with ATH than with standard heparin. The longer half-life of the ATH after its intravenous injection in humans means that a sustained anticoagulant effect can be obtained by administering ATH with a frequency lower than that required in the case of TA and standard heparin without forming complexes. ATH is a thrombin and factor Xa deactivator that is much more effective than AT, and can be effective when used at much lower concentrations than AT in patients with AT deficiency. Also, ATH can access and inhibit thrombin linked to fibrin. Finally, by binding (for example, by means of a covalent bond) to prosthetic surfaces (for example, endovascular grafts), the ATH has been shown to have an in vivo antithromotic activity much greater than that of the AT, bound to the heparin by binding covalent, or hirudin linked by covalent bond.
    [0091] Preterm infants have a high incidence of respiratory distress syndrome (RSD ), a severe lung disease that requires treatment with assisted ventilation. Long-term assisted ventilation leads to the appearance of bronchopulmonary dysplasia (BPD) as a result of lung injury, which allows plasma coagulation proteins to move into the alveolar spaces of the lung. This results in the appearance of thrombin, and subsequently, fibrin. The presence of fibrin in the lung tissue and the air spaces of children deceased by RDS has been frequently observed. This fibrin gel inside the air space makes it difficult to transport fluid outside the pulmonary air spaces, resulting in persistent pulmonary edema. The present technology allows the treatment of said fibrin-mediated diseases of the lung tissue, preventing the formation of intra-alveolar fibrin, maintaining an "anti-thrombotic environment" and / or improving fibrinolysis inside the lung tissue, thereby reducing the Fibrin load in air spaces of the lung.
    [0092] Heparin conjugates can be administered directly into the air spaces of the lung through the respiratory tract prophylactically (before baby 30 breathes for the first time). This guarantees that the antithrombotic agent is directly available at the site of possible fibrin deposits and that the risk of bleeding associated with antithrombotic therapies is avoided. Likewise, the antithromotic agent will already be present in the lung prior to the start of respiratory support associated with the initial lesion, that is, unlike the systemic administration of antithrombin when the crossing of the administered drug into the pulmonary air space does not occur until after the lung injury. Since heparin is linked to AT by means of a covalent bond, it will remain in the pulmonary air spaces. It can also be a coadjuvant therapy of the surfactants currently administered to prevent RDS and BPD. "Pulmonary surfactant" means the soap substance that is normally present in pulmonary air spaces, whose main function is to prevent the collapse of the air space, as well as collaborate in the transfer of gas. The conjugates can also be administered repeatedly through an endotracheal tube or as an inhaled aerosol. It can also be used as adjuvant therapy with asthma medications by inhaler (for example, anti-inflammatory steroids, such as beclomethasone dipropionate), other anti- asthmatics 10 such as cromolyn sodium (bisodic salt of 1,3-bis (2-carboxichrome-5-). iloxi) -2-hydroxypropane, ("INTAL") and bronchodilators such as albuterol sulfate.
    [0093] Many other diseases associated with high thrombin activity and / or the formation of fibrin deposits can be treated by the administration of the conjugates of this invention. The inflammatory processes linked to the respiratory distress syndrome of the adult are basically similar to those seen with the neonatal RDS and can be treated by the antithrombotic therapy described. It has also been observed that spontaneous pulmonary fibrosis activates coagulation / fibrinolytic cascades in pulmonary air spaces. Fibrotic disease of the lung is usually a side effect associated with anticancer chemotherapy, and the antithrombotic administration of covalent heparin conjugates according to this technology can be performed prophylactically prior to cancer chemotherapy to prevent pulmonary fibrosis. Administration is repeated after chemotherapy to make sure that fibrin does not form. A decrease in the activity of antithrombin and an increase in thrombin activity in sepsis is also well documented.
    [0094] 25 Sepsis is the most common risk factor for the development of RDS in adults. Thus, the heparin conjugates according to the present invention can be used to reduce the mortality associated with septic shock.
    [0095] The conjugates according to the present invention can be administered at a therapeutically effective dose, i.e., in an amount that, when administered to a mammal 30 that requires it, is sufficient to effect the treatment, as described above (e.g. treat in another way thrombosis in mammals, or deactivate the thrombin of the clots, or inhibit the accumulation of thrombi). The administration of the active compounds and salts described herein can be carried out through any of the accepted modes of administration for agents that serve similar purposes.
    [0096] In general, an acceptable daily dose is around 0.001 to 50 mg per kilogram of body weight of the recipient per day, around 0.05 to 25 mg per kilogram of body weight of the recipient per day, or around between 0.01 and 10 mg per kilogram of body weight and per day. Thus, for its administration to a person of 70 kg, the dose range can range between about 0.07 mg and 3.5 g per day, between about 3.5 mg and 1.75 g per day, or between 0.7 mg and 0.7 g per day, depending on the individual and the stage of the disease being treated. In the case of ATH, the prolonged half-life allows the compound to be administered less frequently than standard heparin (for example, once or twice a week).
    [0097] Administration can be carried out by any accepted systemic or local route, for example, parenteral, intravenous, nasal, bronchial inhalation (ie, aerosol formulation), transdermal or topical route, in the form of solid, semi-solid or liquids, such as, for example, tablets, suppositories, pills, capsules, powders, solutions, suspensions, aerosols, emulsions or the like, as single dose formats suitable for the simple administration of precise doses. In general, aqueous formulations can be used. The conjugate can be formulated in a non-toxic, inert, pharmaceutically acceptable excipient medium, with a pH of about 3-8 or with a pH of about 6-8. In general, the aqueous formulation may be compatible with the culture or perfusion medium. The compositions will include a conventional pharmaceutical excipient or excipient, and a conjugate of the glycosiaminoglycan, and in addition, may include other therapeutic agents, pharmaceutical agents, vehicles, adjuvants, etc. The vehicles can be selected from different oils, including petroleum derivatives, or those of animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Examples of suitable liquid excipients include water, saline solution, aqueous dextrose or mannitol, as well as glycols, especially in the case of injectable solutions. Among the suitable pharmaceutical vehicles are starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, milk evaporated skim, glycerol, propylene glycol, water, ethanol, and the like. Other suitable pharmaceutical vehicles and their formulations are described in Remington's Pharmaceutical Sciences, by EW Martin (1985).
    [0098] If so desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH regulating agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, oleate triethanolamine, etc.
    [0099] The compounds of the present invention can be administered as a pharmaceutical composition comprising a pharmaceutical excipient in combination with a glycosaminoglycan conjugate. The level of the conjugate in a formulation may vary within the wide range used by any person skilled in the art, for example, between about 0.01 weight percent (% w / w) and about 99.99% p / w. p of the drug, based on the total formulation, and between 0.01% w / w and 99.99% w / w of excipient. In one example, the formulation may be in the range of between 3.5 and 60% by weight of the pharmaceutically active compound, the remainder being suitable pharmaceutical excipients.
    [0100] 10
    [0101] Examples
    [0102]
    [0103] The following examples show the embodiments of the invention that are best known today. However, it is to be understood that as follows there are only 15 examples illustrating the application of the principles of the present invention. The persons skilled in the art can conceive numerous modifications and alternative compositions, procedures and systems, without departing from the espmtu and scope of the present invention. The appended claims seek to cover said modifications and adaptations. Thus, although the present invention has been described above in a particular manner, the following examples provide additional details regarding what are currently considered to be practical embodiments of the invention.
    [0104]
    [0105] Example 1: Elimination of very low molecular weight heparin chains
    [0106]
    [0107] 25 Heparin (0.5 ml of 10,000 IU / ml Leopar Heparin) was filtered on a Sephadex® G-200 chromatography column of 49 cm by 1 cm. Heparin was eluted with 1 M NaCl and fractions of 20 drops (1.27 g per fraction) were collected. The absorbances of each fraction are indicated in Table 1 and Figure 1.
    [0108] Table 1
    [0109]
    [0110]
    [0111]
    [0112]
    [0113] Fractions 24-30 were pooled. These fractions do not include heparin chains with very low plasminogen molecular weights (fractions 31-40). Heparin chains with a higher molecular weight of fractions 1-23 were excluded for the ease of separation of ATH from heparin that has not reacted in subsequent steps. The heparin chains of fractions 24-30 had molecular weights as high as about 18,000 Daltons. The exclusion of the longer heparin chains guarantees that heparin does not overlap with AT and ATH when purifying the product. However, heparin chains with higher molecular weight can be included in the product in other examples.
    [0114] Another gel filtration of heparin (0.5 ml of 10,000 U.I./ml of Heparin Leo®) was carried out on the Sephadex® G-200 chromatography column of 49 cm by 1 cm. One more time, heparin was eluted with 1 M NaCl and fractions of 20 drops were collected (1.23 g per fraction) The absorbances of each fraction are shown below in Table 2.
    [0115] Table 2
    [0116]
    [0117]
    [0118]
    [0119]
    [0120] The results of the chromatography shown in Table 2 were similar in terms of their elution profile to those of the first gel filtration of the heparin of Table 1 above. Fractions 24-30 were pooled and combined with the pooled fractions from the first chromatography, the results of which are given in Table 1. The pooled combined fractions were dialyzed against H2O at 4 ° C and subsequently lyophilized.
    [0121]
    [0122] 10 Example 2: Reaction of heparin with AT
    [0123]
    [0124] It was dialyzed under human AT pressure to a concentration of 13.87 milligrams / ml and subsequently dialyzed once more against 0.02 M phosphate 0.15 M NaCl pH 7.3 at 4 ° C, followed by its storage at - 60 ° C after the dialysis. 19.12 mg of the 15 fractions of the lyophilized heparin of Example 1 above were dissolved in 1 ml of 0.3 M sodium chloride phosphate 1 M NaCl pH 9.5 which had been filtered through a sterile acrodisk with a pore size. 0.2 microns. The resulting solution was placed in a 12 mm by 75 mm plastic test tube and 72 microliters of human TA were added with the mixture. The tube was closed with a plastic cap and the outside of the cap was sealed with parafilm. The tube and its contents were heated in a water bath at 37 ° C for 14 days.
    [0125] After incubation for 14 days, the mixture of heparin and AT was gel-filtered on a 48.5 cm by 1 cm Sephadex® G-200 chromatography column with 1 M NaCl and fractions of 20 drops were collected. The absorbances of each fraction are shown in the Table 3 and in Figure 2. For purposes of comparison, Figure 2 also shows an independent chromatography of the AT alone, performed on the same chromatography column Sephadex® G-200,
    [0126] Table 3
    [0127]
    [0128]
    [0129]
    [0130]
    [0131] Fractions 14-16 containing the ATH product were pooled and dialyzed under pressure against 0.15M NaCl to obtain a final mass of 0.74832 g.
    [0132]
    [0133] Example 3: Inhibition of thrombin activity by the ATH
    [0134]
    [0135] Experiments were performed to evaluate the reaction of thrombin with the pressure dialyzed and pooled fractions of ATH of example 2. In each reaction, 114 microliters of the material being analyzed was mixed with 5.83 microliters of 20 U of IIa (thrombin ) bovine / ml of 0.15 M NaCl in an Eppendorf plastic tube and left at 23 ° C for 10 min. After the 10 minute period, 100 microliters of the solution were mixed with a solution of 875 microliters of 0.036 M sodium acetate, 0.036 M sodium barbital, 0.145 M NaCl pH 7.4 and 25 microliters of 3.125 mg S-2238 / ml H2O in a bucket at 23 ° C when the chronometer starts. S-2238 is the chromogenic substrate of thrombin. The absorbance against H2O at 405 nm of the resulting solution was measured every 10 seconds for 5 minutes. The following reactions were carried out:
    [0136] Reaction 1 (control): 114 microliters of 0.15 M NaCl were analyzed.
    [0137] Reaction 2: 114 microliters of ATH were analyzed.
    [0138] Reaction 3: 28.5 microliters of ATH added to 85.5 microliters of 0.15M NaCl were analyzed.
    [0139] Reaction 4: 11.4 microliters of ATH added to 102.6 microliters of 0.15M NaCl were analyzed.
    [0140] 5 Absorbances at 405 nm are a direct measure of a product cleaved from the S-2238 substrate by any thrombin that remained in the cuvette. The absorbances recorded every 10 seconds for each reaction are shown in Table 4 and in Figure 3.
    [0141] Table 4
    [0142]
    [0143] The data obtained from these four reactions show that even small volumes of the ATH concentrate are able to neutralize thrombin activity in comparison with the control reaction (Reaction 1) with only 0.15 M NaCl.
    [0144]
    [0145] 5 Example 4: Coagulation time with ATH
    [0146]
    [0147] 10 microliters of bovine IIa 20 U (thrombin) / ml 0.15 M NaCl was mixed with: 90 microliters of 0.15 M NaCl, 85 microliters of 0.15 M NaCl plus 5 microliters of the ATH concentrate of Example 2 above, or with 80 microliters of 0.15 M NaCl plus 10 microliters of 10 ATH concentrate. The mixtures were heated at 37 ° C for 1 minute in a plastic tube. Next, 100 microliters of 0.2 g of human fibrinogen / 100 ml of 0.15 M NaCl was mixed when the timer was started. The moment of the first appearance of a clot at the end of a wire ring used as an agitator was recorded. In the successive tests, 90 microliters of pure 0.15 M NaCl yielded about 15 coagulation times of 26.2 seconds, 25.2 seconds, and 26.0 seconds. The 85 microliters of 0.15 M NaCl plus the 5 microliters of ATH concentrate gave coagulation times of 34.0 and 33.8 seconds. The 80 microliters of 0.15 M NaCl plus the 10 microliters of ATH concentrate gave coagulation times of 39.2 and 39.6 seconds. Longer coagulation times indicate a reduction in thrombin activity in the reactions with the ATH concentrate.
    [0148]
    [0149] Example 5: Clotting time with ATH
    [0150]
    [0151] 100 microliters of 0.2 g of human fibrinogen / 100 ml 0.15 M NaCl was mixed with: 90 microliters of 0.15 M NaCl, 85 microliters of 0.15 M NaCl plus 5 microliters of the ATH concentrate of Example 2 above, or with 80 microliters of 0.15 M NaCl plus 10 microliters of ATH concentrate. The mixtures were heated at 37 ° C for 1 minute in a plastic tube. Then 10 microliters of 20 U of bovine IIa (thrombin) / ml 0.15 M NaCl was mixed at the start of the timer. The moment of the first 30 occurrence of a clot at the end of a wire ring used as an agitator was recorded . In the successive tests, 90 microliters of pure 0.15 M NaCl yielded coagulation times of 25.8 and 26.0 seconds. The 85 microliters of 0.15 M NaCl plus the 5 microliters of ATH concentrate gave coagulation times of 30.6 and 31.2 seconds. The 80 microliters of 0.15 M NaCl plus the 10 microliters of ATH 35 concentrate gave coagulation times of 37.2 and 35.2 seconds. The times of Longer coagulation indicates a reduction in thrombin activity in the reactions with the ATH concentrate.
    [0152]
    [0153] Example 6: Lyophilization in a solution with high salt content
    [0154] 5
    [0155] ATH was prepared as described in examples 1 and 2 above. Fractions 13-16 (20 drops per fraction, with an individual weight of about 1.2 g to 1.3 g) containing the ATH eluted from the Sephadex® G-200 column with 1 M NaCl were pooled and lyophilized . The lyophilized material was then suspended in 0.5 ml of water and dialyzed in a 0.15M NaCl solution. The inhibition of thrombin activity in the resuspended ATH was then checked. Three reactions were carried out, using: a buffer solution (0.036 M sodium acetate, 0.036 M sodium barbital 0.145 M NaCl pH 7.4), the resuspended ATH, an AT solution at 13.87 micrograms / ml 0.15 M NaCl. , a heparin solution (similar to that used to produce the ATH) at 10 micrograms / ml 15 of 0.15 M NaCl, a solution of S-2238 at 3.125 mg / ml H2O, and a bovine IIa (thrombin) solution at 10 U IIa / ml 0.15M NaCl.
    [0156] Reaction 1: 114 microliters of buffer solution, 5.83 microliters of IIa. Reaction 2: 104.5 microliters of buffer solution, 9.6 microliters of resuspended ATH, 5.83 microliters of IIa.
    [0157] Reaction 3: 55.0 microliters of buffer solution, 32.9 microliters of AT solution, 26.3 microliters of heparin solution, 5.83 microliters of IIa.
    [0158] The ingredients were added in the order shown for each of the above reactions in a plastic tube, mixing after each addition. After 10 minutes of incubation at 23 ° C, an aliquot portion of 100 microliters of the reaction was taken and mixed in a solution containing 25 microliters of S-2238 plus 875 microliters of buffer solution in a cuvette when the chronometer. Absorbance readings were taken against H2O at 405 nm every 10 seconds. The results of the reactions are shown in Figure 4. The results show that, unlike in the case of mixing of AT plus heparin, the resuspended ATH was not able to inhibit thrombin. There was no significant reduction in thrombin activity when the resuspended ATH was mixed with thrombin, as compared to thrombin alone (shown as open squares in Figure 4).
    [0159] The same resuspended ATH was also tested by combining the resuspended ATH with thrombin and human plasma. A volume of buffer solution, ATH and / or a sample of the heparin fraction (similar to that used to produce the ATH), and a volume of bovine IIa (thrombin) were mixed in a 6 mm borosilicate glass tube per 50 mm at 37 ° C. After 1 minute, a volume of human plasma (heated at 23 ° C immediately prior to use) was added by mixing it when the timer was started. The moment of the first appearance of a coagulum was recorded at the end of the wire used as an agitator. For each reaction, the volume of bovine IIa was 10 microliters of 15 U of 11a / ml of 0.15M NaCl, the volume of human plasma was 100 microliters. The volumes of the other components and the coagulation times are shown in table 5.
    [0160]
    [0161] Table 5
    [0162]
    [0163]
    [0164]
    [0165]
    [0166] These data confirm that, although the resuspended ATH did not itself show thrombin inhibition activity, the heparin chains of the resuspended ATH could catalyze the inhibition of thrombin through the exogenous TA found in human plasma. The coagulation times of the reactions that included the ATH increased enormously up to 120 seconds. Therefore, there was clearly a sufficient amount of ATH present to inhibit thrombin, but ATH did not show any thrombin-inhibiting activity on its own. This data suggests that ATH activity was destroyed by lyophilizing the ATH in a saline solution (high concentration).
    [0167] Example 7: Recycling of the AT
    [0168]
    [0169] The ATH was synthesized by conjugating the AT and unfractionated heparin. The yield of this preparation was 35.28%, defined as a percentage of the initial TA that was recovered as ATH. This left 100 - 35.28 = 64.72% of the original AT not incorporated into the complex. After the conjugation, the rest of unconjugated TA was separated. That unconjugated TA was then used in an additional synthesis in which the recycled TA was reacted with additional heparin to obtain the ATH. Of the recycled TA, 58.59% became ATH. Therefore, of the 64.72% of AT that was not incorporated into the complex in the first ATH preparation, an additional 58.59% became ATH in the second ATH synthesis. In this way, an additional 64.72 x 58.59 / 100 = 37.92% of the original AT 10 used in the first ATH preparation was obtained by recycling the unbound AT in a second ATH synthesis. Finally, when combining the results of the 2 ATH preparations, the total ATH yield in terms of the original TA at the start of the first synthesis was 35.28 37.92 = 73.20%. This total yield of ATH is much higher than the maximum yield of 60% that can be obtained by a single ATH synthesis. Furthermore, it can easily be seen that the unconjugated TA recovered from the ATH synthesis with the recycled TA could be used again for a third ATH synthesis to further raise the conjugate performance in terms of the original AT.
    [0170] It should be understood that the preparations indicated above attempt to illustrate the application of the principles of the present invention. Thus, although the present invention has been described above in connection with the embodiments, it will be apparent to one skilled in the art that numerous modifications can be made and alternative configurations made without departing from the principles and concepts of the invention, such and as set forth in the claims.
    权利要求:
    Claims (1)
    [0001]
    Composition for the prevention of thrombogenesis, comprising: antithrombin and heparin, where at least 50% by weight of the heparin is conjugated with antithrombin, 5 and where at least 98% by weight of the heparin of the composition, either as heparin or in conjugated heparin-antithrombin it has a molecular weight greater than 3,000 Daltons.
    2. Composition of the revindication 1, where the molecular weight is higher than 5,000 Daltons.
    10 3. Composition according to revindication 1, where heparin includes chains containing 18 monosaccharides or more.
    4. Composition according to re v indication 1, where the heparin of the composition has a molecular weight of less than 30,000 Daltons.
    fifteen
    5. Composition according to re v indication 1, where the heparin of the composition has a molecular weight of less than 20,000 Daltons.
    6. Composition according to re v indication 1, wherein the composition also includes a binding agent 20 and heparin is conjugated to antithrombin by the binding agent.
    7. Composition according to re v indication 1, where the heparin of the composition has a molecular weight greater than 3,000 Daltons and less than 30,000 Daltons.
    8. Process for manufacturing a composition according to at least one of the preceding claims, comprising:
    the conjugation of antithrombin with heparin in a solution, where the solution includes: i) water, antithrombin and heparin,
    ii) water, alanine at a concentration of 0.01 to 0.09 molar, antithrombin and heparin, or iii) water, mannitol, and antithrombin and heparin;
    and wherein at least 50% by weight of the heparin content is conjugated to the antithrombin and where at least a portion of the heparin with molecular weight less than 3,000 Daltons is removed prior to the conjugation step so that at least 98% in weight of the heparin of the composition, either as heparin or conjugated antithrombin-heparin, has a molecular weight greater than 3,000 Daltons; Y
    lyophilization of the solution to obtain a lyophilized antithrombin-heparin conjugate where conjugation and lyophilization occur outside a body of a subject.
    9. Process according to claim 8, wherein the portion of the heparin is removed by a process selected from dialysis, diafiltration, gel filtering, electrophoresis, and combinations thereof.
    10. The method of claim 8, further comprising reconstituting the lyophilized heparin antithrombin conjugate with a solvent to form an aqueous solution of antithrombin-heparin conjugate, wherein the antithrombin-heparin conjugate is present at a concentration of 9-11 mg. / ml with respect to the entire volume of the aqueous solution.
    11. The method of claim 8 which has a yield of at least 60% by weight of the antithrombin-heparin conjugate, based on the initial concentration of antithrombin used for the preparation of the antithrombin-heparin conjugate wherein said antithrombin-heparin conjugate is used. yield of at least 60% is achieved, at least partially, by recycling the unconjugated antithrombin and reacting said unconjugated antithrombin with additional heparin.
    twenty
    12. The method according to claim 8, wherein the solution is solution i) and wherein the process additionally comprises introducing a catalyst in Amadori transposition, in solution i) and heating the solution.
    13. Process according to claim 12, wherein the catalyst is selected from the group consisting of 2-hydroxypyridine, tertiary amine salts, ethyl malonate, phenylacetone, acetic acid, and combinations of said products.
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    同族专利:
    公开号 | 公开日
    CA2982832A1|2016-10-20|
    GB2554296A|2018-03-28|
    ES2710313B1|2020-04-07|
    ES2710313R1|2019-07-02|
    US20190030066A1|2019-01-31|
    GB201718876D0|2017-12-27|
    US20170020911A1|2017-01-26|
    WO2016168710A1|2016-10-20|
    JP2018513217A|2018-05-24|
    AU2016249391A1|2017-12-07|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    CA2168038A1|1993-08-19|1995-02-23|Jerald K. Rasmussen|Heparin functional affinity supports|
    US7045585B2|1995-11-30|2006-05-16|Hamilton Civic Hospital Research Development Inc.|Methods of coating a device using anti-thrombin heparin|
    TWI376234B|2005-02-01|2012-11-11|Msd Oss Bv|Conjugates of a polypeptide and an oligosaccharide|EP3294404A4|2015-05-08|2018-11-14|ICU Medical, Inc.|Medical connectors configured to receive emitters of therapeutic agents|
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    US201562148112P| true| 2015-04-15|2015-04-15|
    PCT/US2016/027905|WO2016168710A1|2015-04-15|2016-04-15|Antithrombin-heparin compositions and methods|
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