MXPA97008378A - Compounds and methods to prevent the trombogene - Google Patents

Abstract

The present invention provides compositions and methods for inactivating fibrin-bound troponin in a thrombus or clot, this by the ability of thrombin bound to the clot to catalytically promote further growth is substantially decreased or eliminated. The compositions and methods of the present invention are particularly useful in the prevention of thrombosis in the bypass of the cardiac circuit and in patients on renal dialysis, and for treatment of patients suffering from or at risk of suffering thrombi related to cardiovascular conditions, such as such as unstable angina, acute myocardial infarction (heart attack), cerebrovascular accident (embolism), pulmonary embolism, venal thrombosis, arterial thrombosis, and

Description

COMPOUNDS AND METHODS TO PREVENT TROMBOGENESIS This application is a partial continuation of Series No. 08 / 412,332, filed on March 31, 1995, the teachings of which are incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates generalities of the compounds and methods for the treatment of cardiovascular disease. Particularly, the present invention focuses on modifying thrombus formation and its growth by administration of an agent capable of inactivating thrombin which is also linked to fibrin within a clot or some other surface, but of which only has one minimal inhibitory activity against free thrombin, ie, thrombin in its fluid phase. The invention focuses on the specific cofactor II catalytic agents (HCII) of heparin, which, inter alia, activate HCII by inhibiting thrombin. The activity of the specific HCII catalytic agents of the present invention allows them to inactivate the fibrin-bound thrombin in a patient at concentrations that produce minimal inactivation of free thrombin, thereby decreasing the risk of bleeding. This is a surprising property of the specific HCII catalytic agents of this invention since other typical anticoagulants have a reduced ability to inactivate thrombin bound to fibrin compared to free thrombin (eg, heparin, dermatan sulfate and low molecular weight heparins) , or a substantially equal ability to inactivate thrombin bound to fibrin and free thrombin (eg, hirudin and its derivatives, and inhibitors of the active sites of thrombin inhibitors).

BACKGROUND OF THE INVENTION Thrombosis is a pathological manifestation of clots in blood vessels. Cascade coagulation is a complex biological process which results in the formation of a clot or thrombus composed of platelets and fibrin. Thrombin is bound to fibrin in a clot where it is catalytically activated and capable of amplifying its production over 1000 times by activating coagulant factors in the surrounding blood. The ability of blood to generate thrombin is essential in the prevention of excessive bleeding in some wounds (hemostasis). Thrombin is important in hemostasis because it stimulates platelet aggregation and fibrin formation when a blood vessel is broken. Therefore, an ideal antithrombin would be an agent which can calm the clot by inactivating thrombin bound to fibrin at concentrations that do not produce abnormal bleeding as a result of the inhibition of thrombin production in the general circulation. Thrombosis, which can complicate atherosclerosis, can cause partial or total occlusion of the blood vessel, which is why most cardiovascular complications, including unstable angina, acute myocardial infarction (heart attack), cerebrovascular accidents (stroke) ), pulmonary embolism, cause acute venal thrombosis and arterial thrombosis. These diseases are the biggest cause of disability and mortality in the world, but particularly in the communities of the West. In addition, thrombin and in particular surface bound thrombin play a role in the formation of thrombi in cardiac bypass circuits, after angioplasty and during and after thrombotic therapy for acute myocardial infarction. That is why, patients suffering from these procedures should be treated with high doses of heparin to prevent thrombosis. Although these high doses of heparin can effectively prevent coagulation, they can also cause serious bleeding complications. The clot or thrombi that is formed as a result of activation of cascade coagulation, contains fibrin, platelets and numerous other blood components. Thrombin linked to fibrin remains active and causes clot growth by continuous fibrinogen reproduction and activation of platelets and other clotting factors, such as factor and factor VIII. In addition, unlike free thrombin which is rapidly inactivated by antithrombin factors (for example antithrombin III (ATIII)), thrombin bound to the clot is protected against inactivation. As a result, the clot acts as a reservoir for active thrombin that also favors the growth of the clot. In addition, thrombin induces the proliferation of equal cells and thus may be complicated in proliferative reactions, such as arteriosclerosis and restenosis followed by angioplasty or atheromylectomy. Since thrombin becomes critical to the formation of thrombi, the use of thrombin inhibitors for the treatment of thrombosis and thrombotic complications has been proposed for many years. A large number of partial effectiveness inhibitors have been used for years. Heparin, for example, can be used as an anticoagulant and antithrombin agent to inhibit fibrin formation, platelet aggregation and thrombus formation. However, heparin has certain limitations. For example, it has biophysical limitations, since it acts as an anticoagulant by activating ATIII, it is even relatively ineffective to the inactivation of thrombin bound to fibrin in safe doses, thus continuing thrombus growth by thrombin linked to fibrin in pre-existing thrombi. In sum, the doses required to produce an antithrombotic effect are completely unpredictable and therefore the administration of the drug must be monitored closely. Low molecular weight heparin (LMWH) can be used as an anticoagulant and antithrombin agent to inhibit fibrin formation, platelet aggregation and thrombus formation. LMWHs act by activating the ATIII and as such have the same biophysical limitations as heparin. However, LMWHs produce a more predictable anticoagulant than heparin. Thus, both heparin and LMWH have the limitation of not being inactivated in thrombin bound to a surface. The consequences of this are (a) the high concentrations that are necessary to obtain an antithrombin effect, which can lead to excessive bleeding and (b) once the agents circulate, the thrombin bound to a surface can reactivate coagulations. The inactivation of thrombin bound to the clot may be carried out with another set of compounds known as direct thrombin inhibitors. Such inhibitors include hirudin and its derivatives and inhibitors of the active sites of thrombin, as well as argatroban and PPACK (Chloromethyl arginyl D-phenylalanyl-L-propyl-L-arginyl ketone). Hirudin is an antithrombin substance extracted from the salivary glands of leeches. Related compounds include hirulog which is a small, synthetic analogue of hirudin. While these substances are capable of inhibiting thrombin bound to the clot, they have the following limitations. First, they typically do not inactivate thrombin selectively but do so at the same concentrations that are required to inhibit free thrombin. Second, the inhibition of thrombin is generally stoichiometric and, then if very high concentrations are not used, the inhibitory effect can be saturated by the large amount of thrombin that is generated in the places where thrombin bound to a surface accumulates ( for example in bypass circuits, or venous or arterial vein thrombosis). As a result of the two limitations already mentioned, high concentrations of direct thrombin inhibitors (e.g., hirudin) must generally be administered by interaction with the free thrombin inhibitor generated by thrombin bound to the coagulum. Such high concentrations of inhibitor may, however, cause undesirable bleeding. In addition, direct thrombin inhibitors (e.g., hirudin, its analogs and LMW inhibitors of active sites in thrombin, as they argatroban) are generally reversible and, then, the inhibitory effect is lost when the drug is cleared from the blood. Unfortunately this reversible inhibitor can leave a recursive activation of coagulation. In sum, the inactivation of thrombin bound to the clot can be carried out with a third class of compounds which bind reversibly or irreversibly at the site, i. e., catalytic, active thrombin. PPACK is an example of an irreversible active site inhibitor. Such inhibitors, however, generally lack certain specificity for thrombin and until today their efficacy is questioned. In addition, these inhibitors have the same limitation as hirudin, which is why they regularly have the same reaction against clots and free thrombin. This is problematic because it evidently indicates that the total inhibition of free thrombin using irreversible active site inhibitors can cause excessive bleeding. For obvious reasons, it is necessary to improve the compounds and methods that have been used, for example, to inhibit thrombogenesis associated with cardiovascular disease. In particular, it would be convenient to supplement the compounds and methods capable of reducing and eliminating the thrombin activity bound to the fibrin of the existing thrombin without the cardiovascular system. Such elimination or reduction of thrombin bound to the clot should be essentially irreversible so that the increase in clots does not increase after the treatment is administered and thus stops it. As such, compounds and methods are necessary and which are capable of inactivating thrombin within the clot or thrombus with an agent that does not strongly inactivate free thrombin, since such an agent would not adversely affect the control of bleeding (hemostasis). The present invention fills these and other needs. Hirudin derivatives that block the active sites of thrombin are described in U.S. Patent Nos. 5,240,913 and 5,196,404. A bifunctional antithrombotic composition which includes an inhibitor of the glycoprotein group llb / Illa and one of thrombin are described in WO 92/10575. Glycoprotein Illa analogue peptides by inhibition of thrombogenesis described in WO 90/001178. Factor X and / or Xa inhibitors described in U.S. Patent Nos. 5,239,058 and 5,189,019, and PCT publications WO 93/09803, WO 92/04378 and WO 92/01464. Inhibitors of the factors VII and / or VIII are described in U.S. Patent Nos. 5,223,427 and 5,023,236 and WO 92/06711. Anti-adhesive platelets and related antibodies are described in WO 92/08472. For a review of the structure and function of thrombin, see, Stubbs and Bode, Thrombosis Research 69: 1-58 (1993). For a review of the limitations of heparin and the potential benefits of new anticoagulants such as antithrombotic drugs, see, Hirsh, Circ. 88: 1-C (1993). In short, we have numerous modified heparin compounds, as well as other glycosaminoglycans and their derivatives that have been developed. For example U.S. Patents Nos. 5,296,471, 5,280,016 and 5,314,876 describe the desulfation of heparin, periodic oxidation of heparin / heparan sulfates followed by a reduction of the result of aldehyde groups and high molecular masses N, 0 -sulfurized heparosans, respectively. Fractions of low molecular weight heparins have been used for several years (see, Mascellani, et al., Thrombosis Research 74: 605-615 (1994), and Shhehan, et al., J. Biol. Chem. 289: 32747 -32751 (1994).

SUMMARY OF THE INVENTION The present invention provides compounds and methods for the treatment of cardiovascular diseases. The invention relates, inter alia, to agents capable of effectively inactivating thrombin bound to the clot. Particularly, in one aspect, the present invention provides heparin-specific cofactor II (HCl I) catalytic agents capable of selectively inactivating thrombin which is bound to fibrin in a clot or other surface, but which has only minimal inhibitory activation against free thrombin. The selective activity of the specific HCII catalytic agents of this invention allow the fibrin bound thrombin to be inactivated in a patient at concentrations that produce minimal inactivation of free thrombin, which is why it decreases the risk of bleeding. Preferably, the inactivation of fibrin or external thrombin is essentially irreversible in such a way that the increase in clots will not substantially restart after such specific HCII catalytic agents (eg, activation of agents that activate, catalyze or induce HCII by inactivation of thrombin bound to fibrin) are free of blood.

In one embodiment, the agents of the present invention are low molecular weight heparin (LMWH) preparations that have been modified such that they contain less than 5% and, preferably, less than 3% of the antithrombin III (ATI 11) by catalyzing the activity or, interchangeably, activating the activity of standard or unmodified heparin LMWH, but more ATI! I activating the activity that the sulfate dermatan. In addition, due to their reduced long chain compared to standard heparin, the agents of the present invention have much less activity as inactivation catalysts by the free thrombin HCII that both the simple heparin or the sulfate dermatan. Thus, the agents of this invention have a very weak activity when contrasted against free thrombin in a coagulation time test, while it would not be predicted to be effective anti-thrombotic agents. Surprisingly, however, the agents of the present invention are capable of efficiently catalyzing the inactivation by the HCII of bound thrombin to a surface, e. g., thrombin bound to fibrin or thrombin bound to the exchangeable clot. External thrombin is activated, generally, by the formation of a covalent, irreversible complex of thrombin HCII, as well, in contrast to typical antithrombi and other anticoagulants (eg, dermatan sulfate, heparin, low molecular weight heparin (LMWHs) , hirudin and other direct inhibitors to thrombin), the HCII specific catalyst agents of this invention have the ability to irreversibly select and inactivate thrombin bound to fibrin without having major effects against thrombin in its fluid phase. Without being bound by a given theory, this ability is explained by the observation of this invention that they produce in exchange in the HCII which allows it to efficiently bind the thrombin when the enzyme is immobilized on a surface, but which lacks the size to bind thrombin when it is free in its liquid phase. The agents of this invention can be used in various methods, for example, to modify the formation of thrombi in a patient without taking it to a state of greater severity in systemic bleeding. Such methods include the provision to the patient of pharmaceutically acceptable doses of an agent capable of inactivating thrombin bound to the clot without minimal inactivation of the free thrombin. The agents are preferably characterized by a specific antifactor Ha by activation by cofactor heparin II from about 2 to about 5 units / mg in an antifactor assay, an antithrombin III of specific catalytic reaction from about 0.2 to about 1.5 units / mg in an anti-Xa assay, and a solubility in aqueous medium of about 150 to about 1, 000 mg / ml. In addition, these agents have an effective anticoagulant which is attributed to a mechanism by HCII and ATIII, and by a mechanism that is independent of both HCII and ATIII. In sum, the agents are preferably polyionionically carbohydrate in less than 24 monosaccharin units, generally with heparin preparations of molecular weight between 3,000 and 8,000 Daltons (+1,000 Daltons). The present invention includes heparin preparations which are prepared from heparin using a variety of techniques. In one of the modalities, heparin, i. e., the simple, non-fractional heparin is first reduced in the size of its molecule by chemical depolymerization and the fractions with molecular weights in a range between 3,000 to 8,000 Daltons (+ 1, 000 Daltons) that are isolated. In a preferred manner at present, heparin is depolemized using nitrous acid. Subsequently, the resulting low molecular weight heparin is chemically modified to reduce its affinities with antithrombin III. This can be carried out chemically or, alternatively, through the use of an antithrombin II in an affine column. In a preferred embodiment, this is carried out chemically by an oxidation reaction followed by a reduction reaction. In this preferred embodiment, heparin low affinity, and low molecular weight is a mixture of heparin molecules having a native sugar (eg, a uronic acid residue) as the non-reducing terms and a residual 2,5-anhydromannitol as the reducing terminal, and having molecular weights of about 3,000 to 8,000 Daltons. In this formation it is credible that the reduction of the molecular size bound with the reduction of the activity of activation of the ATI If originating the agents of this invention with its capacity to effectively inactivate the thrombin bound to the clot with only a minimum inactivation of the free thrombin . As an alternative of heparin preparations, the agents of this invention can be, for example, negatively charged polysaccharides different from heparin, negatively charged polyanions, electronegative organic molecules with affinity to HCII and the like. Such substances will generally bind to the HCII with an affinity of at least 10 6 M, preferably close to 10 8 M or stronger, but with a weak affinity to thrombin, preferably weaker than about 10 6 M. desired and depending on the use, the pharmaceutical compositions used to prevent thrombogenesis without substantially preventing normal coagulation in a patient, can be prepared by condensing the following compounds: i. about 90 to 99.9% by weight of an agent capable of preventing coagulated thrombin, especially the specific HCII compound, which has a minimum of affinity, or is free from it, the binding affinity of ATI 11 is able to displace and inactivate thrombin bound to fibrin and ii. about 0.1 to 10 percent of a catalytic agent ATI 11 or, exchanging it to an active agent ATIII, such as heparin or a lower molecular weight heparin (LMWH), capable of inactivating the thrombin passage. In a currently preferred embodiment, the catalytic activity of the HCII or, interchangeably, the activation of the HCII activity and optionally mixing its composition is about 2 to 5 units / mg and, preferably, about 3 to 4 units / mg. In another embodiment, the present invention provides a method for preventing thrombin bound to the clot and thrombin in its liquid phase in a patient without aggravating its blood system, the method comprising administering to the patient an acceptable dose of (i) an agent Specific catalytic HCII capable of inactivating thrombin bound to the clot, the catalytic agent HCII has a minimum affinity with antithrombin III (ATI II) and a factor II of heparin against a factor II of heparin of 2 to 5 units / mg in a sample of testing in an antifactor lia and (ii) an ATIII catalyst, i. e., activating an agent capable of flowing thrombin passage, activating the ATI II agents available for the use of this method, but not limiting them to heparin and lowering its molecular weight. In this method the catalytic agent HCII and the catalytic agent ATIII, i. e., activating the ATI agent li can be administered to the patient simultaneously or sequentially. When administered to the patient, the catalytic agent HCII and the catalytic agent ATI 11 can be administered in a single or compound solution or alternatively as two solutions or compounds. In another embodiment, the present invention provides a product comprising (i) an agent capable of inactivating thrombin bound to the clot and (ii) a catalytic agent ATIII capable of inactivating thrombin in its liquid phase as a combined preparation or simultaneously separated or in sequential use, without clinically inducing the blood system. The catalytic agents ATI 11 available for use in this product, do not limit heparin and its low molecular weight. In this product, the thrombin inactivating agent and the catalytic agent ATI 11 can be administered to the patient simultaneously. When administered to the patient, the inactive agent of the thrombin and the catalytic agent can be supplied as a single or composite solution, alternatively, as two different solutions or compounds.

Preferred agents in the present invention are obtained from heparin.

One class of products included in the invention are preferred as agents and, as a separate subclass, the heparin preparations of the present invention, as products in both subclasses have not been sulphated into residues of uronic acid and in the open form they are substantially free from the groups aldehydes. Exemplary members of this class have about 30% of their uronic acid residue in open form. Another class of these agents and products of heparin preparations have a native sugar (e.g., uronic acid residue) as the non-reduced terminal in a 2.5 anhydromannitol residue as the reduced terminal. Some particularities of these products belong to both classes already mentioned. Another aspect of the invention resides in a polyanionic carbohydration capable of effectively inactivating thrombin bound to the clot in favor of free thrombin, obtained from heparin. They have non-sulfated uronic acid residues in open form, and are substantially free of aldehyde groups. Also product of this invention is one capable of inhibiting thrombin bound to the clot, in a specific catalytic agent HCII obtained by: i. simple depolymerization of unfractionated heparin by depolymerized nitrous acids; I. oxidation of heparin depolymerized with sodium processed in an aqueous medium for 24 hours at 4 ° C, and stopping the oxidant action by the sum of ethylene glycol excesses followed by an extensive dialysis against distilled water using a dialysis tube with 500 MW of cut. iii. reducing the oxidized product by the sum of sodium borohydrate and then allowing the mixture for 25 hours at 23 ° C, adjusting the pH of the reaction of the mixture to 3.0 with HCl to destroy excess borohydrates as soon as it increases to pH 7.0 by NaOH aggregation; iv. dialyzing the product resulting extensively against distilled water; and V. coating the product by lyophilisation; and optionally vi. By passing the product on an antithrombin lll affinity column, the product obtained has the following properties: * molecular weight of 3,000 to 8,000 (± 1, 000) * specific activity of about 2 to 5 units / mg antifactor Ha * specific activity of less of 1.5 units / mg of antithrombin. In another aspect a process for preparing a specific catalytic agent HCII having an unreduced native sugar as a final group in an unreduced anhydromannitol 2.5 sugar as the other final group is carried out, said process comprising: (i) depolymerization of unfractionated heparin; (ii) oxidation of the result of heparin with low molecular weight; and (iii) reducing oxidation, heparin with low molecular weight; where the optional weight includes additional purification and other procedures when obtaining said specific HCII catalytic agent. The products and agents of the invention are preferably for pharmaceutical use. This invention also includes the use for the manufacture of a medicament for the treatment of a cardiovascular disease of any of the products or agents of the invention, such as for example an agent to activate the HCII by inhibiting thrombin to the exclusion of the significant ATI 11 by inhibiting thrombin. Other products and agents of the invention which may be used include the catalytic agents HCl I, LMWH polyanionic carbohydrate preparations and products obtainable as above and the product V18 described in the examples (or a substance similar to the product V18), as well as also substances with the same inhibition characteristics or pharmacological properties as the above-mentioned products and agents. The invention further includes the use for the manufacture of a medicament for the treatment of thrombosis by prophylaxis or therapy of a derivative of heparin having its uronic acids unsulfated in an open form and substantially free of aldehyde groups. In addition to the above, the pharmaceutical compositions are provided with an agent or product whose binding to HCII and allowing it to interact with a local non-fibrin binding on thrombin. The present invention provides pharmaceutical compositions of a catalytic agent HCII and a catalytic agent ATI II, ie, and an active agent ATIII, and composite agents to select the inactivity of the bound surface or thrombin bound to the clot and a thrombin inhibitor in the phase of fluid. Such pharmaceutical compositions, which may be in the form of mixtures, are useful for the treatment of numerous cardiovascular conditions. In sum, such compositions are useful in conjunction with conventional thrombosis treatments, such as the administration of plasminogen activating tissue (tPA), streptokinase, and the like, as well as with an intravenous intervention, such as angioplasty, atherotomy, and the like. A further understanding of the nature and advantages of the invention may become apparent with the portions of the description and drawings BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates the mechanisms proposed by means of which heparin binds to HCII and facilitates the HCII and thrombin approach, as well as an irreversible covalent bond can form a Leu-Ser link in HCM between the catalytic center of thrombin.

FIGS. 2 and 3 illustrate the proposed mechanism by means of which the HCIi catalytic agents of the present invention are capable of selective activity against bound surface thrombin.

FIGS. 4 and 5 show the results of a series of experiments comparing the relative ability of several heparins and a LA-LMWH HCII specific catalytic agent, i. e., V18, to inhibit and displace fibrin thrombin using a filter. There is a dose that responds for both exposure and inactivation with 85% inactivation and about 60% exposure 4 μM. Analysis of gels represented in the thrombin exposure and the residual inactivity of bound thrombin indicating that the HCII is covalently assigned to thrombin, indicating that thrombin is permanently inactivated.

FIGS. 6A and 6B illustrate the ability of dermatan sulfate and a specific catalytic LA-LMWH HCII of the present invention, i. e., V18, respectively, for free inactivation and thrombin-fibrin using the "hanging clot" test.

FIG. 7 illustrates the elution contour of V18 on a Sephadex G-50.

FIG. 8 illustrates the range of labeled thrombin diffusion of the fibrin clot.

FIG. 9 illustrates the effect of 0.25 μM HCII, with and without 60 μg / ml of one of the HCII catalytic agents of the present invention (i.e., V18), on the activity of bound thrombin in purified fibrinogen.

FIG. 10 illustrates the recirculation of the extracorporeal circuit used to examine the ability of a coagulating agent throughout the blood system.

FIG. 11 illustrates the comparative effects of dermatan sulfate and one of the LA-LMWH HCII specific catalytic agents of the present invention (i.e., V18) also have aliquots of the same human blood that was taken from the extracorporeal recirculation circuit.

FIG. 12 illustrates the results when using a mixture of a LA-LMWH HCII specific catalytic agent, i. e., V18, and a catalytic agent ATI 11 (e.g., heparin).

FIG. 13 illustrates the effects of 1 anti-Xa unit / ml and 2 anti-Xa units / ml of the unmodified LMWH on the clot of the filter and on coagulation thrombin (TCT).

FIGS. 14 and 15 illustrate the comparative effects of dermatan sulfate and one of the specific catalytic agents LA-LMWH HCM of the present invention, i. e., V18, which have on the clot in the extracorporeal recirculation circuit in a concentration range of 120 μg / ml to 960 μg / ml.

FIG. 16 illustrates the effects of hirudin on the blood clot in an extracorporeal recirculation circuit with a concentration range of 10 U / ml at 20 U / ml.

FIG. 17 illustrates the effects of heparin in combination with V18 in a deviant simulation model.

FIG. 18 illustrates the effect of one of the specific catalytic agents LA-LMWH HCII of the present invention, i. e., V18, on a coagular weight after a damaged balloon.

FIG. 19 illustrates the effect of simple heparin on a coagulated weight after a damaged balloon.

FIG. 20 illustrates the effect of the simple heparin and a specific catalytic agent LA-LMWH HCII of this invention, i. e., V18 on the coagular weight after a damaged balloon.

FIG. 21 illustrates the effect of various anticoagulants on a clot lysis using the Rabbit Venous Thrombosis Treatment.

FIG. 22 illustrates several anticoagulants on the increase of clots using the Rabbit Venous Thrombosis Treatment.

FIG. 23 illustrates the effects of V18 on the 'TCT (A), the APTT (B), and the Xa factor of the clotting time (Xa temporal clot) (C) in normal plasma (¡¡), ATI 11 deficient plasma (m) and HCII deficient plasma (").

FIG. 24 illustrates the effect of LMWH on TCT (A), APTT (B), and factor Xa temporal clot (Xa temporal clot) (C) in normal plasma (J¡), ATM deficient plasma (m) and poor plasma HCII (~:).

FIG. 25 illustrates the effects of dermatan sulfate on TCT (A), APTT (B), and temporal coagulant factor Xa (Xa temporal clot) (C) in normal plasma (¡), ATTII deficient plasma (a), and HCII deficient plasma (*).

FIG. 26 illustrates the effects of Fraction 1 on the TCT (A), of the APTT (B), and the temporal coagulant factor Xa (Xa temporal clot) (C) in normal plasma (¡), ATI 11 deficient plasma (a), and HCII deficient plasma (m).

FIG. 27 illustrates the effects of Fraction 2 on the TCT (A), the APTT (B), and the temporal coagulant factor Xa (Xa temporal clot) (C) in normal plasma (! ¡), ATIII deficient plasma (:), and HCII deficient plasma (m).

FIG. 28 illustrates the effects of Fraction 3 on TCT (A), APTT (B), and temporal coagulant factor Xa (Xa temporal clot) (C) in normal plasma (¡), defective plasma ATIII (), and HCII poor plasma (m).

FIG. 29 illustrates the comparative additive effects of heparin (0.1 U / ml) on the prolongation of thrombin produced by DS and V18.

FIG. 30 illustrates the effect of increasing doses of heparin on the complete clot to thrombin (A) (m), PPACK (B) (~) clot and coagular thrombin (C) (i) that is aggregated.

FIG. 31 illustrates the effect of increased doses of dermatan sulfate on the complete clot to thrombin (A) (m), PPACK (B) () and coagular thrombin (C) (¡) that is aggregated.

FIG. 32 illustrates the effect of increased doses of V18 on the complete clot to thrombin (A) (m), PPACK (B) (~) clot and coagular thrombin (C) (¡) that is added.

FIG. 33 illustrates the effect of increased doses of Fraction 1 on the complete clot to thrombin (A) (m), clot PPACK (B) (") and coagular thrombin (C) (¡) that is added.

FIG. 34 illustrates the effect of increasing doses of Fraction 2 on the complete clot to thrombin (A) (m), clot PPACK (B) (]) and coagular thrombin (C) (¡) which is added.

FIG. 35 illustrates the effect of increasing doses of Fraction 3 on the complete clot to thrombin (A) (m), clot PPACK (B) () and coagular thrombin (C) (¡) that is added.

FIG. 36 compares the effects of heparin ((A), upper panel), Fraction 1 ((B) middle panel) and the combination of heparin and 10 of Fraction 1 ((C), lower panel) on the temporary clot filled to said thrombin (), a PPACK () clot and a coagular thrombin (¡) that is added.

FIG. 37 Illustrates the effect of increased doses of a combination of simple heparin and dermatan sulfate (120 μg) on the temporary clot filled with said thrombin (m), a PPACK clot () and a coagulating thrombin (¡) that is added.

FIG. 38 illustrates the effect of increased doses of a combination of dermatan sulfate and simple heparin (0.5 U / mL) on the temporal full clot to said thrombin (A) (m), a PPACK (B) (:) clot and a coagular thrombin (C) ( | ) that is added.

FIG. 39 illustrates the effect of increased doses of a combination of V18 and simple heparin (SH) (0.5 U / mL) on the temporary clot filled with said thrombin (A) (u), a PPACK (B) () clot and a thrombin coagulate (C) (¡) that is added.

FIG. 40 lustrates the effect of heparin on preventing the clot in closed circuits.

FIG. 41 compares the effectiveness of Fractions 1, 2 and 3 when combined with 1.5 units / ml of heparin in the closed circuit.

FIG. 42 places in front of it a sum of active anti-Xa inhibitors in a spiral system with or without ATI 11 present.

FIG. 43 illustrates the prolongation of temporal coagulation thrombin in a spiral system containing measures of fibrogen in the presence and absence of ATI 11 or HCII of several GAGs.

FIG. 44 illustrates the relative effects of simple heparin (A), LMWH (B), V18 (C) and Fraction 1 of V18 (C) in inactivation Xa, IXa and Xla in a plasma system to which each of these coagulated enzymes they were added.

FIG. 45 illustrates the effect of platelets on the active anticoagulant of V18 (A), Fraction 1 of V18 (B), LMWH (C) and SH (D).

FIG. 46 illustrates the results of the recalcification time using several GAGs in platelets-substantial plasma (PRP), platelets-low plasma (PPP) and platelets-free plasma (Pit-Free).

FIG. 47 illustrates the mechanism of a weak group. FIG. 48 illustrates the Factor Xa group on a phospholipid surface. FIG. 49 illustrates the consequences of a weak group. FIG. 50 illustrates the mechanism of a weak group.

DETAILED DESCRIPTION AND PRESENTATION OF THE INVENTION The present invention provides compositions and methods for the inactivation of thrombin linked to fibrin within a thrombus or clot, therefore, the ability of thrombin bound to the clot catalytically promotes the increase of the clot which it diminishes it or eliminates it substantially. The compositions and methods of this invention are used particularly to prevent thrombosis in the circuit of cardiovascular devices and in patients suffering from renal dialysis and for the treatment of patients suffering from a risk of cardiovascular thrombosis, such as unstable angina, myocardial infarction acute (heart attack), cerebrovascular accident (stroke), pulmonary embolism, severe venal thrombosis, arterial thrombosis, etcetera. This invention is not limited to such uses, however; in the compounds and methods described, uses may be found in other in vitro or in-vivo situations while it is effective to inhibit clots or increase thrombi or eliminate clots or thrombi. For example, the compounds of the present invention can be used as anticoagulants to inhibit thrombin bound to the clot in several in-vitro studies, assays and the like. "Proteoglycan" includes as a reference a protein, which is next to one or more glycosaminoglycan chains. Proteoglycans are polyanionic compounds that have properties that reflect both the protein and the glycosaminoglycan chains. "Glycosaminoglycan" includes as a reference a polysaccharide compound of repetitive disaccharin units. The disaccharins always contain an amino sugar (ie, glucosamine or galactosamine) and some other monosaccharin, which can be a uric acid (ie, glucuronic acid or iduronic acid) as in hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate or sulfate dermatan or a galactose like keratan sulfate. The glycosaminoglycan chain that can be sulfated or divided from repetitive disaccharins. With the exception of hyaluronic acid, all glycosaminoglycans are covalently linked by an "Initial Protein", i.e., which is formed as proteoglycans. "Heparin" (or, alternatively, "standard heparin" (SH) or "unmodified heparin") is included as a reference to a mixture of polysaccharide chains composed of artificial repetitive disaccharides of an uronic acid residue (D-glycocid glucurinoco or L - iduronic acid) and a D-glucosamine and residue. Many of these disaccharides are sulfated into uronic acid residues and / or glucosamine residues. Generally, heparin has a molecular weight of 6,000 Daltons at 20,000 Daltons, depending on the heparin acid and the methods used to isolate it. The structural form of the repetitive disaccharide units of heparin is generalized as follows: In the above formula, the "squiggle" attached to the C5-C6 of uronic acid indicates that the uronic acid may also be D-glucuronic acid (carboxyl above) or L-iduronic acid (carboxyl below). In sum the Ri can also be H or S03, and R2 can be H, Ac (acetyl) or S03. "The low molecular weight of heparin" (LMWH) includes references to the preparation of heparin having a molecular weight of about 3,000 Daltons to 8,000 Daltons. "Low affinity heparin" (LAH) (or, alternatively, "simple low affinity heparin" (LASH)), as used herein, includes references to the preparation of heparin whose binding which binds to antithrombin III ( ATI 11) with an affinity of 10-6 M, generally, weaker than 10-5 M.

"Low affinity and low molecular weight heparin" (LA-LMWH), includes reference to the preparation of heparin having a molecular weight of 3,000 to 8,000 Daltons and whose binding to ATII with an affinity of 10-6 M, preferably, weaker 10-5 M. The preparation LA-LMWH can be a mixture of heparin molecules having a molecular weight range of 3,000 to 8,000 Daltons (± 1, 000 Daltons). This mixture of molecules LA-LWMH is generally referred to as V18 or, alternatively, as L18. Alternatively, the mixture of heparin molecules can be separated using several components, for example excluding the chromatographic gel. The LA-LMWH mixture can be separated into three fractions of the LA-LWMH by means of the chromatographic gel. Fraction 1 has a molecular weight of 8,000 Daltons, Fraction 2 has a molecular weight of about 5,000 Daltons and Fraction 3 a molecular weight of about 3,000 Daltons. A "heparin preparation" includes references to a simple preparation of unfractionated heparin. In a previous embodiment, the HCII specific catalytic agents of this invention are modified heparins which have been prepared from unfractionated single heparin using the following chemical reactions: (1) depolemirization of unfractionated heparin nitrous acid to produce low molecular weight heparin (LMWH); (2) oxidation of the LMWH result to open the structures of the divided uronic acid without sulfation using, for example, sodium periodate; and (3) reduction of the oxidized LMWH to reduce the aldehydes (to alcohols) formed during the depolymerization and the oxidation steps, for example, sodium borohydrate.

"Sulphated heparin" (HS), is given as a reference to a glycosaminoglycan which contains a repeated desiccated unit similar to heparin, but which has more N-acetyl groups, a few N-sulfate groups, and a higher level. low of O-sulphate groups. "Sulfate Dermatan", (DS), is included as a reference in a heterogeneous glycosaminoglycan containing repeated disaccharide units consisting of N-acetyl-D-galactosamine and D-glucuronic and repeated disaccharin units in N-actyl-D-galactosamine and acid L-iduronic. Uroonic acids occur with varying sulfation levels. "Hirudin" is included as a reference to an antithrombin substance that is extracted from the salivary glands of leeches. "The hirulog" is included as a reference to a small synthetic analog of hirudin. "Monosaccharins" refers to a polyhydric alcohol also containing a group of aldehydes or a ketone, i.e., a simple sugar. Monosaccharins are included as a reference to a natural event of simple sugars, even though these sugars have been chemically modified. Modified monosaccharins include, but are not limited to, increasing or decreasing sulfation or having carboxyl modified amino or hydroxyl groups. Monosaccharins can be chemically modified by: N-desulfation (see, e.g., Inoue, Y., et al., Carbohydrate Res. 46, pp.87-95 (1976)); N-result (see, eg, Lloyd, AG, et al., Biochem Pharmacol., 20, pp. 637-648 (1971)), N-acetylation (see, eg, Danishefsky, I., et al., Biphys. 90 pp. 114-121 (1970); N-succinylation (see, eg, Nagasawa, K., et al., J. Biochem 81 pp. 989-993 (1977)); N-deacetylation (see, eg, Dimitriev, BA, et al., Carbohydr Res. 40, pp. 365-372 (1995); O-desulfation (see, eg, Jacobson, I., et al., J. Biol. Chem. 255, pp. 5084-5100 (1980)), carboxy reduction, methylation of free hydroxyl or amino groups, etc. "Polysaccharide", as used herein, refers to a linear or branched polymer of more than 10 monosaccharides that are chained by medium of glycosidic bonds "Polyanion", as used herein, refers to molecules that have a large number of negative charges. "Polyanionic carbohydrates" is included as a reference to carbohydrates that have a large number of negative charges. " An additive heparin ", as used herein, includes references to a heparin or compound similar to heparin which is associated with an agent prior to administration to the patient. After incorporation, the additive heparin is also an unfractionated heparin or the fraction isolated from the third part of a low molecular weight of unfractionated heparin. "Neighbor alcohol groups" is included as a reference to two hydroxyl groups on adjacent carbon atoms. Particularly, "close alcohol groups" are used herein to refer to the two hydroxyl groups on carbon atoms C2 and C3 of the heparin preparations of this invention. "Oxidation agent" or, alternatively, "oxidant", is included as reference (1) Oxygen after redituting it, (2) removes the hydrogen from a compound, or (3) attracts negative electrons. Includes oxidation reagents allowed but not limited to sodium periodate, under reactions and conditions known to those that work ably, lead tetraacetate. "Reductive agent" or alternatively "reducing agent" is included as a reference to a substance that is oxidized by reducing another substance. It includes agents that allow to reduce, but are not limited to, sodium borohydrogen, and other hydrogen metals, under known reactions and conditions to those that work skillfully, lithium aluminum hydrogen. "Affinity" is expressed in terms of the dissociation constant (Kd). As such, in each time an affinity is mentioned, it refers to the Kd, not the Ka. "An increase in clinical insecurity in systemic bleeding", as mentioned herein, includes a preferred incorporation, referring to a clot activated in less than 400 seconds and to a thrombin clot in less than 100 seconds as long as the agent is used at the highest and most effective concentrations. The "antifactor test Ha", as used here, is a catalyst test HCII which is carried out as follows: a fixed amount of human thrombin is added to plasma containing a chromogenic synthetic thrombin substrate.

After incubation with the compound of interest, the amount of residual thrombin is determined by measuring the absorbance at 405 nanometers. The "Xa antifactor test", as used here, is a catalyst test ATI 11 which is carried out as follows: a fixed amount of factor Xa is added to the plasma which contains a synthetic chromogenic factor of the subtracted factor Xa.

After incubation with the component of interest, the amount of the residual factor Xa in activation is determined by measuring the absorption at 405nm. The "clot test sample", as mentioned herein, includes as a reference a test used to examine the inhibitory effect of agents against the fluid phase and the clot linked to the thrombin activity, a thrombin (0.2-4.0 nM) is incubated with citro-plasma for 60 minutes at 37 ° C in the presence or absence of heparin at the indicated concentrations. At the end of the incubation period, plasma levels of Fibrinopeptide A (FPA) are determined, and the percentage of inhibition of FPA generation are calculated by each inhibitor in concentration. To determine the inhibitory effect against the thrombin activity bound to the clot, the washed fibrin clots are incubated in plasma citrate for 60 minutes at 37 ° C in the presence or absence of varying heparin concentrations. At the end of the incubation period, the plasma levels of FPA are determined, and the percentage of clot inhibition induced in the generation of APF is then calculated by each inhibitor in concentration. A "plate-taking test" (or, alternatively, "filtration-based test"), as mentioned herein, includes as a reference an analysis used to test the ability of a component to displace and inactivate bound thrombin to a fibrin. This analysis is carried out using the registry of the examples mentioned below. It has been shown that the binding of fibrin placed in thrombin (anion bound to exosite 2) is different from the local fibrinogen binding of thrombin (anion bound to exosite 1). What it is, when thrombin is bound to fibrin, as is the case in the clot or matrices of thrombin, the local fibrin binding will be occupied, whereas the local fibrinogen binding residues are arranged to bind to the fibrinogen and Orient the bound fibrinogen by interaction with local catalytic thrombin which promotes the conversion of fibrinogen to fibrin. The active local enzyme and exosite 1 are available to bind and split other substrates as well as, include factor V, factor VIII and the thrombin receptor on platelets. Thus, it is possible to break or interfere with the binding between the local fibrin binding on thrombin and / or the binding of local thrombin on the fibrin in order to separate thrombin from the clot or thrombi within a fenced aqueous environment, generally within of blood in the vascular medium. While the fibrin bound to thrombin in the clot or thrombi is protected by inactivation of heparin and endogenous antiproteins, thrombin isolated from the clot or thrombi becomes susceptible to inactivation of endogenous antiproteins and / or suitable medicinal therapies. It is known that free thrombin, i.e., thrombin in its liquid phase, is irreversibly inactivated by two plasma inhibitors, antithrombin III (ATIII) and cofactor heparin II (HCII), with ATIII being the primary inhibitor. Both inhibitors give an increased marked activation in the presence of certain classes of sulfated polysaccharides. What results from the activity of ATII is catalyzed by heparin and by low molecular weight heparin (LMWH), while the activity of the HCII is catalyzed by high concentrations of heparin and dermatan sulfate. Although heparin (but not LMWH) is a potent HCII catalyst, the predominant effect of heparin at therapeutic concentrations is an ATIII catalyst, because heparin has a high affinity for ATI 11 than at such concentrations of HCII. In addition, the plasma concentration of ATI II is more than twice that of HCII (2.4 mM and 1 mM, respectively) and, therefore, heparin catalyzes both ATI 11 and HCII only when administered at very high concentrations. The HCII inactivates thrombin by acting as a pseudo-substrate of the enzyme. Thrombin divides HCl I, resulting in the formation of a complex enzyme-inhibitor covalent. The formation of this complex is very slow in the absence of a catalytic. Thus, in the presence of dermatan sulfate or heparin, the HCII value by inhibiting thrombin increases by about 2,000 folds (see, Tallefson, Ann., NY Acad. Sci. 714: 21-31 (1994)). A model by HCII catalysis by dermatan sulfate or heparin is carried out in a process of two steps. First, the sulfated polysaccharin binds to a region of positive charges in HCII and causes unfolding of the N-terminal, negative charge segment of the HCII molecule (FIG 1). This electronegative N-terminal segment then interacts with the region of positive charges on thrombin known as exosite 1. The interaction of the HCII as altered with thrombin increases the inhibition value by approximately 50 folds. If the sulfated polysaccharin chain is sufficiently long, a second step comes which strongly attributes the HCII value by inactivating thrombin.

In this second step, the sulfated polysaccharide binds the electropositive region to the thrombin known as exosite 2. The sulfated polysaccharins that can bind both the inhibitor and the enzyme effectively approximate the HCII and thrombin, thereby increasing the HCII value by inactivation of thrombin over 2,000 times. The activity of the specific HCII catalytic agents of the present invention is based, in part, on inactivating thrombin bound to fibrin without the need for displacement. Particularly, the postulated mode of activity of the specific HCII catalytic agents of the present invention is based on the following observations. First, because thrombin bound to fibrin via exosite 2, exosite 2 is available to interact with the altered HCII. As a result, the altered HCII can inactivate thrombin bound to fibrin without the need to displace it. Second, it has been discovered that certain sulfated polysaccharides, which are poor catalysts of inactivation by thrombin HCII in solution because they are of insufficient chains to bind to HCII and to thrombin, are able to effectively catalyze the inactivation of fibrin bound to the thrombin by HCM. Therefore, the specific catalytic agents of this invention are useful as selective inhibitors of clot-bound thrombin in vivo because they inactivate fibrin-bound thrombin without inducing a marked system of anticoagulant system. Without attempting to be limited to a particular theory, it has been thought that this selective inhibition occurs due to the requirements for catalysis, ie, activation, inactivation by HCII of free thrombin are different from those needed by activation of inactivation by fibrin-bound HCII. the thrombin. Therefore, according to the present invention, it has been identified that polyanions are more effective in inactivating fibrin linked to thrombipase than free fibrin. These polyanions are unable to fully catalyze the inactivation by HCII of free thrombin since they are also of insufficient chain, or because they lack of negative charges necessarily to bind to HCII and to thrombin. Such agents, however, are capable of inactivating fibrin bound to thrombin since, in theory, they are bound (ie, binding of HCII and thrombin) is not a prerequisite by inactivation by efficient HCII of thrombin when the enzyme is immobilized on the surface of fibrin. Instead of a conformational alteration of HCII, for example, sulfated polyanion is sufficient to promote inactivation. The ability of sulfated polysaccharides to bind HCII and thrombin depends on their size. Sulphated polysaccharides which contain less than 24 monosaccharide units are generally of insufficient length to bind thrombin and, from here, do not dramatically increase the thrombin inhibition value by HCII. However, because these agents can still bind to the HCII and induce a conformational change, they will increase the inhibition value of thrombin to a modest extent. In addition, when thrombin is bound to fibrin to exosite 2 and immobilized, polysaccharin will not be required to bind to thrombin and, therefore, thrombin bound to the clot can be easily inactivated by the HCM that the inhibitor has been modified. by low molecular weight polysaccharin (FIGS 2 and 3). What has been thought is a possible mechanical explanation for the selectivity of the specific catalytic agents of this invention against thrombin bound to the clot. The compositions and methods of the present invention may provide for complete or semi-complete inactivation of the clot or thrombi bound to thrombin, while amplification by coagulation thrombin is substantially inhibited or prevented. Generally, at least 60% inhibition of the clot or thrombi linked to thrombin can be carried out, preferably at least 90% inhibition, and still more at least 95% inhibition (by "inhibition", in this case). context, tries that the thrombin activity substantially and irreversibly inactivated so that the thrombin molecule can not promote the formation and increase of the clot or thrombi; Generally, inhibition refers to a reduction in activity. Several catalytic tests of HCM or, alternatively, active assays of HCM are well known to those who work skillfully. An example of the active assay of HCII is the anti-factor assay (see, Ofoso, Blood 64: 742-747 (1984)). Other active assays of HCII include those described in U.S. Patent Application. Series No. 08/175, 211 (file December 27, 1993). Similarly, several ATI 11 catalytic assays or, alternatively, ATM I activation assays are well known to those who work well. An example of such an ATI 11 activation assay is the anti-Xa assay (see, Teien, et al., Throm. Res. 8: 413-416 (1976)). In sum, several anticoagulant assays are known as those which are effective, such as thrombin bound to the temporal clot, temporal coagulating factor Xa, partial temporal thromboplastin and temporary active coagulant. The following assays are generally described in, eg, Low-Molecular-Weight Heparin in Prophylaxis and Therapy of Thromboembolic Diseases (H. Bounameaux (ed.); Marcel Dekker, Inc.; New York, New York (1994)), the teachings of which are incorporated herein by reference for all purposes. The products and agents of the invention will be described in more detail, only in an exemplary manner, with illustrative reference to the specific HCII catalytic agents of the present invention. The preferred properties described below in relation to the specific HCM catalytic agents are also applicable to the agents and products of other aspects of the invention. The HCII specific catalytic products and agents of this invention will preferably exhibit one or more of the following characteristics: i) a specific HCM catalytic activity or, alternatively, a specific HCM activation activity of 2 to 5 units / mg in an antifactor assay chromogenic agent and, preferably, about 3 to 4 units / mg; I) a specific catalytic activity ATI 11 or, as well, a specific activation activity ATI f I of 0.2 to 1.5 units / mg in a chromogenic antifactor Xa assay and, preferably, about 0.5 to 1.3 units / mg; iii) a solubility in aqueous medium space of 150 to 1,000 mg / ml, preferably of about 200 to 750 mg / ml or about 400 to 500 mg / ml. iv) at concentrations that perform an in vivo antithrombic effect, a plasma anticoagulant activity as measured by temporary coagulant thrombin for 20 to 80 seconds (with a 20 second control), preferably 25 to 45 seconds, and even more 30 seconds; and v) an anticoagulant effect which is contributed to a mechanism through HCM and ATIII, and by a mechanism which is independent of HCH and AT III. The HCM specific catalytic agents of the present invention are preferably glycosaminoglycans. In particular, the specific catalytic agents HCIIs are preferably polyanionic carbohydrates of about 14 to 20 monosaccharin units. Generally the HCII specific catalytic agents are heparin preparations having a molecular weight of about 3,000 to 8,000 Daltons (± 1, 000 Daltons). As such, in one scheme, heparin is a preferred acid of the HCM specific catalytic agents of this invention. Heparin, as has been explained, is a high sulfated dextrorotative compressed mucosacarin of D-glucosamine and D-glucuronic acid or L-iduronic residues. Generally, heparin has a molecular weight of about 6,000 to 20,000 Daltons, depending on the heparin acid and the methods used to isolate it. In a preferred scheme, the HCII specific catalytic agents of the present invention are heparin preparations using the following chemical reactions: 1) depolymerization of unfractionated heparin by producing LMWH; 2) oxidation of the LMWH result; 3) reduction of oxidized LMWH; 4) which is apparently easy for those who work skillfully what the order of these steps can be modified, e.g., unfractionated heparin can be oxidized, depolymerized and reduced, provided for the final step that is at a reduced pace. In sum, the heparin preparations are, however, the specific HCII catalytic agents of the present invention which can be, for example, negatively charged polysaccharides to heparin others, polyanions with negative charges, electronegative organic molecules with an affinity of HCII and the like. Such substances generally bind to HCM with an affinity of at least 10-6 M, preferably at least 10-8 M or stronger, but with weak affinity for thrombin, preferably weaker than 10-6 M. In one of the embodiments, the HCII specific catalytic agents of this invention can have the following formula: In the above formula, R1 is a related member of the group of H, D-residue glucosamino acid, D-residue glucuronic acid and L-residue idurónico acid consistent. R2 is a select member of the group consisting of H, D-residue glucosamino acid, D-residue glucuronic acid and L-residue iduronic acid and anhydromannitol. R3, in the above formula, is a select member of the group consisting of H and S03 finally, R4 is a select member of the group consisting of H, S03 and CH3CO. In the above formula, the indices "n" are independent and can have linear values from 0 to 14. The values of "n" are selected such that the specific HCII catalytic agents of the present invention will have molecular weights of about 3,000 Daltons to 8,000. Daltons (± 1, 000 Daltons). In another scheme, the heparin derived from the HCM catalytic agents of this invention may have the following formula: In the above formula, R1 and R2 are independent members selected from the group consisting of H, D-residue glucosamino acid, D-residue glucuronic acid and L- iduronic acid residue. R3, in the above formula, is a select member of the group consisting of H and S03 finally, R4 is a select member of the group consisting of H, S03 and CH3CO. In the above structure the sequences have a pentasaccharide heparin sequence by ATI 11 that is substantially removed by ATIII chromatographic affinity. As mentioned, the HCII catalytic agents of this invention will generally have molecular weights of about 3,000 to 8,000 Daltons (± 1, 000 Daltons). Specific catalytic agents HCM, i.e., catalytic agents which selectively catalyze the inactivation by HCM of thrombin-bound fibrin, obtained from the heparin preparations can be prepared using a number of different methods. As mentioned above, in a preferred scheme, the HCM catalytic agents of the present invention are heparin preparations using the following chemical reactions: 1) depolymerization of unfractionated heparin by producing LMWH using nitrous acid; 2) oxidation of the LMWH result using sodium periodate; and 3) reduction of the oxidized LMWH using sodium hydrate. In this scheme, the result of low affinity heparin in low molecular weight is a mixture of heparin molecules having a native sugar (eg, uronic acid residue) as the non-reduced terminal and an anhydromannitol 2.5 residue as the reduced term, and having molecular weights of about 3,000 to 8,000 Daltons. Particularly, heparin with low affinity of ATM I is prepared from an unfractionated simple heparin (specific activity 150 to 160 antifactor Xa and antifactor lia units / mg) or low molecular weight heparin chemically also by an oxidation reaction followed by a reaction reduced or, alternatively, by a chromatographic affinity ATI II. In a preferred scheme, the low affinity heparin of ATI 11 is chemically prepared. Such chemical modifications involve a treatment of alcohol-vicinal groups present in the preparation of heparin with an oxidizing agent followed by a reducing agent according to the protocol placed ahead in an exemplary section S.E. properly the oxidizing agents included, but not limited to, sodium periodate and, under certain known reactions to those that work well, lead tetracetate. Properly reducing agents included, but not limited to, sodium hydrate, other hydrated metals and, under a reaction certainly known to those that work well, hydroxide lithium aluminum. These reactions split the neigh i.e., C2-C3 units to a critical non-sulfated glucuronic acid residue found without the pentasaccharide heparin sequence for ATIM. The division of this ligature markedly reduces the affinity of heparin for ATIII. Alternatively, the chromatographic affinity ATIII can be used to select those heparin chains with little affinity for ATI 11 when prepared by another technique, the low affinity of heparin (LAH) has the following characteristics: a) It is essentially free of ATIII catalysis, ie, active reaction ATIM (with antifactor Xa 1.5 units / mg), but retains the antifactor activity Ha (about 2 to 5 units / mg) because this ability to catalyze, ie, active, HCII. b) Compared with the running material, the LAH or LA-LMWH has reduced the anticoagulant activity in plasma (as measured also by activated partial temporal thromboplastin or thrombin bound to the temporary clot), but retains this activity by catalyzing it in activation of spiral thrombin containing physiological concentrations of HCM (see table 1).

TABLE 1 c) In a filter containing nickel-plated assays, LAH is as effective as simple heparin in promoting displacement by HCM (FIG.4) and inactivation (FIG.5) or fibrin bound to thrombin. Both simple heparin and LAH inhibit fibrin bound to thrombin to a greater extent than LAH displaces it, consistent with the concept that HCM is able to bind and inactivate thrombin that is bound to fibrin via exosite 2. d) The majority of the thrombin displacement also by single heparin or LAH is covalently composed of the HCH as determined by SDS-PAGE analysis (data not shown). e) Based on the extent of clot inhibition induced fibrinopeptide A (FPA) generation in plasma, LAH is as effective as simple and unfractionated heparin in inactivating fibrin bound to thrombin (FIGS 6A and 6B). In a suspended coagulate assay, both agents produce only a minimal thrombin displacement because it is linked to fibrin via exosite 2, starting at exosite 1 and the free local active when interacting with the HCII. A fraction of low molecular weight heparin (LMWH) can be prepared using a variety of techniques, including simple depolymerization using nitrous acids, enzymatic degradation with heparinase and gel filtration. In a given scheme, the LWMH is prepared by simple and unfractionated heparin using depolymerization of nitrous acid. The result of LMWH is usually a mixture of heparin molecules having a molecular weight of about 3,000 to 8,000 Daltons (± 1, 000 Daltons). This mixture of materials can be used directly or can be separated into its various components using, for example, chromatographic gel exclusion. For example, the LMWH mixture can be arbitrarily separated into three fractions by exclusion of chromatographic gel. Fraction 1 has a molecular weight of about 8,000 Daltons, Fraction 2 has about 5,000 Daltons and Fraction 3 a molecular weight close to 3,000 Daltons. As illustrated in the examples hereinabove, these three fractions have properties which are different from each as well as from the mixture of materials, i.e., the mixture of heparin molecules have a molecular weight of about 3,000 to 8,000 Daltons. Therefore, depending on whether the mixture of materials, Fraction 1, Fraction 2 and Fraction 3 or various combinations of these materials are used, one can take advantage of different properties. However, the LA-LMWH, e.g. V18 is less effective than simple heparin in displacing thrombin from the fibrin-coagulated filter (FIG 4), which effectively inhibits thrombin binding (FIG 5). For example, 4 mM of V18, a specific catalytic agent which is a mixture of LA-LMWH molecules having molecular weights of about 3,000 to 8,000 Daltons, displaces only 60% of the bound thrombin, but produces 85% more than inhibition of the activity of the bound enzyme. However, V18 causes less inhibition of thrombin in its liquid phase because 4 mM of V18 produces a thrombin bound to the temporary clot of only 50s, while 4 mM of V18 of single heparin produces a thrombin bound to the excessive temporal clot. . The LA-LMWH of the present invention has the following characteristics: a) Its specific activity is about 0.2 to 1.5 units / mg anti-factor Xa and about 2 to 5 units / mg anti-factor Ha. B) Compared with simple heparin without fractionation and LAH, LA-LMWH has a lower anticoagulant activity in plasma (as measured by thrombin bound to the temporal clot) and produces less catalysis, ee, activation, of thrombin inactivation by HCII in physiological concentrations containing HCII coils (see , Table, suppra). c) In a filter containing nickel-plated assays, LA-LMWH is less effective than simple heparin or LAH in promoting the displacement by HCM of fibrin-thrombin, but is capable of promoting the inactivation of thrombin bound to a similar extent. (FIGS 5 and 6). d) Based on its ability to inhibit the clot-induced fibrinopeptide A (FPA) generation in plasma (FIGS 6A and 6B), LA-LMWH is substantially as effective as simple and unfractionated heparin and LAH in inactivating fibrin bound to thrombin. In contrast, LA-LMWH is less than about one-tenth as effective as heparin and LAH in inactivating free thrombin in a thrombin assay bound to the temporal clot. Therefore compared to single and unfractionated heparin and LAH, LA-LMWH selectively promotes the inactivation of fibrin bound to thrombin. e) The LA-LMWH of this invention has an anticoagulant effect which is contributed to a mechanism by HCM and ATIII, and by a mechanism which is independent of the HCM and the ATIII.

Therefore, the specific HCM catalyst products and agent of this invention preferably have one or more of the following characteristics. In one scheme, the present preferred invention provides heparin preparations that have been chemically modified in two ways. First chemically have a depolymerized use, eg, nitrous acid and, therefore, diminished in sides of about a third of the size of heparin of molecular origin and, generally has molecular weights of 3,000 to 8,000 Daltons (± 1, 000 Daltons ). This modification results in a remarkable loss of its ability to catalyze the activity of heparin cofactor II (HCII) against free thrombin, while retaining its ability to catalyze the activity of HCII against the surface of thrombin. Second, they are subject to oxidation and reduction reactions which result in a loss of about 95 to 99% of their activity linked to antithrombin III. Particularly, they have weakness to the catalytic activity ATI 11 or, well, activating reaction ATI 11 as evidenced by a level of antifactor Xa of approximately 0.2 to 1.5 units per ml, which represents about 95 to 99% of decrease in activity compared to unmodified low molecular weight heparin. Therefore, the result of the specific HCII catalytic agents of this invention have a weak activity against free thrombin. These two properties, especially, have a relatively greater ability than other catalytic HCII or, HCII activators (such as sulfate dermatan) to inactivate the bound surface of free thrombin and some, although markedly diminished, the activity of antifactor Xa and the dependent ATM, are responsible for its improved antithrombic effects compared to heparin, dermatan sulfate and hirudin in a blocked circuit, which is a measure of the surface of bound thrombin, at concentrations which have a lesser effect than these anticoagulants in the inactivation of free thrombin as measured by the prolongation of thrombin bound to the temporal clot. In addition, surprisingly, such agents have an anticoagulant effect which is contributed in a mechanism by HCM and ATIII. As a result of the following properties, the HCII-specific catalytic products and agents of this invention, ee, catalysts which are capable of displacing and / or inactivating thrombin bound to the clot, can be used to prevent thrombus formation and / or block the growth of thrombi in a patient without causing excessive bleeding, ie, if it produces a disease in the blood system. Particularly, the specific catalytic agents of this invention can be administered in doses which inactivate the surface of thrombin, but which have a minimal effect on the inhibition of free thrombin. As such, in another scheme, the present invention provides a method by inhibiting thrombus formation in a patient, preferably without leading to a clinical disease in the blood system. The method comprises the step of administering to the patient a pharmacologically acceptable dose of a heparin specific cofactor II catalyst agent capable of inactivating thrombin bound to the clot, the specific catalytic agent HCII being characterized by: (i) a heparin specific activity cofactor II against the heparin cofactor II from 2 to 5 units / mg in an antifactor assay; (ii) a specific antithrombin III (ATIII) activity against a factor Xa of 0.2 to 1.5 units / mg in a Xa antifactor assay; and (iii) a solubility in aqueous medium of 150 to 1,000 mg / ml. The specific catalytic agents HCII (and other thrombin activators) of this invention are effective when used alone or, when used in low doses of heparin or low molecular weight heparin which is required by inactivating free thrombin. In conditions of resistant heparin (i.e., disorders of very high doses of heparin), for example, the HCM specific catalytic agents of this invention can be used as a heparin-sparing agent as such, in another scheme, the present invention provides combinations and bonds of a specific catalytic agent HCII and a catalytic agent ATI II, ie, an activation agent ATIII. Such bonds are useful for preventing thrombogenesis in a patient without substantially preventing normal coagulation. Therefore, the compositions or linkages of this invention are useful for preventing the formation of thrombi in a patient without inducing a clinical disease in the blood system. In sum, such compositions or unions are useful for prophylaxis, for the treatment of veins or arterial thrombosis and for the prevention of coagulants in extra corporal circuits. The mixtures of this invention are generally given from 90 to 99.9% by weight of an HCM agent further, from 95 to 98.5% by weight; and from 0.1 to 10% by weight of an ATIII agent, i.e., an active ATI 11 of 0.5 to 5% by weight. Generally, all the HCII catalytic activity of the bonds will be from 2 to 5 units / mg and from 2 to 4 units / mg. The catalytic ATI 11 or activates are suitable for use in the joints of this invention shown, but not limited, heparin and LMWH. In sum, other agents can be added to modify the binding, including, for example, LMWH (0.1 to 5% by weight), (heparin 0.1 to 5% by weight) direct thrombin inhibitors of active factor X, etc. In another embodiment, the invention provides a method for preventing thrombin bound to the clot and thrombin in its liquid phase in a patient without leading to a clinical discomfort of the blood system, the method comprising the step of administering to the patient a pharmacologically acceptable dose. of (i) an HCII agent capable of preventing thrombin bound to the clot in HCM agent has a minimal affinity for antithrombin III (ATM) and a heparin of specific activity cofactor II of about 2 to 5 units / mg in an antifactor assay; and (ii) an ATI II agent capable of inactivating thrombin in its liquid phase. Properly the agents ATIII, i.e., AT lll activating agents, including but not limited, heparin, heparin in low molecular weight, direct inhibitors of thrombin and direct inhibitors of activated factor X. Also in this method, the HCM agent and the ATIII, or activating agent can be administered to the patient simultaneously, separately or sequentially. When the patient is administered simultaneously, the HCM agent and the ATI 11 can be administered together or in a single or composite solution, they can even be administered separately as two different or compound solutions. The HCII agents and mixtures of this invention can be incorporated as components in pharmaceutical compositions which are useful for the treatment of cardiovascular conditions described above. Such compositions will also be useful in conjunction with conventional thrombotic treatments, such as administration of plasminogen activator tissue (tPA), streptokinase, and the like also as an intravascular intervention, such as angioplasty, arterectomy, and the like. Suitable pharmaceutical compositions will contain a dose of therapeutic effect of one or more active products of the invention, a catalytic agent HCII in a pharmaceutically acceptable charge. Other pharmaceutical compositions will contain a dose of therapeutic effect of a binding of an active product of the HCH agent and an ATIM agent. By a "dose of therapeutic effect" it is given that a sufficient amount of HCII agent or, alternatively, a combination or binding of. for example, an HCII agent and an ATI 11 agent will be presented in order to inactivate thrombin bound to the clot i / o by preventing the augmentation or thrombi when treating a cardiovascular thrombosis condition, such as those described above.

Generally, the active product of the HCII agent will be presented in the pharmaceutical compound at a spaced concentration of about 200 mg per dose at 2g per dose and, preferably, at a concentration of 500 mg per dose at 1 g per dose. The daily doses can vary widely depending on the activity of the particular HCM agent used, but will generally be presented in a concentration space 30 mg per kg of weight per day at 500 mg per kg of weight per day and, even more, to a space of concentration of 50 mg per kg of weight per day to 200 mg per kg of weight per day. With respect to the combined or pharmaceutically bound compositions, the active product or HCII agent will be presented in a concentration space of 3 mg / kg per dose of 30 mg / kg per dose and, to a concentration space of 3 mg / kg per dose. dose at 10 mg / kg per dose, and agent ATIII shall be presented in a concentration space of 10 U / kg per dose at 500 U / kg per dose and, a concentration space of 15 U / kg per dose at 100 U / kg per dose. Daily doses may vary widely depending on the activity of the particular agent HCM and ATI II, or agents actively employed. Generally, the HCM agent will be presented in a concentration space of 3 mg per kg of weight per day to 30 mg per kg of weight per day and, a concentration space of 3 mg per kg of weight per day to 10 mg per kg of weights per day, while agent ATI 11 will usually be presented in a concentration space of 10 U / kg of weight per day at 500 U / kg of weight per day and, a concentration space of 15 U / kg of weight per day at 100 U / kg of weight per day. The acceptable pharmaceutical carrier can have any compatible, non-toxic substance upon arrival of the HCM agent or the binding of HCII and ATI II to the patient. Sterile water, alcohol, nutrients, waxes and inert solids can be used as carriers. Assistants, pharmaceutically acceptable, spiral agents, dispersed agents and the like can be incorporated into the pharmaceutical compositions. Such compositions will be suitable for an oral, nasal, respiratory or parenteral administration, or, subcutaneous vascular or intravenous, can even occur when the substances provided by this invention arrive by transdermal administration. In view of the following, it is apparently easy for those HCII agents which have good function and which can be used to effectively inhibit the clot or fibrin bound to thrombin without leading to a clinically serious disease in the blood system. In sum, the HCM agents of this invention can be effective when used in combination with other catalytic ATIII, activating agents, (heparin or LMWH) to inhibit thrombin bound to the clot and thrombin in its liquid phase without giving a serious disease in the blood system. As such, the HCII agents of this invention can be used alone or in combination with other ATMI catalysts in treating a significant number of cardiovascular complications, including unstable angina, acute myocardial infarction (heart attack), cerebral vascular accidents (stroke), pulmonary embolism , acute venal thrombosis, arterial thrombosis, etc. In summary, being useful in pharmaceutical compositions for the treatment of cardiovascular conditions described above, it will be readily appreciated that the active products or HCM agents and combinations of this invention can be used as reagents to explain the in vitro coagulation mechanism. Having produced heparin fractions or their derivatives having there the properties described above, other thrombin inactivators or HCII agents having specific catalytic affinity can be produced by a variety of well-known methods of their good function. For example, Fodor, et al., U.S. Patent No. 5,143,854 discloses a technique called "VLSIPS" in which diverse collection of short peptides are formed by selecting positions on solid substrate. Such peptides are then out by the ability to alternate according to the HCM, such residues optionally in competition with the heparin fractions. Short peptides can also be produced and subtracted using a phage display technology (see, e.g., Devlin, WO 91/18980). Optionally, this polypeptides or variants are produced by other variegated methods, by obtaining an improved binding affinity for HCM (see, eg, Ladner, US Patent No. 5,223,409, which is incorporated by reference in its entirety for all purposes) . The peptide analogues of HCII activators can be prepared by a conventional solid phase synthesis or recombinant techniques, both of which are well described in their function. The synthesis available solid phase techniques are based on the sequential addition of amino acids to a growth chain on a solid substrate phase, as first described in Merriefield, J. A. m. Chem. Soc. 85: 2149-2156 (1963). Commercial systems for automated solid phase synthesis are now widely available to provide, such as Applied Biosystems, Inc.; Foster City California. In addition, the recombinant polypeptide production techniques that are best described in the Technical and Scientific Literature. See, for example, Molecular Cloning: A Laboratory Manual, Sambrook, et al., Eds; Cold Spring Harvor Laboraty Press, Cold Spring Harvor, New York (1989), vols. 1 -3. The small molecule mimetics of the HCM agent of this invention can be carried out through the use of techniques known for that work in the area of drug design. Such methods included, but not limited to, the analysis of the consistent field itself (SEF), interaction configuration analysis (Cl) and dynamic computation programs in normal mode, all of which are available. See, Rein, et al., Computer-Assisted Modeling of Receptor-Ligand Interactions, Alan Liss, New York (1989) and Navia and Marcko, "Use of Structural Information in Drug Design", in Current Opinion in Structural Biology, Vol. 2, No. 2, pages 202-210, 1992. The preparation of compounds identified by these techniques will depend on their structure and other characteristics and can normally be carried out by simple chemical synthesis methods as described in the available texts such as Furniss, et al., Vogel's Textbook of Practical Organic Chemistry., John Wiley & Sons, New York 1992 and Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York 1989. The invention will be described in more detail in the form of specific examples. The following examples offer illustrative purposes and none is intended to limit or define the invention in any way. In the examples the thrombin inactivators are illustrated by heparin derived from HCII agents, but the invention is not limited to such agents.

EXAMPLES A. Preparation of the material.

Heparin is a glycosaminoglycan (GAG) isolated from natural acids (usually from the intestinal mucosa of the pig). One aspect of this invention is to prepare a low molecular weight heparin fraction that is essentially free of antithrombin III activity, yet retains the ability to catalyze the heparin cofactor II. As a consequence of its ability to catalyze the heparin cofactor II, this modified (GAG) can inactivate the thrombin bound to fibrin. Generally, low molecular weight heparin is prepared from simple unfractionated heparin by benzylation followed by alkaline depolymerization; depolymerization of nitrous acid; enzymatic depolymerization with heparinase; or perioxidative depolymerization. In this case the low molecular weight heparin is prepared from simple heparin using depolymerized nitrous acid. Low molecular weight heparin is then modified by reducing its affinity for antithrombin III. This may be chemically accomplished, or through the use of an antithrombin III affinity column. In this case it is chemically carried out by an oxidation reaction followed by a reduction reaction. The result of the low affinity of low molecular weight heparin is generally a mixture of heparin molecules having molecular weights of 3,000 to 8,000 Daltons. This range corresponds to 10 to 24 monosaccharide units. The specific activity of this material is 2 to 5 U / mg antithrombin and less than 1.5 (starting 100 anti-Xa) anti-Xa U / mg. The mixture of LA-LMWH can be used directly or, it can be separated within several compounds using for example chromatographic exclusion gel. The fraction of LA-LMWH generally produces three fractions of LA-LMWH when they are separated by chromatographic exclusion gel. Fraction 1 has a molecular weight of 8,000 Daltons, Fraction 2 has a molecular weight of 5,000 Daltons and Fraction 3 has a molecular weight of 3,000 Daltons FIG. 7 Illustrates the elution contour of the LA-LMWH mixture of materials using a G-50 SEPHADEX column in various concentrations of glucuronolactones. As with the LA-LMWH mixture of materials the specific activity of these three fractions LA-LMWH is 2 to 5 units / mg antithrombin and less than 1.5 (beginning, 100 anti-Xa) anti-Xa units / mg. The following examples illustrate experimental protocols which can be used to prepare HCM agents of this invention. As mentioned the main chemical reactions to the preparation of HCM agents of this invention are: (1) depolymerization of nitric acid of heparin by producing heparin in low molecular weight; (2) oxidation of heparin periodate in low molecular weight by opening the circular structures of uronic acid split in half without sulfation; and (3) reduction of aldehyde borohydrate (alcohols) formed during nitrous acid and periodate steps. 1. Example 1: Preparation of V-18, Lot # 7-1 a. Step 1: Isolation of heparin LMW. Lot # 7 Porcine intestinal heparin (250 grams) was dissolved in 5 liters of purified water (5% solution). The sodium nitrite was added and dissolved in the solution giving a concentration of 10 mM. The temperature of the solution was between 18 and 24 degrees Celcius. Over the course of 2 minutes, 30 ml of 37% HCl were added and the pH adjusted from an initial value of 6.32 to a final pH of 3.00. The result of the solution was then of a duration of 2 hours at room temperature by allowing the depolymerization to occur. At the end of the 2 hour incubation, the pH was adjusted to a value of 6.75 by slow addition of 18 ml of 50% NaOH solution. The sample was then diluted to a volume of 12.5 liters and ultrafiltered against 10 volumes of purified water using an apparatus equipped with Millipore Pellicon ultrafiltration with 10,000 cut-off molecular weight membranes (1.5 m 2). The penetration of the ultrafiltration was then concentrated and dialyzed using a Millipore Pellicon ultrafiltration equipped unit with 3,000 cut molecular weight membranes (1.5 m2). At the same time the 125 liters of penetration were concentrated of about 6 liters, was then dialyzed against 3 volumes of purified water. The retention was frozen dry when giving heparin LMW, Lot # 7. Product = 106 grams (42%). The molecular weight characteristics of heparin LMW intermediate # 7 (above) were determined by their high performance to the chromatographic exclusion in conjunction with laser multi-angle scattering (HPSEC-MALLS and are compiled in Table 2.

TABLE 2 - HPSEC-MALSS Results for intermediate Heparin & the product V18 Fraction with weight above a given molecular weight, expressed in percent. / '/' Average molecular weight weight //; Average number of molecular weight IV Polydispersity b. Steps 2 v 3: Oxidation and LMW Heparin Reduction. Lot # 7 LMW heparin, lot # 7 (100 grams) was dissolved in one liter of purified water at 4-10 Celcius degrees. One liter (1 liter) of a 200 mM sodium acetate solution, at a pH of 5.0 was added to the dissolved sample. Two liters (2 liters) of a 200 mM sodium periodate solution was added to the buffered LMW-heparin solution. The resulting solution was incubated in the dark at 4-10 degrees C for 48 hours with gentle agitation. At 48 hours, the excess sodium periodate was destroyed by the addition of glycerol (70 ml). The pH of the resulting reaction mixture was brought to neutrality with -10 ml of a 50% solution of NaOH and the resulting solution dissolved in about 5.5 liters. This solution was then dialyzed (ultrafiltrated) using the Millipore Pellicon system equipped with 3,000 MWCO membranes (as in step 1). After ~ 7 volumes of permeate were collected, the intermediate solution of oxidized LMW heparin was concentrated to a volume of -3 liters. The reduction of the oxidized intermediate was carried out in the following manner: 63 g of sodium bicarbonate was added and mixed in the 3 It of solution obtained above to give a solution -0.25 M NaHC03. This solution was kept cold (4-10 ° C) while a solution (1.2 liters) containing 22.8 g of NaBH. (also at 4 to 10 ° C) was gently added with agitation. Once all the NaBH4 solution was added, the reaction was maintained at 4-10 ° C for 3.5 hours. The reaction pH was then adjusted to 4.0 with 6M HCl (-230 ml), mixed for -30 minutes, then adjusted up to a pH of 6.2 with addition of 50% NaOH. This sample was then ultrafiltered once more with the 3,000 MWCO membrane using the Pellicon Millipore system (7 volumes) and finally filtered sterilely through a 0.2 μ filter Sterivex (millipore). The resulting solution was cold-dried to give V18, Lot # 71. Yield = 76.6 grams. The characteristic molecular weight of the product was determined by HPSEC-MALLS and is compiled in table 2, supra. 2. Example 2: Preparation of V18, Batches # 9-1 and 9-2 a. Step 1: Isolation of heparin LMW, Lot # 9 Porcine intestinal heparin (250 gr) was dissolved in 5 liters of purified water (5% solution). The sodium nitrite (8.62 g) was added and dissolved in the solution to give a concentration of 25 mM. The solution temperature was between 18 and 24 degrees Celcius. During the course of about 2 minutes, 33 milliliters of a solution at 37% HCl was added and the pH adjusted from an initial value of 6.32 to a final pH of 3.01. The resulting solution was then stirred for two hours at room temperature to allow depolymerization. At the end of the two hour incubation, the pH was adjusted up to a value of 6.28 by slow addition of a 50% NaOH solution. The sample was then diluted to a volume of 10 liters and ultrafiltered against 7 volumes of purified water using a Millipore ultrafiltration instrument Pellicon equipped with 3,000 MWCO membranes (1.5 m2). The sample was then concentrated to -3.5 liters and cold-dried to give heparin LMW Lot # 9.

Yield 120.4 gr (48%). b. Steps 2 v 3; Oxidation v Reduction of V18, Lots 9-1 v 9-2 Two equal but separate samples of 50 gr (50 gr) of LMW heparin, Lot # 9 was treated as follows: LMW heparin (50 gr) was dissolved in 500 ml of purified water (4 to 10 ° C). A sodium acetate buffer (500 ml of 200 mM, pH . 0) was added to each identical sample. Then sodium periodate was added (1,000 ml of a 200 mM solution) to each reaction and the resulting solution was each incubated in the dark and cold for 72 hours. After 72 hours of incubation, 40 ml of glycerol was added to each sample to destroy the excess periodate. The pH of the reaction, from each sample, was then brought to -6.5 and each ultrafiltered against 7 volumes of purified water using the Millipore Pellicon equipped with 3,000 MWCO membranes. The reduction of each of the oxidized LMW intermediate results and the other steps in were exactly as indicated in sample 7-1 in example 1 above. The yields of V18, lots # 9-1 and 9-2 were: 37.4 gr and 36.7 gr respectively. The molecular weight characteristics of V18. lots # 9-1 and 9-2 were determined by HPSEC-MALLS and is compiled in table 2, supra. 3. Example 3: Preparation of V18, Lot # 20-1 a. Step 1: Isolation of Heparin LMW, Lot # 20 In 500 liters of purified intestinal water, 500 grams of porcine intestinal heparin were dissolved. Sodium nitrate (9.0 gr) was added and dissolved in this solution to provide a concentration of 12.8 mM. The temperature of the solution is handled between 18 and 24 degrees Celcius. During the course of about 2 minutes, 59 ml of a 37% solution of HCl was added and the pH was adjusted from an initial value of 6.32 to a final pH of 3.0. The resulting solution was then stirred for two hours at room temperature to allow depolymerization. At the end of the two-hour incubation, the pH was adjusted up to a value of 6.57 by slow addition of a 50% NaOH solution. The sample was then diluted to a volume of 17 liters and ultrafiltered against 10 volumes of purified water using a Millipore Pellicon 11 ultrafiltration instrument equipped with 8,000 MWCO (1.5 m2) membranes. The permeate from the ultrafiltration was then concentrated using a Millipore Pellicon II ultrafiltration unit equipped with 3,000 MW Cut Off (1.5 m2) membranes. Once the 170 liters of the permeate was concentrated to around 8.5 liters, it was then dialyzed against 7 volumes of purified water. The retentate was then dry dried to give heparin LMW Lot # 20. Yield 171.3 gr (34%). The molecular weight characteristics of V18. lots # 9-1 and 9-2 were determined by HPSEC-MALLS and is compiled in table 2, supra. b. Steps 2 and 3: Oxidation v Reduction of V18. Lots 20-1 LMW heparin (170 g of lot 20) was dissolved in 1700 ml of purified water (4 to 10 ° C). A sodium acetate buffer solution (1700 ml of 200 mM, pH 5.0) was added and mixed. Sodium periodate (3,400 ml of a 200 mM solution) was added to the mixture to initiate the reaction and the resulting solution was incubated in the dark and cold for 72 hours. After this incubation time, glycerol (15 ml) was added to destroy the excess periodate. The pH of the reaction was then brought to -6.5 and the oxidized LMW heparin was ultrafiltered against 7 volumes of purified water using the Pellicon II system equipped with 3,000 MWCO membranes.

The reduction of the resulting oxidized LMW intermediate and all other step in the process was exactly as indicated for sample 7-1 in Examples (above). The performance of V18 Lot # 20-1 was 131 gr. The molecular weight characteristics of V18. lots # 9-1 and 9-2 were determined by HPSEC-MALLS and is compiled in table 2, supra.

B- Filtration-Based Test In summary, to examine the ability of several heparin derivatives to catalyze the inactivation by HCII of fluid thrombin, its ability to displace and inactivate fibrin bound to thrombin was studied. In this system, a fibrin clot was formed in a covered filter bed of a 96-well plate by adding 25 μl of buffer containing 8 μM fibrinogen, 8 mM CaCl 2 and 8 nM 125 I-labeled thrombin in each well. After incubation for 30 minutes at room temperature, the resulting clot was washed twice with 150 μl of buffer and the bound thrombin was quantified by radioactivity counting. Varying the concentrations of the materials derived from heparin was added with or without HCM. In each case, these components were added in a volume of 20 μl, incubated for 20 minutes and then aspirated through the filter plate. This step was repeated and the clot was then washed twice with 50 μl of buffer. The amount of thrombin displaced from the clot was determined by counting the wash buffer, then the amount of thrombin remaining bound to the fibrin was quantified by counting the filter. The enzymatic activity of the bound bound thrombin was determined by the addition of a synthetic thrombin substrate and by monitoring the changes in the fluorescent signal due to the reaction in a 3 minute interval. Finally, the washing with the buffer was collected and the filters were punctured, then heated in SDS-containing buffer, subjected to SDS-PAGE followed by autoradiography to determine the extent to which thrombin was complexed to HCM. As illustrated in Figure 4, 1 μM HCII (that the physiological concentration of HCM) displaces only small amounts of thrombin. SH, LASH, LMWH and a fraction of heparin with a molecular weight of 3,000 Daltons does not displace thrombin without HCM. However, when used in conjunction with 1 μM HCII, these substances all displace thrombin (Figure 4), with SH and LASH producing more displacement than LMWH (which has an average molecular weight of around 5,500 Daltons). However, all of these agents inhibit thrombin that remain bound to fibrin (Figure 5), with SH and LASH producing more or less complete inhibition at high concentrations and LA-LMWH, for example, V18, producing slightly less inhibition . Analysis by gel electrophoresis of the material that remains attached to the filter indicates that most of the thrombin is complexed to HCM, explaining its lack of chromogenic activity. These findings suggest that thrombin that remains attached to fibrin is bound via exosite 2, this leaving exosite 1 and the site available to interact with HCM.

C. Hanging Clot Test The hanging clot test is used to examine the inhibitory effects of agents against fluid-based thrombin activity and clot-bound thrombin. To determine inhibitory effects against fluid phase thrombin activity, a-thrombin (0.2-4.0 nM) was incubated with cited plasma for 60 minutes at 37 ° C in the presence or absence of heparin at the indicated concentration. of incubation, the level of fibrinopeptide A (FPA) in plasma was determined, and the percentage of inhibition of generation of FPA was then calculated for inhibitory concentration.To determine the inhibitory effect against thrombin activity bound to the clot, washed fibrin clots were incubated in plasma coded for 60 minutes at 37 ° C in the presence or absence of variable concentrations of heparin.At the end of the incubation period, the plasma levels of FPA was determined, and the percentage of inhibition of generation of FPA inducers was determined. The clots were then calculated for each inhibitory concentration, each bar representing the average of three separate experiments (each made by duplicate). ado), while the lines above the bars represent the SD. Figures 6A and 6B illustrate comparative studies of the ability of dermatan sulfate (the HCM activator prototype) and one of the specific LA-LMWH HCII catalysts of the present invention, eg, V18, to inactivate free thrombin and bound thrombin. clot using the clot clot tests. At 5 μg / L, LA-LMWH was much more selective than dermatan sulfate (dermatan sulfate was only half as effective in inactivating fibrin-bound thrombin, while LMWH was more or less effective in inactivating bound thrombin. fibrin and free thrombin). At 20 μg / ml, the advantage of LA-LMWH was still apparent until thinking that this was more or less obvious. At high concentrations, both are about 90% as effective in inactivating bound thrombin and free thrombin. Other results of the hanging clot test also indicate that the LA-LMW HCM activators of the present invention attached to and inactivated thrombin bound to the thrombus or clot. In these experiments, 250 μl containing thrombin was suspended in 10 ml of solution containing either 150 mM NaCl or 2 M NaCl. One of the following was added to the isotonic buffer: HCII 0.25 or 0.5 μM 100 μg of the catalytic agent LA-LMWH HCII-specific, or the combination of HCII and the catalytic agent LA-LMWH HCII-specific. The following measurements were made: (1) the diffusion of thrombin out of the thrombus; (2) the thrombin catalytic activity in the thrombus, which was calculated by measuring the production of fibrinopeptide A, removing the thrombus and adding it to a fibrogen solution; and (3) the magnitude of thrombin / HCM complex formation, which was determined by solubilizing the thrombus and carrying out SDS-PAGE and autoradiography. Figure 8 illustrates the diffusion rate of thrombin labeled from the clot fibrin. After 6 hours, about 40% of the thrombin remains in the thrombus suspended in 150 mM NaCl. Neither the addition of HCM alone or in combination with the catalytic agent LA-LMWH HCII-specific, for example V18, influenced the diffusion rate. In effect, the combination of HCM and the catalytic agent LA-LMWH HCM-specific actually reduces the diffusion rate to a small amount. In contrast, the diffusion rate of thrombin was increased markedly increased when the clot was suspended in 2 M NaCl. Figure 9 compares the catalytic activity of the thrombin bound to the thrombus, measured by removing the thrombus after five hours of incubation with the initial solutions and suspending it in a fibrinogenic solution. Thrombi were suspended in 150 nM NaCl in the presence or absence of HCII or the catalytic agent LA-LMWH HCM-specific, eg, V18, only generated around 6000 nM FPA. By contrast, the production of FPA by thrombi that was suspended in 150 mM containing the combination of HCM and the catalytic agent LA-LMWH HCM-specific was suppressed in approximately 95%. Thrombi that were suspended in 2 M NaCl generated about 4000 nM FPA, consistent with the fact that they contain about half of the thrombin found in thrombi incubated in buffers low in salts with or without HCII. These findings indicate that the HCM-specific catalytic agent LA-LMWH of the present invention, eg, V18, inactivates thrombin bound to the thrombus in the presence of HCII, but as illustrated in FIG. 8, does this without displacing the thrombin of the clot. This supports the concept that thrombin bound to fibrin via exosite 2, leaving exosite 1 available to interact with HCM (see, patent application serial number 08 / 175,211 (filed December 27, 1993), teachings of which are incorporated here by reference).

The analysis of the gels indicates that a high percentage of the thrombin coated from the thrombi that have been suspended in a buffer containing the combination of HCM and the catalytic agent LA-LMWH HCII-specific, for example, V18, was complexed to the HCM. In contrast, predominantly not complexed, thrombin was recovered in the thrombi suspended in buffer containing the HCII alone or the catalytic agent LA-LMWH HCII-specific only. These findings indicate that the catalytic agent LA-LMWH HCII-specific, for example, V18, produces permanent inactivation of the thrombin bound to the thrombus by means of the complex formation of covalent catalysis between thrombin and HCM. D. Extracorporeal Recirculation Circuit The selective ability to inactivate thrombin bound to fibrin was demonstrated by the use of an extracorporeal closed circuit containing human blood to measure the ability of several anticoagulants to inactivate bound thrombin to the surface, and time Coagulation of thrombin was used to measure the ability of several anticoagulants to inactivate thrombin in the fluid phase. A catalytic agent LA-LMWH HCM-specific of the present invention, for example, V18, was compared to the following anticoagulants: dermatan sulfate, low molecular weight heparin, heparin and unfractionated hirudin. 1. The Extracorporeal Closed Circuit An extracorporeal closed circuit diagram is shown in Figure 10. The circuit consists of Tygon tubing (3/16 * x 1/16") connected to a 40 micron transfusion filter (Pall SQ40S). a three-way stopcock for blood samples A surgical pressure transducer connected to a pressure recorder (Hewlett-Packard) is connected in line with the filter in such a way that this coagulation of the filter causes a non-violent increase of the The entire coded blood taken from healthy normal volunteers is used.The circuit is fed with 50 ml of whole blood located in a container and kept in a temperature controlled bath at 37 ° C. The anticoagulant to be tested is added to the circuit. , the coagulate is initiated by means of recalcification (0.8 ml of M CaCh), and the blood is recirculated at a constant flow rate through the thrombogenic surface using a pump. roller head (modular Cole-Parmer MFLX operated with a large F / 6 roll head cartridge). The thrombosis of the filter is monitored by a change in the pressure in a meter inserted in the circuit, close to the filter. Progressive obstruction to flow occurs because fibrin forms on the surface of the filter and, in addition, because thrombin bound to the clot or thrombus promotes more coagulation.

The inactivation of bound thrombin to the surface and the thrombin in fluid phase can be measured simultaneously using this system. More particularly, the inactivation of bound thrombin to the surface is quantified by recording the delay of the circuit coagulation. This delay is contributed to, to a greater or lesser degree, the inactivating effect of the anticoagulant in the fluid phase of thrombin, whose generation is enhanced by thrombin bound to the clot. The inactivation of thrombin in the fluid phase is quantified by recording the coagulation time of the plasma thrombus prepared from the blood removed from the circuit just before calcification. 2. Results The following points summarize the results of the experiment in which: a) one of the two activators of HCM, for example the catalytic agent LA-LMWH HCII-specific (V18) or dermatan sulfate, was added to the blood alone ( Figure 11); b) the activators were added in combination with a low concentration (less than 2% by weight) of an unmodified low molecular weight heparin (LMWH) (Figure 12); c) the unmodified LMWH was added alone (Figure 13); d) dermatan sulfate was added alone (Figure 14); LA-LMWH was added alone (Figure 15); and f) hirudin was added alone (Figure 16). Figures 14 and 15 illustrate the comparative effects that dermatan sulfate and the catalytic agent LA-LMWH HCM-specific, for example V18, have in the coagulation in the circuit over the concentration range of 120 μg / ml to 960 μg / ml. As can be seen in Figure 8, the catalytic agent LA-LMWH HCII-specific, for example, V18, produces a dose-dependent inhibition of coagulation in the circuit. More particularly, coagulation occurs in the circuit in about 20 minutes with the catalytic agent LA-LMWH HCII-specific in a dose of 240 μg / ml, and is counteracted with a dose of LA-LMWH of 960 μg / ml. The coagulation times corresponding to thrombin are shown in Table 1, supra. Table 3, infra, shows the comparative effects of various concentrations of LMWH or mixtures of LA-LMWH with LMWH or dermatan sulfate. The coagulation time by thrombin was 26 seconds at a concentration of LA-LMWH of 240 μg / ml and 43.6 seconds by a concentration of 960 μg / ml of LA-LMWH. In contrast, as shown in Figure 15, coagulation in the circuit occurs in less than 10 minutes with a dose of dermatan sulfate of 240 μg / ml, even though the corresponding coagulation time corresponding to thrombin was greater than 500 seconds (see table 1, above). In addition, coagulation occurs in less than 20 minutes with a dose of dermatan sulfate of 960 μg / ml, although the corresponding coagulation time of thrombin was 841 seconds (see table 1, supra). TABLE 3 THROMBIN COAGULATION TIME (2 UNITS) 0. 5 units / mL LMWH + > 500 240 μg / mL DS 1 unit / mL LMWH + 38 240 μg / mL LA-LMWH 1 unit / mL LMWH + > 500 240 μg / mL DS LMWH Low molecular weight heparin LA-LMWH Low molecular weight, low affinity heparin (V18) DS Dermatan sulfate.

In addition, the relative effects of the catalytic agent LA-LMWH HCII-specific, for example V18, and dermatan sulfate were compared when aliquots of the same human blood sample was tested in the model (figure 11). At a basis weight, the catalytic agent LA-LMWH HCII-specific is more effective than dermatan sulfate in circuit thrombosis. Then, 480 g / ml of the catalytic agent LA-LMWH HCM-specific was able to completely prevent coagulation for 90 minutes, while an 8-fold increase in the amount of dermatan sulfate may not completely prevent coagulation in the circuit.

You found, detractan sulfate is a much more effective inhibitor of thrombin in the fluid phase (see table 1 above). For example in this experiment at 240 micrograms / ml, the coagulation time of thrombin by the catalytic agent LA-LMWH HCM-specific is about 26 seconds (control 21 seconds) compared to more than 500 seconds by the dermatan result . Figure 13 illustrates the effects of one unit per ml of anti-Xa and two units per ml of anti-Xa of unmodified LMWH in the coagulation of the filter and in the coagulation time of the thrombin. At these concentrations, the LMWH has minimal effects in the prevention of thrombosis at a time when the coagulation time of thrombin was prolonged to 37 and 64 seconds, respectively (see table 3, infra). Figure 12 illustrates the results of using a mixture of a catalytic agent LA-LMWH HCM-specific, e.g., V18, and an efficient ATIM catalyst (e.g., LMWH). At concentrations of 0.5 units per ml of anti-factor Xa of LMWH (which produces a clotting time of thrombin of 20 seconds), the unmodified LMWH was not effective in preventing coagulation of the filter, but when the same amount of LMWH was used in conjunction with 240 micrograms per ml of the catalytic agent LA-LMWH HCM-specific for example V18, the mixture was highly effective in inhibiting the coagulation of the filter. The mixture of LMWH and dermatan sulfate was also very effective. However, the corresponding thrombin coagulation time for the LMWH / dermatan sulfate combination was greater than 500 seconds while the corresponding thrombin coagulation time for the LMWH / LA-LMWH HCM-specific catalyst mixture was only 20 seconds (see table 3). These findings clearly indicate that compared to dermatan sulfate, the catalytic agent LA-LMWH HCM-specific of the present invention, eg, V18, has selective activity against thrombin bound to fibrin. Then, it is more effective than dermatan sulfate in preventing coagulation of the filter, but it produces much less prolongation of the coagulation time of thrombin because it has less inhibitory activity against thrombin in the fluid phase. In contrast to the catalytic agent LA-LMWH HCM-specific, hirudin is much less effective in preventive thrombosis in the circuit at concentrations that prolong the coagulation time of thrombin to more than 500 seconds, at concentrations that prolong the coagulation times of thrombin less than 500 seconds (see figure 16 and table 1), and when the combination of the catalytic agent LA-LMWH HCM-specific and LMWH are used to prolong the clotting time of thrombin to less than 30 seconds ( see Figure 13 I Table 1. Figure 17 illustrates the effect of heparin or heparin in combination with V18 in a bypass simulation model, and Table 4 establishes the relationship between ACT and fibrinogenic deposition. in a bypass filter In Table 4, heparin alone is compared to heparin in combination with V18 TABLE 4 RELATIONSHIP BETWEEN ACT AND FIBRINOGENIC DEPOSITION IN BYPASS FILTERS: COMPARISON OF HEPARIN ONLY WITH A COMBINATION OF HEPARIN AND V18 ACT Deposition of Fibrinogen (s) (%) Heparin (2.0 U / mL) 651 36 Heparin (1.5 W / mL) 452 84 Heparin (1.5 U / mL) and 453 5 V18 (0.2 mg / mL) E. Animal Disease Prevention Model Venous Thrombosis Example I 1. Surgical Procedures Studies were done on New Zealand male white rabbits weighing between 3 and 4 kilograms. The left and right jugular veins were exposed through a ventral incision in the neck. A 2-centimeter segment of each jugular vein was identified and the lateral branches were ligated. After a venous blood sample was collected for coagulation studies, the animals were randomized to receive an intravenous bolus either of standard low affinity heparin, low affinity low molecular weight heparin, or a saline solution. Four minutes after this treatment, a second blood sample was collected for coagulation studies. A French balloon catheter 4 was then inserted into the right jugular vein and the balloon was inflated and the endothelium in the 2-cm jugular vein segment was damaged by passing the inflated balloon 5 times. The stasis was then induced in the 2-centimeter segment by placing 2 tourniquets approximately 2 centimeters apart around the blood-filled segment. As soon as this was done, an intravenous bolus of thromboplastin (7mg / kg) was injected into the left jugular vein and stasis in this vein was induced by placing 2 tourniquets 2 centimeters apart around a segment filled with blood. After 15 minutes of venous occlusion, a blood sample was taken for coagulation studies and the clots of the venous segments were weighed. 2. Results The HCM-specific LA-LMWH catalytic agents of the present invention, for example, V18, has been studied in a model rabbit simulating prophylaxis in high-risk states. A dose response relationship was established. At 8 mg / kg (about 100 μg / ml which is about five times smaller than required to prevent thrombosis in the bypass circuit), there was complete inhibition of thrombosis (see Figure 18). To test the synergistic effect of the combination of the catalytic agent LA-LMWH HCM-specific of the present invention, for example, V18, with a catalyst ATIII, the combination of heparin at a concentration of 10 U / kg (a concentration that there is no effect on the formation of thrombi (see Figure 19)) and the catalytic agent LA-LMWH HCII-specific, for example V18, at a concentration of 2 mg / kg and 4 mg / kg was studied. Over 60% inhibition of thrombosis was seen when 2mg / kg of the HCII-specific LA-LMWH catalytic agent was used (a dose that has virtually no effect when used alone), and 100% inhibition of thrombosis was seen when use 4mg / kg of the LA-LMWH HCII-specific catalytic agent (a dose that produces less than 40% inhibition when used alone) (see figure 20). The coagulation time of thrombin was not increased when 8 mg / kg of LA-LMWH was used alone, or when it was used in combination with 10 U / kg of heparin, indicating that the effectiveness is seen with minimal inhibitory effects in free thrombin. SH and LMWH with low affinity for ATHI (LASH and LA-LMWH, respectively) were prepared from SH (specific activity, 160 anti-Xa and anti-lla units / mg) and LMWH (specific activity, 100 anti-Xa U / mg), respectively, by dates of oxidation periods and subsequent reduction as described in Casu, B., et al., "Retention of antipilemic activity by dates of oxidation period of non-coagulating heparins", Arzneim-Forsch / Drug Res. 36: 637-42 (1986). The anticoagulant activities of the low affinity derivatives were compared with those of the starting materials and dermatan sulfate (DS). As illustrated in table 5, when compared in equivalent amounts by weight, LASH and LA-LMWH, for example V18, are essentially devoid of anti-Xa activity, indicating that these can not greatly enhance the inhibition of factor Xa by antithrombin III. In addition, these low affinity derivatives have less effect on thrombin coagulation time (TCT) than the other compounds of the same family. Thus, eliminating its ability to catalyze ATIII, its Anticoagulant activity was reduced.

TABLE 5 GAG (concentration) Anti-Xa _ SH (0.5 U / ml) 0.3 SH (2.0 U / ml) 1 .3 SH (10.0 U / ml) 6.0 LASH (3 μM / ml) 0.07 LASH (1 1 μM / ml) 0.06 LASH (50 μM / ml) 0.06 LMWH (0 05 U / ml) 0.6 LMWH (2.0 U / ml) 2.8 LMWH (10.0 U / ml) 1 1 .0 THE LMWH (5 μM / ml) (V18) 0.04 THE LMWH (20 μM / ml) ( V18) 0.05 LMWH (100 μM / ml) (V18) 0.06 DS (1 μM / ml) 0.05 DS (10 μM / ml) 0.06 DS (100 μM / ml) 0.05 When the activity of the fractions of heparin of different molecular weights were compared, a fraction of 9,000 Daltons has activity close to that of the SH, in addition a fraction of 5,000 Daltons has an activity close to that of the LMWH. In contrast, a fraction of 3,000 Daltons has much less activity (Figures 5 and 6). These findings are, once again, consistent with the concept that the maximum activation of the FCII occurs with the heparin chain containing 20 or more units of onoscarids, corresponding to a molecular weight of 8,000 Daltons or more.

Example II 1. Anesthesia New Zealand white rabbits, free of specific pathogens, were anesthetized with an intramuscular injection of ketamine (50 mg / kg) and Xylasin (2 mg / kg). Once anaesthetized, the cervical ventral area was shaved and prepared with alcohol and iodine solutions. A 22-gauge catheter (Becton-Dickinson, Sandy, UT) was inserted into the right central atrial artery and marginal atrial vein, to collect a blood sample and for venous administration of fluids and anticoagulants, respectively. Once these procedures were carried out, the rabbits were transferred to the interior of the operating room. After a brief exposure to the mixture soflurane (1 to 4%), oxygen (1 L / minute) and nitrous oxide (0.5 L / min) by face mask, the rabbits were intubated with an endothelial tube No. 2 Fr and kept in the same mixture of anesthetics inhaled until the end of the procedure. 2. Creation of Thrombi The right external jugular vein and facial vein were exposed through an incision in the skin of the cervico-ventral part. A segment of the facial vein was occluded with two 4-0 silk sutures positioned 0.5 cm apart. All lateral branches of the jugular vein were ligated to two centimeters in length. After introducing a Fogarty Fr No. 3 thrombectomy catheter into the jugular vein through the occluded segment of the facial vein, the balloon was inflated and the lining of the endothelium in the 2-cm segment of the jugular vein was damaged by 15 steps of catheter. Two silk sutures of number 40 were placed around the damaged vein with a separation of 1.5 cm. The Fogarty catheter was removed and replaced with a # 60 polyethylene tube within the isolated jugular vein segment. The blood was evacuated from the damaged vein and the segment was occluded with the sutures. The vein segment was rinsed with 500 U of bovine thrombin diluted in 0.5 ml of saline for five minutes, taking care that the vein segment was completely occluded to prevent the systemic release of thrombin. The thrombin solution was removed and the segment was then rinsed with a saline solution. Approximately 1 ml of arterial blood was rapidly withdrawn into a 1 ml syringe and mixed with approximately 1 μCi of labeled fibrinogen l 25l. 0.2 ml of this mixture was injected into the segment of occluded jugular vein. The catheter was then removed, the facial vein was ligated and the thrombus allowed to mature for 30 minutes. At the same time, two aliquots of 0.2 ml of the same blood were allowed to coagulate in test tubes for 30 minutes at 37 ° C Since pilot tests showed that the thrombi formed ex vivo are equivalent in weight and radioactivity to those formed In situ in the jugular vein, the mean weight and radioactivity of the clots were used as an index of the mass and radioactivity of the in situ clots Twenty minutes after the thromb injection into the jugular vein segmentwere given to animals or (a) heparin, 70 U / kg IV bolus and 280 U / kg sc 96 h X 2, (b) dermatan sulfate, 2 mg / kg IV bolus and 8 mg / kg sc q6h X 2, (c) V-18, 2 mg / kg sc q6h X 2, (d) a combination of hepapna and dermatan sulfate in the same dose, or (e) a combination of hepapna and V-18 in The same dose Control animals received equivalent volumes of saline water in the same intervals After 30 minutes of maturation of the thrombus, the tourniquets were removed and two 4-0 silk sutures were placed through the wall of the jugular vein in The thrombus to prevent its migration The occlusive tourniquets were then removed and the blood flow was restored through the jugular vein segment. The cervical incision was rinsed with 0 5 ml of penicillin and the skin was routinely closed. The rabbits were left to recover breathing 100% oxygen When they recover the gag reflex the endotracheal tube is removed and the animals on transferred to the recovery room 3. Endpoints After 24 hours of creation of the thrombus, the animals were euthanized and the jugular segment was opened and the residual thrombus was weighted and counted by radioactivity. By comparing these results with the weight and radioactivity of the clots formed ex vivo, the percentage change in the weight of the clots (growth of the clots) and radioactivity (lysis of the clot) was calculated (See figures 21 and 22).

F. The Coagulation Profile of V18 and its Fractions, Compared with Low Molecular Weight Heparin and Dermatan Sulfate In Normal and Deficient Plasmas in ATM and HCII.

The coagulation studies were carried out to characterize the anticoagulation profile of V18 and three fractions of V18 which were separated by exclusion gel chromatography to obtain Fraction 1 (Average molecular weight 8,000), fraction 2 (average molecular weight of 5,000) and fraction 3 (average molecular weight of 3,000). In addition, because V18 is a modified LMWH with some characteristics of Dermatan Sulfate (DS), the anticoagulant profile of V18 was compared with those two sulfated polysaccharides. Such comparisons were made using the thrombin coagulation time (TCT) assay, the activated partial thromboplastin time (APTT) assay and the factor Xa coagulation time assay. These three tests were carried out in normal plasma, plasma deficient in ATM and plasma deficient in HCM. Figure 23 illustrates the effects of V18 on APTT (A), TCT (B) and coagulation time of factor Xa (coagulation time Xa) (C) in normal plasma, plasma deficient in ATM and plasma deficient in HCM . From Figure 23A, it is apparent that a dose-dependent increase in APTT which is both dependent on ATM and HCM, since a similar dose-dependent prolongation is seen in normal plasma, reduced plasma in ATM and in HCM. Figure 23B illustrates that there is a prolongation of the TCT above a concentration of about 120 μg / ml. From this, there is a pronounced increase in TCT in normal plasma and reduced plasma in HCM. Such findings indicate that V18 has weak activity against free thrombin and that this activity is highly dependent on HCM. Figure 23C illustrates that there is a dose-dependent prolongation in the coagulation time of Xa, which is both HCM and ATM dependent. These findings indicate that in addition to its activity against clot-linked thrombin, V18 has weak activity against fluid phase thrombin, which is dependent on HCII and additional anti-Xa activity, which is dependent on the ATIM. For comparative purposes, Figures 24 and 25 illustrate the anticoagulant profiles of LMHW (Figure 24) and DS (Figure 25). In contrast to V18, the inhibitory effect of LMWH on TCT (Figure 24A), APTT (Figure 24B) and Time Xa (Figure 24C) is mainly ATM dependent, with a minor contribution of HCM to TCT prolongation and time For. In addition, in contrast to V18, the inhibitory effects of DS on TCT (Figure 25A), APTT (Figure 25B) and time Xa (Figure 25C) is entirely dependent on HCII. Figures 26, 27 and 28 illustrate the anticoagulant profiles of the three fractions of V18 which were separated by size using gel exclusion chromatography. Fraction 1 has an average molecular weight of about 8,000, fraction 2 has an average molecular weight of about 5,000 and fraction 3 has an average molecular weight of 3,000. The inhibitory effects of fraction 1 in the TCT (Figure 26A) is mainly dependent on the HCM, in the APTT (Figure 26B) dependent on both HCII and ATM and in time Xa (Figure 26C) mainly dependent on the ATM. The inhibitory effects of fraction 2 on the TCT (Figure 27A) is dependent on the HCII, on the APTT (Figure 27B) dependent on both the HCII and the ATM and on time Xa (Figure 27C) mainly dependent on the ATM. In contrast to fraction 1, fraction 2 is much less potent against thrombin in the fluid phase and less potent in the APTT and time Xa assays. Fraction 3 has minimal activity in the TCT assay (Figure 28A), and weak activity in the APTT assays (Figure 28B) and Xa time (Figure 28A), and weak activity in the APTT assays (Figure 28B) and Xa time ( Figure 28 C). Table 6 is a summary table showing the concentrations of the various glycosaminoglycans (GAGS) required to double APTT and TCT.

TABLE 6 At a basis weight, the DS is much more potent than V18, and DS has potency similar to fraction 1. V18 fractions lose potency with molecular weight reduction. Figure 29 illustrates the comparative additive effect of heparin (0.1 U / mL) on the prolongation of thrombin time produced by DS and V18.

G. Complete Blood Clotting Time Trial (WBCT1 To Test Inhibition of Free Thrombin and Clot Attachment Studies were conducted to compare the effects of the various GAGS in fluid phase thrombin and thrombin bound to the clot in a modified whole blood coagulation time trial. The clots were prepared around polystyrene hooks by adding 250 μl of CaC to 10 ml of poorly deficient plasma (final concentration 0.025 M). The clots or thrombi were allowed to mature for 1 hour at 37 ° C. These were then removed, attached to the hooks and washed four times for 30 minutes. Control hooks with thrombin bound to the clot inactivated by PPACK were prepared by incubation of the clots for the first 30 minutes of incubation with 10 μM PPACK. The coagulation times of the whole blood was carried out by adding 950 μl of human blood obtained from a normal donor to 50 μl of the anticoagulant (or control buffer). The clots (thrombin attached to the clots) or clots PPACK (thrombin attached to the inactivated clot) which were attached to the polystyrene hook, or free thrombin was added in a sufficient concentration to reduce the time of coagulation of base in order to obtain thrombin bound to the clot (free thrombin) to the blood tubes that were incubated at 37 ° C and the clotting time was using the inclined tube method, and recorded. Figure 30 illustrates the effect of increasing heparin doses in the clotting time of whole blood to which free thrombin (A), a PPACK (B) clot and thrombin bound to a clot (C) were added. IC 50 was 0.3 units / ml, 0.6 units / ml and 0.9 units / ml for free thrombin, PPACK thrombus and thrombin bound to the clot, respectively. Then, the ratio of the relative sensitivity of this whole blood system to the effects of free thrombin (A), to thrombin bound to a clot (C) is around 3, and the ratio of the relative sensitivity of the blood Complete blood for the effects of the PPACK clot (B) and thrombin attached to a thrombus (C) is around 1.5. Figure 31 illustrates the effect of DS dose increases on the clotting time of whole blood to which free thrombin (A), a PPACK (B) clot and thrombin bound to a clot (C) are added. The IC 50 was 480 μg / ml of the free thrombin, and it was > 1440 for both PPACK thrombus and for thrombin bound to the thrombus. Thus, the ratio of the relative sensitivity of the whole blood system to the effects of free thrombin (A), and thrombin bound to a clot (C) is > 3.0, and the ratio of the relative sensitivity of the whole blood system to the effects of the inactivated PPACK clot (B) to thrombin bound to the clot (C) is > 3.0. Figure 32 illustrates the effect of increasing the dose of V18 in the coagulation time of whole blood to which free thrombin is added.

(A), a PPACK clot (B) and thrombin attached to a clot (C). IC 50 was 90 μg / ml, 180 μg / ml and 250 μg / ml for free thrombin, PPACK thrombus and thrombin bound to the clot, respectively. Then, the ratio of the relative sensitivity of this whole blood system to the effects of free thrombin (A), to thrombin bound to a clot (C) is around 3, and the ratio of the relative sensitivity of the blood whole blood for the effects of the clot PPACK (B) and thrombin attached to a thrombus (C) is about 1.5. Figure 33, 34 and 35 illustrate the effects of the dose increase of the Fractions 1, 2 and 3 of V18, respectively, in the clotting time of the whole blood to which free thrombin (A) is added, a PPACK (B) clot and thrombin bound to a clot (C). For fraction 1, IC 50 was 22 μg / ml, 25 μg / ml and 37 μg / ml free thrombin, PPACK thrombus and thrombin bound to the clot, respectively. Then, the ratio of the relative sensitivity of this whole blood system to the effects of free thrombin (A), to thrombin bound to a clot (C) is around 1.7, and the ratio of the relative sensitivity of the Complete blood system for the effects of the inactivated PPACK clot (B) and thrombin attached to a thrombus (C) is around 1.6. For fraction 2, IC 50 was 90 μg / ml, 240 μg / ml and 250 μg / ml free thrombin, PPACK thrombus and thrombin bound to the clot, respectively. Then, the ratio of the relative sensitivity of this whole blood system to the effects of free thrombin (A), to thrombin bound to a clot (C) is around 2.7, and the ratio of the relative sensitivity of the Complete blood for the effects of the inactivated PPACK clot (B) and thrombin attached to a thrombus (C) is around 1.0. For fraction 3, IC 50 was 150 μg / ml, 320 μg / ml and around 600 μg / ml free thrombin, PPACK thrombus and thrombin bound to the clot, respectively. Then, the ratio of the relative sensitivity of this whole blood system to the effects of free thrombin (A), to thrombin bound to a clot (C) is around 4.0, and the ratio of the relative sensitivity of the whole blood for the effects of the inactivated PPACK clot (B) and thrombin attached to a thrombus (C) is around 1.9.

Figure 36 compares the effects of heparin (Figure 36A, upper panel), Fraction 1 (Figure 36B intermediate panel) and the combination of heparin and 10 μg / ml of Fraction 1 (Figure 36C, lower panel) in the WBCT . The results for heparin and for fraction 1 have been described above and have been established in figure 36 for purposes of comparison with the combination of heparin and fraction 1. The IC 50 of the combination (Figure 36C, lower panel) for free thrombin was 0.3 units / ml (for example, the same value as that of heparin (figure 36A, upper panel)). Similarly, IC 50 for the PPACK clot is of the same value as for heparin, for example, about 0.55 units / ml. In contrast, the IC 50 for thrombin bound to the clot is the same as for the PPACK clot, for example 0.55 units / ml. Then, in contrast to heparin, the ratio of the relative sensitivity of this whole blood system to the effects of free thrombin (A), to thrombin bound to a clot (C) is around 2.0, and the ratio of the relative sensitivity of the whole blood system to the effects of the inactivated PPACK clot (B) and thrombin attached to a thrombus (C) is around 1.0. Figure 37 illustrates the effect on the WBCT of the combination of heparin and DS. In contrast to V18, 120 μg / ml of DS did not influence the coagulation time in the presence of PPACK clot or thrombin bound to the clot. However, when the concentration of DS that was added to 0.5 units / ml of heparin was increased (figure 38), it was possible to calculate a relationship for the relative IC 50 values. The relative sensitivity ratios of this whole blood system to the effects of free thrombin (A), to thrombin bound to a clot (C) is around 1.1, and the ratio of the relative sensitivity of the whole blood system to the effects of the inactivated PPACK clot (B) and thrombin attached to a thrombus (C) is around 1.0. Figure 39 illustrates the effect in the WBCT of the combination of heparin and V18 on the coagulation time of whole blood to which free thrombin (A), a PPACK (B) clot and thrombin attached to a clot are added ( C). IC 50 was 50 μg / ml, 45 μg / ml and 45 μg / ml free thrombin, PPACK thrombus and thrombin bound to the clot, respectively. Then, the ratio of the relative sensitivity of this whole blood system to the effects of free thrombin (A), to thrombin bound to a clot (C) is around 3, and the ratio of the relative sensitivity of the Complete blood for the effects of the inactivated PPACK clot (B) and thrombin attached to a thrombus (C) is around 1.0. A summary of the effects of the various GAGS on the WBCT in the presence of free thrombin, the PPACK clot and the thrombin bound to the clot and, in addition, the relationships of the corresponding IC 50 have been established in Table 7.

TABLE 7 GAGS CONCENTRATIONS REQUIRED TO PROLONG THE COAGULATION TIME (IC50) IN THE ABSENCE AND PRESENCE OF A COAGULO In addition, Table 18 provides a summary of the synergy between V18 and heparin against thrombin bound to fibrin.

TABLE 8 SYNERGY BETWEEN V18 AND HEPARINE AGAINST TROMBINE LINKED TO FIBRINE IN COMPLETE BLOOD Time of Coagulation V18 μ / ml 0 6 60 14 120 20 240 75 Heparin 0.5 U / ml 30 0.5 U / ml heparin plus 60 μ / ml of V18 150 H. Studies of Prevention of the Coagulation of a Bypass Circuit Figure 40 illustrates the effect of heparin on the prevention of coagulation in a bypass circuit. Coagulation occurred, with 84% of fibrinogen consumption, at a heparin concentration of 1.5 units / ml. Coagulation was partially prevented by a concentration of 2.0 units / ml, although there was still 36% consumption of fibrinogen up to this concentration. V18 prevents coagulation in the circuit at a concentration of 960 μ / ml when used alone and at a concentration of around 120 to 240 μ / ml when used in combination with 1.5 units / ml of heparin. Fraction 1 prevents coagulation in the circuit at a concentration of about 120 μ / ml. Figure 41 illustrates the design of elusions of Fractions 1, 2 and 3 from a column of Sepharose G50. In addition, Figure 42 compares the effectiveness of Fractions 1, 2 and 3 when combined with 1.5 units / ml of heparin. Fraction 1 prevents coagulation in the circuit at a concentration of around 60 μ / ml, considering that Fraction 2 was partially effective at a concentration of around 60 μ / ml and fraction 3 was ineffective. These experiments showed that of the three fractions, fraction 1 is the most effective in the Bypass circuit and fraction 3 is the least effective. The minimum effective concentration of GAGS when used alone or, alternatively, in combination with 1.5 units / ml of heparin have been established in tables 9 and 10.

TABLE 9 EFFECTIVE COMBINATION IN THE CIRCUIT (MINIMUM CONCENTRATION) 1 .5 U / mL SH 120 μ / ml L18 1 .5 U / mL SH + 60 μ / ml FRACTION "1" TABLE 10 MINIMUM EFFECTIVE DOSE BYPASS CIRCUIT Weight μ / ml SH 12 LMWH 80 V18 960 FRACTION 1 240 DS > 3840 The relative concentration of the GAGS required to prolong the APTT and TCT, to prevent the coagulation of whole blood in the presence of a clot, and to prevent coagulation in the bypass is shown in Table 1 1.

TABLE 11 CONCENTRATION OF GAGS REQUIRED TO PROLONG THE TIME OF COAGULATION The IC 50 for the TCT and the corresponding doses required to prevent coagulation in the circuit and the relative relationship are shown in Table 12.

TABLE 12 COMPARISON OF IC50 FOR TCT AND EFFECTIVE DOSE IN BYPASS I. Measurement of the Second Order Relationship Constant A summary of the increase in the second order relationship constants (K2) of the factor Xa catalysis and the inhibition of thrombin by the ATM, and the inhibition of thrombin by the HCM in concentrations of 6, 60, and 300 μg / ml are shown in Table 13.

TABLE 13 INCREASE OF Ks The increase of the K2 at therapeutic concentrations of these GAGS is shown in Table 14 TABLE 14 INCREASE OF THE K2 OF GAGS At therapeutic concentrations (for example around 6 μg / mL), heparin (SH) and LMWH produce their anticoagulant effect by activating ATM, with only a weak contribution of the HCM catalyst. In contrast, at therapeutic concentrations (around 60 to 300 μg / mL), V18 produces its anticoagulant effect by activating both ATM and HCM. In addition, at therapeutic concentrations (around 300 μg / mL), DS produces its anticoagulant effect by activating HCM. It is interesting to note that in both tests in which coagulation is through the action of fibrin bound thrombin (for example the clotting time of whole blood with plasma clot and the bypass circuit), concentrations of DS are required much higher than V18 to prevent less coagulation, including V18 has about 50 times less rate of increase in thrombin inhibition by HCII. The binding affinity (Kd) of the different GAGS for thrombin (lia), factor Xa, and ATM are shown in table 15.

TABLE 15 INTRINSIC MEASUREMENTS OF JOINTS OF GAGs TO SERENE PROTEASE AND ITS INHIBITORS J. Experiments to Determine if V18 Has Anti-alcholastic Effects That Are Independent of HCII or ATM The experiments were carried out to determine whether V18 has coagulation effects that are independent of ATM or HCM. These experiments were designed to determine: 1) whether V18 inhibits factor Xa independently of ATM; 2) if V18 imparts fibrin polymerization; 3) if V18 inactivates factors IXa or Xla independently of ATM; and 4) if V18 is as effective in platelet-rich system as in poor systems in this. 1. Inhibition of ATM-independent factor Xa In a buffer system, the activity of the anti-factor Xa was measured in the presence and absence of ATM. V18 does not activate factor Xa in the absence of ATM (Figure 43). 2. Inhibition of fibrin polymerization To determine whether V18 interfered with fibrin polymerization, the prolongation of the coagulation time of thrombin in a fibrinogen-containing buffer system was measured in the absence of ATM or HCM. For comparison, the experiments were carried out with heparin (SH) and DS. None of these GAGS prolongs the coagulation time of thrombin in the absence of ATM or HCM. The effect of V18 was dependent on HCII as was the effect of DS. On a weight basis, the DS was about 20 times more potent than V18. In contrast to V18 and DS, which required HCM and non-ATM to inhibit the coagulation of fibrinogen by thrombin, heparin acts through both co-factors, although ATM was a more effective co-factor (Figure 44). 3. Inactivation of IXa and Xla These experiments were carried out to determine whether V18 or fraction 1 of V18 prolong APTT by inactivating IXa or Xla by mechanisms independent of ATM or HCII. The relative effects of heparin, LMWH, V18 and fraction 1 of V18 on inactivation of Xa, IXa and Xla were studied in a plasma system in which each of these coagulation enzymes was added (figure 44) . Each of the coagulation enzymes was added at a concentration that produces a coagulation time of between 40 and 50 seconds. Heparin (Figure 44A) prolonged the clotting time IXa more than the coagulation time Xa and had relatively less effect on the coagulation time Xla. In contrast, all other GAGS, including V18 and fraction 1 of V18, have similar effects on the coagulation time of Xa and IXa. These results indicate that V18 (Figure 44C) and fraction 1 of V18 (Figure 44D) do not have the same effects on IXa or Xla that are independent of ATM or HCM.

K. Effect of Platelets on the Anticoagulant Activity of V18 and Fraction 1 The coagulation time of factor Xa was carried out in platelet-rich, platelet-poor and platelet-free plasma in the presence of heparin, V18, LMWH and Fraction 1 of V18. In all cases, the coagulation time was shorter in platelet-rich plasma, indicating that factor Xa is protected against inactivation by these GAGs, including V18 and Fraction. 1 (Figure 45). However, the anticoagulant effects of V18 appear more resistant to platelets than heparin or LMWH. The results of the recalsification times with all these GAGS were much shorter in platelet-rich plasma than in platelet-poor plasma, indicating that factor IXa in the tenacious complex is protected from inactivation for these GAGs (Figure 46). 1. V18 has anticoagulation effects that are independent of HCII or ATIH Although V18 has minimal anti-factor Xa activity when tested in vitro on a standard chromogenic factor Xa test, it has appreciable anticoagulant activity when tested in HCM plasma without platelets. This is summarized in Table 16 which describes the effect of V18 and heparin on the recalcification coagulation time (coagulation of platelet-platelet-free plasma with citrates by the addition of calcium and phospholipids [cephalin]). In normal plasma, V18 at a concentration of 500 μg / mL increases the 9.6-fold recalsification time. The addition of this concentration of V18 increases the recalsification time 3.6 times in ATM-plateletless plasma (the "HCII effect") and 4.1 times in platelet-free HCII plasma (the "ATM" effect). In addition, V18 increases the re-labeling time 2.9 times in plasma without platelets of both HCM and ATM (double platelet-free plasma). In contrast, the effect of heparin prolongation of the recalsification time was virtually entirely dependent on the ATM. Thus, the increase in the recaalsification time with heparin was only 9.5 in normal plasma, 1.2 in ATM plasma without platelets (the "HCM effect") 6.0 in platelet-free HCII plasma (the ATM effect) and 1.3 in platelet-free plasma both of HCM and ATM. These findings indicate that the anticoagulant effect of V18 is attributed to both mechanisms by HCII and ATM, and by mechanisms that are independent of ATM and HCM. In contrast, more or less all the anticoagulant effects of heparin is through ATM.

Without trying to restrict any particular theory, it can be thought that two interrelated mechanisms are responsible for the activity of V18, independent of the HCII. Both are related to the fact that V18 interferes with the assembly of factor Xa on the surface of platelets. The process of blood coagulation occurs on the surface of platelets as a result of the assembly of various coagulation factors on the surface. Then factor IXa forms a complex with the factor Villa to form the tenacious complex that activates factor X to factor Xa, and factor Xa in turn forms a complex with factor Va on the surface of the platelet (the "prothrombinase complex") that converts prothrombin into thrombin. Factor Va is the factor Xa receptor on the surface of the platelet. The activation of factor V to factor Va is through the surface of thrombin. In the thrombus, which consists of aggregated platelets in a fibrin mesh, the activation of factor V is by thrombin that is bound to fibrin in close proximity to the platelets (figure 47). Factor Xa is protected from inactivation by its natural inhibitors, the ATM when this coagulation enzyme is linked to the Va factor on the surface of the platelet. Therefore, agents that prevent the binding of factor Xa to the surface of platelets markedly improve the activation rate of factor Xa by ATM (Figure 49). V18 interferes with the binding of factor Xa to the surface of the platelet by two different but interacting mechanisms (figure 50). First, by inactivating thrombin bound to fibrin through a mechanism that includes HCII, V18 provides activation of factor V to factor Va, this reducing the density of receptor factor Xa on the surface of the platelet. Second, as shown, V18 competes with factor Xa attached to the phospholipid surface (platelet surface). Both mechanisms act to inhibit the binding of factor Xa to the platelet surface, this by turning factor Xa vulnerable to inactivation even by a relatively weak weak ATM catalyst such as V18 (Figure 49). Interference with the assembly exhibited by the V18 is new. The process not only targets Xa factors, but, as shown in Figure 49, other coagulation enzymes. Other discoveries have attempted to compete with the Xa and IXa junctions, using inactive analog proteins. However, the antithrombotic mechanism described above for V18 is unique because it combines an HCM activator with thrombin selectivity bound to fibrin with a single process to interfere with the assembly of factor Xa on the plaquetal surface that exposes unprotected factor Xa to inactivation by ATM. By combining HCM inactivation of thrombin bound to fibrin with an inhibition of thrombin generation by the ATM (through the inactivation of factor Xa), V18 modulates thrombogenesis by inhibiting the two critical pathways (Figure 50).

TABLE 16 Normal asthma Pl astma AT111 without Plasma HCI I without Pl astma double without Platelets Platelets Platelets Time in Minutes CONTROL 1/5 OF Cefalma 3 3 2 3 2 3 2 3 RELATIONSHIPS V18 200 ua / ml 1/5 of cephalin 2 8 1 8 2 0 1 5 V18 300 μq / ml 1/5 of cefalma 4 6 2 3 2 8 2 3 V 18 500 μq / ml 1/5 of cefalma 9 6 3 6 4 1 2 9 Hepapna 0 5 U / ml 1/5 of cefalma 9 5 1 2 6 1 3 M. Comparison of V18 (MW 3,000 to 8,000) v SPL (MW 1,000 to 3000) in a Bypass Circuit Cardiopulmonary Simulator This example compares the activity of V18 with SPL, a material similar to V18 having a molecular weight of 1, 000 to 3,000 Daltons, in a cardiopulmonary bypass circuit. As stated in table 17, infra, in contrast with V18, the SPL is substantially inactive. 1. Preparation of SPL. A Material Similar to the V18 with a Weight Molecular from 1,000 to 3,000 Daltons Two grams of SPL product (1, 000 to 3,000 Da) was dissolved in 50 ml of distilled water. An equal volume of 0.2 M sodium metaperiodate (freshly made) was added. The solution was mixed and maintained at 4o C for 24 hours. % .0 ml of ethylene glycol was added and the solution was placed in a dialysis tube (MWCO 500) and dialyzed against several changes of distilled water for 24 hours. The solution was returned to a beaker and 800 mg of potassium borohydrate was added with constant stirring. The solution was kept in the dark for 2.5 hours at room temperature. The pH was adjusted to 3.0 with 1 N HCl and quickly readjusted to a pH of 7.4 with 1 N NaOH. The solution was dialyzed once more in the same manner as described and the lyophilized yielded the final product. 2. Comparison of V18 v SPL in the Bypass Circuit Cardiopulmonary Simulator A comparison of V18 and SPL was carried out in the cardiopulmonary simulator bypass circuit described above. The obtained results established that in contrast to the V18, the SPL is substantially inactive (See table 17, infra). It is for this reason that the fraction of the heparin molecule having a molecular weight below 3,000 Da was removed from V18. By removing such molecules from V18, the potency of V18 increased substantially.

TABLE 17 N. Comparison of the Efficacy and Safety of Hirudin with the Combination of V18 and Heparin in a Bypass Circuit Cardiopulmonary Simulator This example compares the efficacy and safety of hirudin with the combination of V18 and heparin in the cardiopulmonary simulator bypass circuit described above. The results obtained clearly establish that in contrast to hirudin, V18 has a very good APTT in concentrations that do not have problems of hemorrhages (See table 18).

TABLE 18 ACT Treatment (according to two) Time to Failure of Fibrin Deposited Treatment% Hirudin 340 30 > 90 Hirudina 480 60 > 90 Hirudina 610 75 > 90 Hirudina > 1500 > 90 9 V18 358 > 90 8 V18 and Heparin 370 > 90 6 Heparin 360 35 91 SALINA 180 10 > 90 O. Comparative Effects of V18 + Demin in Thrombin in Phase Fluid and Thrombin Linked to the Clot This example compares the effects of V18 and desmin, for example, Low molecular weight dermatan sulfate, in fluid phase thrombin and thrombin attached to a clot using the hanging clot test described previously. The The results set forth in table 19, infra, unequivocally establish that the desmin is less selective than V18 and, in addition, less powerful than V18 for a factor of about 4.

TABLE 19 It is understood that the description made is intended to be illustrative but not restrictive. Many modalities will be apparent to those with expertise in the technical field, when reading the description. The scope of the invention may, however, be determined not with reference to the description but instead, with reference to the attached clauses, together with the full scope of equivalents to which said claims are directed. The disclosure of all articles and references, including patent applications and publications, are incorporated as a reference for any purpose.

Claims (66)

R E I V I N D I C A C I O N S
1. A l-specific (HCII-specific) cofactor catalytic agent of heparin, which inactivates thrombin bound to a clot, characterized by: (i) a heparin cofactor II of specific activity against heparin cofactor II of about 2 to 5 units / mg in an anti-factor test; (ii) an antithrombin III (ATM) of specific activity against factor Xa from about 0.2 to about 1.5 units / mg in a Xa antifactor test; and (iii) a solubility in aqueous medium in a range from about 150 to about 1,000 mg / ml.
2. The agent claimed in claim 1 characterized by one or both of the following characteristics: (i) it has an affinity with the anti-male III of less than about 3% of that of an unfractionated heparin; (ii) is a polyanionic carbohydrate of about 10 to about 24 units of monosaccharides.
3. The agent claimed in 1 or 2, which is a heparin preparation having a molecular weight of between about 3,000 and about 8,000 Daltons.
4. The agent of claim 3, wherein the heparin preparation: (i) consists essentially of the fraction of the lowest molecular weight isolated from unfractionated heparin; or (ii) is produced from unfractionated heparin by chemical reduction of the molecular weight range of between about 3,000 to about 8, 000 Daltons.
5. The agent of claims 3 or 4, wherein the heparin preparation: (i) has an average molecular weight of about 8,000 Daltons; (ii) has an average molecular weight of about 5,000 Daltons; or (iii) has an average molecular weight of around 3,000 Daltons
6. The agent of any of claims 3 to 5, wherein the heparin preparation is produced from unfractionated heparin by chemical reduction of the molecular weight range of from about 3,000 to about 8,000. Daltons and has a reduced antithrombotic activity by treatment of neighboring alcohols present in the preparation of heparin with an oxidizing agent followed by a reducing agent and optionally where the oxidizing agent is selected from sodium periodate, dimethyl sulfoxide, anhydric acid, tetraacetate of lead and ascorbic acid, and the reducing agent is selected from sodium borohydride, lithium aluminum hydride, metal hydrides and hydrazine.
7. 7. The agent of any of clauses 3 to 6, wherein the preparation of heparin consists essentially of material without ATM affinity, said material being prepared by affinity purification on a solid phase to which ATM is immobilized and the effluent retained.
8. 8. The agent of claim 1 having formula (a): wherein: - R1 is a member selected from the group consisting of H, D-glucosamine acid residue, L-iduronic acid residue; - R2 are members independently selected from the group consisting of H, D-glucosamino acid residue, L-iduronic acid residue, and anhydromannitol. - R3 is a member selected from the group consisting of H and S03; - R4 is a member selected from the group consisting of H, S03 and CH3CO; the n indexes are independently selected and can have values in the range from 0 to 14; where: the specific catalytic agent HCII has a molecular weight in the range of 3,000 to about 8,000 Daltons; or the formula (b): 0 wherein: Ri and R2 are members independently selected from the group consisting of H, D-glucosamino acid residue, D-glucuronic acid residue, L-iduronic acid residue; - R3 is a member selected from the group consisting of H and S03; and 5 - R. is a member selected from the group consisting of H, SO3 and CH3CO; where: the specific catalytic agent HCM has a molecular weight in the range between about d 3,000 and about 8,000 Daltons.
9. 9. The agent of any of claims 1 to 8, which is characterized by one or both of the following characteristics: (i) a cofactor II of heparin has a specific activity of at least about 3 to 4 units / mg in anti-Factor Ha activity; (ii) an ATM cofactor of specific activity of about 1 unit / mg in an anti-Xa test.
10. 10. The agent of any of clauses 1 to 9, where: (i) the agent is combined with a heparin additive, or (ii) the agent is combined with a heparin additive and the weight ratio of the specific catalytic agent HCII to the Heparin additive is larger than about 2 to 1, where optionally the heparin additive is unfractionated heparin or is the third lowest molecular weight fraction isolated from unfractionated heparin.
11. 11. A pharmaceutical composition for inhibiting thrombogenesis in a patient, the composition comprising: (i) from 90 to about 99.9% by weight of a specific HCII catalytic agent that inactivates thrombin bound to fibrin, with the specific HCII catalytic agent only minimal affinity for antithrombin III (ATM); (I) from 0.1 to about 10% by weight of a catalytic agent that inactivates thrombin in the fluid phase, where; the catalytic activity of the HCM of said composition is about 2 to about 5 units / mg.
12. The composition of claim 1, wherein the specific HCH catalyst agent is (i) a polyanionic carbohydrate of from about 10 to about 24 units of monosaccharides; or (ii) the polyanionic carbohydrate is from about 10 to about 24 units of monosaccharides and is a heparin preparation having a molecular weight between about 3,000 to about 8,000 Daltons.
13. The composition of claim 1, wherein the preparation of the heparin is further defined as well as the characteristics expressed in part i of one or more of claims 4, 5, 6, 7, 8 and 9.
14. The composition of claim 1 or claim 1 wherein the ATM catalyst agent is unfractionated heparin, or is the third fraction of the lowest molecular weight isolated from unfractionated heparin.
15. A product comprising: (i) an agent that inactivates thrombin bound to the clot, said agent having a minimal affinity with antithrombin III (ATM) and a heparin cofactor II of about 2 to about 5 units / mg in a Haifa antifactor test; (ii) an ATM catalytic agent that inactivates thrombin in the fluid phase as well as a preparation for simultaneously separating or sequentially using the inhibition of thrombin bound to a clot and thrombin in the fluid phase in a patient.
16. The product of claim 15, wherein the thrombin inhibiting agent is (i) a specific HCII catalyst agent as defined in item i of one or more of claims 1 to 8, or (ii) a polyanionic carbohydrate as defined by the specific features expressed in claim 12 ( i) or (ii), and additionally or alternatively has a heparin cofactor II with specific activity of at least about 3 or about 4 units / mg in anti-Ha factor activity.
17. The product of claim 15 or claim 16, wherein the ATM catalytic agent is unfractionated heparin, or is the third lowest molecular weight fraction isolated from unfractionated heparin.
18. A low molecular weight heparin preparation characterized by: (i) a molecular weight of or between about 3,000 to 8,000 Daltons (± 1, 000); (ii) a heparin cofactor II of specific activity against heparin cofactor II from about 2 to about 5 units / mg in an anti-factor assay. (iii) an antithrombin III (ATM) of specific activity against factor Xa from around 0.2 to about 1.5 units / mg in an anti-Xa test. (iv) a solubility in aqueous medium within a range between about 150 to about 1,000 mg / ml; and (v) a residue of uronic acid or other native non-reducing sugar as an end group and a non-reducing sugar 2,5-anhydridomannitol as the other end group.
19. A polyanionic carbohydrate capable of selectively inactivating thrombin bound to a clot by favoring free thrombin, obtainable from heparin, having its non-sulphated uronic acid residues in the form of an open ring, and substantially free of aldehyde groups.
20. A carbohydrate of claim 19, which has a non-reducing native sugar as an end group and a reducing 2,5-anhydridomannitol sugar as the other end group, and / or where about 30% of the uronic acid residues are in Open ring shape.
21. 21. A preparation of claim 18 or a carbohydrate of claim 19 or claim 20, which is further characterized by one, two or 3 of the essential features of claim 1 and / or by the essential features of claims 2 to 9.
22. 22. A product capable of selectively inhibiting thrombin bound to the clot obtainable by: (i) unfractionated heparin depolyzed using nitrous acid; (ii) isolating and solubilizing the depolyzed fractions having a molecular weight between 3,000 and 8,000 (± 1,000) to provide a low molecular weight fraction; (iii) splitting the low molecular weight fraction using periodate; (iv) reducing the fraction broken down using sodium borohydrate, where optionally a column with affinity to antithrombin III is used to reduce the affinity of the depolyzed heparin, and the low molecular weight fraction has a specific activity of about 2 to about of 5 units / mg of antithrombin and less than about 1.5 units / mg of anti-factor Xa.
23. 23. A product obtainable by: (i) depolyzation of standard unfractionated heparin depolyzed by nitrous acid; (ii) oxidation of heparin depolyzed with sodium periodate in an aqueous medium for 24 hours at 4o C, and stop the oxidation reaction by the addition of an excess of ethylene glycol followed by extensive dialysis by means of distilled water using piping of dialysis with a cut to 500 MW. (iii) reduce the oxidized product by the addition of sodium borohydride and, after allowing the reaction mixture to remain 25 hours at 23 ° C, adjust the pH of the reaction mixture to 3.0 with HCl to destroy the excess borohydrate which immediately increases the pH to 7.0 by the addition of NaOH; (iv) extensively dialyzing the resulting product with distilled water; and (v) recovering the product by lyophilization; and optionally (vi) passing the product in a column with affinity to antithrombin III, the product obtained having the following properties; • molecular weight of 3,000 to 8,000 (± 1, 000) • specific activity of about 2 to about 5 units / mg of anti-factor lia • specific activity of less than about 1.5 units / mg of antithrombin lia.
24. A catalyst agent of any of claims 1 to 10, a preparation of claim 18 or claim 21, a carbohydrate of any of claims 19 to 21, or a product of claim 22 or claim 23 for pharmaceutical use.
25. 25. A pharmaceutical product comprising as an active ingredient a catalyst agent of any of claims 1-10, a preparation of claim 18 or claim 21, a carbohydrate of any of claims 19 to 21, or a product of claim 22 or claim 23 and optionally a pharmaceutically acceptable excipient, diluent or support, the pharmaceutical optionally further comprises a fluid phase thrombin inhibitor for simultaneous, separate or sequential administration with said active component.
26. 26. A process for preparing a specific HCII catalyst agent having a non-reductive native sugar as one of the end groups and a non-reductive 2,5-anhydromannitol sugar as the other end group; said process comprising: (i) depolyzation of unfractionated heparin; (I) oxidation of the resulting heparin of low molecular weight; and (iii) reducing oxidized low molecular weight heparin; wherein said process optionally includes an additional purification or other processes to obtain said specific HCM catalyst agent.
27. 27, A process according to Claim 26 wherein unfractionated heparin is depolymerized using nitrous acid and / or low molecular weight heparin is reduced using sodium borohydride.
28. 28. The use of the catalyst agent of any of claims 1 to 10, a preparation of claim 18 or claim 21, a carbohydrate of any of claims 19 to 21, or a product of claim 22 or claim 23 or of a catalyst agent Specific HCH obtainable using a process of claim 26 or claim 27 for the manufacture of a medicament for the treatment of cardiovascular disease, the use additionally further includes a fluid phase thrombin inhibitor for the manufacture of the medicament.
29. 29. The use for the manufacture of a drug for the treatment of thrombosis as prophylaxis or therapy, of a derivative of heparin having its residual uronic acids in the form of an open ring and substantially free of aldehyde groups.
30. 30. A method for inhibiting thrombus formation in a patient without inducing a clinically unsafe increase in systemic bleeding, said method comprising the step of administering to the patient a pharmaceutically acceptable dose of a heparin-specific cofactor II catalyst agent that inactivates thrombin bound to the patient. clot, said HCM catalyst agent characterized by: (i) a cofactor II of heparin with specific activity against heparin cofactor II, from about 2 to about 5 units / mg in an anti-Ha factor test; (ii) an antithrombin III (ATM) of specific activity against factor Xa from around 0.2 to about 1.5 units / mg in an antifactor test For; and (iii) a solubility in aqueous medium in a range of about 150 to about 1,000 mg / ml.
31. 31 A method of claim 30, wherein said specific HCII catalyst agent has an anti-iris III with affinity of less than 3% that of unfractionated heparin.
32. 32. A method of claim 31, wherein said specific HCM catalyst agent has an anticoagulant effect which is attributed to both a mechanism through the intervention of ATM and HCII as well as to a mechanism that is independent of both HCM and ATM.
33. 33. A method of claim 31, wherein said specific HCM catalyst agent is a polyanionic carbohydrate of from about 10 to about 24 units of monosaccharides.
34. 34. A method of claim 33, wherein said polyanionic carbohydrate is a heparin preparation having a molecular weight of between about 3,000 to about 8,000 Daltons.
35. 35. A method of claim 34, wherein said preparation consists essentially of the third lowest molecular weight fraction isolated from unfractionated heparin.
36. 36. A method of claim 34, wherein said heparin preparation is produced from unfractionated heparin by chemical reduction of molecular weight in a range of between about 3,000 and about 8,000 Daltons.
37. 37. A method of claim 36, wherein said heparin preparation has an average molecular weight of about 8,000 Daltons.
38. 38. A method of claim 36, wherein said heparin preparation has an average molecular weight of about 5,000 Daltons.
39. 39. A method of claim 36, wherein said heparin preparation has an average molecular weight of about 3,000 Daltons.
40. 40. A method of claim 36, wherein the affinity of the antithrombin III of said heparin preparation is reduced to less than about 3% of ia corresponding to unfractionated heparin.
41. 41. A method of claim 40, wherein said reduction in the affinity of thrombin III consists in the treatment of vicinal alcohol groups present in said heparin preparation, with an oxidizing agent followed by a reducing agent.
42. 42. A method of claim 41, wherein said oxidizing agent wherein the oxidizing agent is selected from sodium periodate, dimethyl sulfoxide, anhydric acid, lead tetraacetate and ascorbic acid, and said reducing agent is selected from sodium borohydride, lithium and aluminum hydride, metal hydrazine hydrido.
43. 43. A method of claim 34, wherein said heparin preparation consists essentially of material without affinity for ATM, said material is prepared by affinity purification in a solid phase to which the ATM is immobilized and the effluent is retained.
44. 44. A method of claim 30, wherein said specific HCII catalyst agent has the formula wherein - R1 is a member selected from the group consisting of H, D-glucosamine acid residue, D-glucuronic acid residue and acid residue of L- iduronic; - R2 is a member selected from the group consisting of H, D-glucosamino acid residue, D-glucuronic acid residue, L-iduronic acid residue, and anhydromannitol. - R3 is a member selected from the group consisting of H and SO3; - R is a member selected from the group consisting of H, SO3 and CH3CO; and the indices n are independently selected and can have values in the range from 0 to 14; where: the specific catalytic agent HCM has a molecular weight in the range of 3,000 to about 8,000 Daltons
45. 45. A method of claim 30, wherein said specific HCM catalyst agent has the formula wherein Ri and R2 are members independently selected from the group consisting of H, D-glucosamino acid residue, D-glucuronic acid residue, L-iduronic acid residue; - R3 is a member selected from the group consisting of H and S03; and - R4 is a member selected from the group consisting of H, S03 and CH3CO; where: the specific catalytic agent HCII has a molecular weight in the range between about d 3,000 and about 8,000 Daltons.
46. 46. A method of claim 30, wherein said HCM specific catalyst agent has a specific activity of heparin cofactor II of at least about 3 to about 4 units / mg in anti-Ha factor activity.
47. 47. A method of claim 30, wherein said specific HCII catalyst agent has a specific ATM activity of about 1.0 units / mg in an anti-Xa test.
48. 48. A method of claim 30, wherein said HCM specific catalyst agent is mixed with a heparin additive prior to administration to the patient.
49. 49. A method of claim 48, wherein the weight ratio of said specific catalytic agent HCII to said heparin additive is greater than about 2 to 1.
50. 50. A method of claim 48, wherein said heparin additive is unfractionated heparin.
51. 51. A method of claim 48, wherein said heparin additive is one third of the fraction of lower molecular weight isolated from unfractionated heparin.
52. 52. A pharmaceutical composition for inhibiting thrombogenesis in a patient, said composition comprising: (i) from 90 to about 99.9% by weight of a specific HCII catalytic agent that inactivates fibrin-bound thrombin, with the catalytic agent HCM specific only minimal affinity for antithrombin III (ATM); (ii) from 0.1 to about 10% by weight of a catalytic agent that inactivates thrombin in the fluid phase, wherein: the catalytic activity of said composition is about 2 to about 5 units / mg.
53. 53. A composition of claim 52 wherein said specific catalytic agent HCII is a polyanionic carbohydrate of from about 10 to 24 units of monosaccharides.
54. 54. A composition of claim 53 wherein said polyanionic carbohydrate is a surrounding heparin preparation having a molecular weight of between about 3,000 and about 8,000 Daltons
55. 55. A composition of claim 52 wherein said ATM catalytic agent is unfractionated heparin.
56. 56. A composition of claim 52 wherein said ATM catalytic agent is one third of the fraction of lower molecular weight isolated from unfractionated heparin.
57. 57. A method to inhibit thrombin bound to the clot and thrombin in fluid phase in a patient without inducing a clinically unsafe increase in systemic bleeding, said method comprising the steps of administering to the patient a pharmacologically acceptable dose of: (i) a specific catalytic agent HCM that inactivates thrombin bound to the clot, said specific catalytic agent HCM having minimal affinity with antithrombin III (ATM) and a cofactor II of heparin of specific activity against heparin cofactor II of around 5 units / mg in an antifactor test Ha; and (ii) an ATM catalyst agent that inactivates thrombin in the fluid phase.
58. 58. A method of claim 57, wherein said HCM specific catalyst agent and said ATM catalytic agent are administered to the patient simultaneously.
59. 59. A method of claim 57, wherein said HCM specific catalyst agent has an anticoagulant effect which is attributed to both an ATM mechanism and a mechanism by HCM, and by a mechanism that is independent of both HCM and ATM.
60. A method of claim 57, wherein said specific catalyst agent is a polyanionic carbohydrate of from about 10 to about 24 units of monosaccharides.
61. A method of claim 57, wherein said polyanionic carbohydrate is a heparin preparation having a molecular weight of between about 3,000 and about 8,000 Daltons.
62. A method of claim 57, wherein said specific HCII catalyst agent has the formula: wherein R1 is a member selected from the group consisting of H, D-glucosamine acid residue, D-glucuronic acid residue, and L-iduronic acid residue; - R2 is a member selected from the group consisting of H, D-glucosamino acid residue, D-glucuronic acid residue, L-iduronic acid residue, and anhydromannitol. - R3 is a member selected from the group consisting of H and SO3; - R4 is a member selected from the group consisting of H, S03 and CH3CO-; and the indices n are independently selected and can have values in the range from 0 to 14; where: the specific catalytic agent HCM has a molecular weight in the range of 3,000 to about 8,000 Daltons
63. 63. A method of claim 57, wherein said HCM specific catalyst agent has the formula: Ri and R2 are members independently selected from the group consisting of H, D-glucosamino acid residue, D-glucuronic acid residue, L-iduronic acid residue; - R3 is a member selected from the group consisting of H and SO3; and - Ro is a member selected from the group consisting of H, SO3- and CH3CO-; where: the specific catalytic agent HCII has a molecular weight in the range between about d 3,000 and about 8,000 Daltons.
64. 64. A method of claim 57, wherein said specific HCII catalyst agent has a cofactor II of specific activity of at least about 3 to about 4 units / mg in anti-Ha factor activity.
65. 65. A method of claim 57, wherein said ATM catalyst agent is unfractionated heparin.
66. 66. A method of claim 57, wherein said catalyst agent is one third of the lowest molecular weight fraction of unfractionable heparin. SUMMARY The present invention provides compositions and methods for inactivating fibrin-bound thrombin in a thrombus or clot, this by the ability of thrombin bound to the clot to catalytically promote further growth is substantially decreased or eliminated. The compositions and methods of the present invention are particularly useful in the prevention of thrombosis in the cardiac circuit bypass apparatus and in patients on renal dialysis, and for the treatment of patients suffering from or at risk of suffering thrombi related to cardiovascular conditions, such as unstable angina, acute myocardial infarction (heart attack), cerebrovascular accident (embolism), pulmonary embolism, venal thrombosis, arterial thrombosis, etc.