Combined application of factor vii polypeptides and factor viii polypeptides

FIELD: medicine, pharmaceuticals.

SUBSTANCE: invention relates to pharmaceutical agent containing factor VII or factor VII-related polypeptide and factor VIII or factor VIII-related polypeptide. Disclosed are application of factor VII or factor VII-related polypeptide and factor VIII or factor VIII-related polypeptide in production of drug, kit for episodic bleeding treatment, as well as methods for prophylaxis and treatment of episodic bleeding in subject.

EFFECT: accelerated coagulation of FVIII-deficit plasma.

43 cl, 1 tbl, 2 dwg, 6 ex

 

The FIELD of INVENTION

This invention relates to a pharmaceutical composition comprising a preparation of factor VII or the preparation of a polypeptide, a related factor VII, and the preparation of factor VIII or preparation of the polypeptide, a related factor VIII. The invention is also related to a set of components for the treatment of bleeding episodes, including the preparation of factor VII or the preparation of a polypeptide, a related factor VII, and the preparation of factor VIII or preparation of the polypeptide, a related factor VIII. The invention is also related to the use of the preparation of factor VII or preparation of the polypeptide, a related factor VII, and a preparation of factor VIII or preparation of the polypeptide, a related factor VIII to obtain drugs. In addition, the invention relates to methods for the treatment of bleeding, reduce clotting time, enhance hemostasis, reducing the number of injections of the protein clotting factor needed to achieve stop bleeding (hemostasis), reducing the number of input protein coagulation factor required to achieve hemostasis, prolongation of the time of the dissolution of a blood clot (fibrinolysis), increasing the strength of the blood clot and increase in the production of a fibrin clot.

The BASIS for the creation of INVENTIONS

Factor VII clotting is a plasma is the first coagulation factor (FVII). Activated factor VII (FVIIa) initiates the normal process of hemostasis by forming a complex with tissue factor (TF)exposed due to damage of the vascular wall, which subsequently activates factors IX and X (FIX, and FX), putting them in their activated form, factors IXa and Xa (FIXa and FXa). Factor Xa converts a limited number of prothrombin to thrombin on the surface of cells bearing tissue factor. Thrombin activates platelets and factors V and VIII in factors Va and VIIIa (FVa and FVIIIa), both of which serve as cofactors in the further process, leading to the full release of thrombin (full thrombin burst). This process includes the production of factor Xa under the action of factor IXa (in complex with factor VIIIa) and occurs on the surface of activated platelets. Finally, thrombin converts fibrinogen into fibrin, which leads to the formation of a fibrin clot.

Factor VII exists in the plasma mainly in the form of single-stranded zymogen, which is cleaved by the action of factor Xa, turning in its double-stranded activated form, FVIIa. Recombinant activated factor (rFVIIa) develops in prodemokraticheski agent. The introduction of rFVIIa leads to a rapid and highly efficient prognosticeskom response in hemophilia patients with bleeding, which cannot be treated with products of the coagulation factors is C-process for the antibody productions. Also patients with bleeding with deficiency of factor VII, or patients with normal clotting system, but with increased bleeding, can effectively be subjected to treatment with factor FVIIa. In these studies there was no unwanted side effects rFVIIa (in particular, the occurrence of thromboembolism).

Factor VIII clotting is a glycoprotein (MV 330000), which circulates in the blood. It is secreted by the liver and endothelium and secreted into the plasma, where and circulates in the form of a complex with factor a background of Villebranda. Factor VIII functions as a cofactor in blood coagulation, where it accelerates the conversion of factor X to factor Xa in the presence of factor IXa, calcium and phospholipid. Despite the fact that it is synthesized as single-chain polypeptide in the plasma it circulates mainly in the form of a double-stranded molecule. Activation of FVIII in the active cofactor requires additional proteolysis by the action of thrombin or some other proteases. Reducing the amount or activity of factor VIII in the blood stream leads to the development of hemophilia A. the reduction of the activity of factor VIII is directly proportional to the severity of the disease. Traditional treatment of hemophilia a is the replacement of the missing protein obtained from plasma or rekombinantnymi VIII (the so-called FVIII-substitution, or substitution treatment or therapy).

Deficiency of coagulation factor (e.g., FVIII deficiency) reflects genetic defects of different types. In cases where there is severe genetic damage, such as deletions, or shift the reading frame mRNA is not produced and the result is a (heavy) deficiency. Less severe forms of genetic damage, such as non-critical image localized point mutations that lead to the secretion of a protein with reduced biological activity. Non-inherited option is recessive and X-linked nature, meaning that it affects only men, who have only one X chromosome. The severity of the defect, coagulation may be weak or pronounced. The severity depends on the concentration of functional factor VIII in plasma. The goal of replacement therapy is to increase the level of activity of the coagulation factor in a patient (hereinafter called "level factor") until, until it is approximately restored hemostasis, and then maintain this level until the end of the treatment, when it comes to recovery. If initiation of effective treatment is delayed wound healing may be delayed, and then will need treatment more intensive than usual. The duration and intensity of treatment depends on the t concentration of coagulation factor in the plasma, required for hemostasis, it can be restored in blood and time-life material transfusion.

The level of factor VIII may also be more or less reduced in some individuals (for example, women who are carriers of the disease), which are heterozygous for this gene defect. Such entities may have an increased tendency to bleeding in comparison with those patients who are moderately affected by hemophilia and can be subjected to appropriate treatment.

Some patients receiving substitution therapy of factor VIII (affected by hemophilia A), observed the formation of antibodies against the injected factor VIII. However, in patients born with a normal level of factor VIII (who do not have congenital deficiency of factor VIII)may, for unknown reasons at a later age to develop the formation of autoantibodies against factor VIII (acquired hemophilia A). In both cases, antibodies can be represented in a small, moderate or large number. In the case when patients have low or average titer inhibitors (i.e. antibodies), they sometimes can be treated with factor VIII.

Show hemophilia can have very different degree of severity. Patient with factor VIII is not defined or its level is less than 1%, generally considered arelim patients with acute lesions, which at the slightest injury, and sometimes accidentally, you may experience bleeding in the muscle tissue and joints. A small amount of factor VIII gives significant protection, so that patients with a level of factor VIII, comprising 1-5% of normal, usually only suffer posttraumatic bleeding and less severe bleeding in the tissues of the muscles and joints, etc. and is often considered to be sick, diseased-to-moderate disease. Patients in whom the level of factor VIII is more than 5%, usually prone to bleeding in cases of major trauma or surgery, and these patients are generally considered to be ill-affected to a moderate degree. It should be noted that this classification is not applicable in all cases. Some patients with very low levels of factor VIII in rare cases, prone to bleeding, while others, where the level of factor VIII is even higher than 5%can be again subjected to bleeding in the "target tissue", the original damaged by traumatic hemarthrosis, and in these patients the disease is considered "hard" pronounced. However, the usual symptoms of bleeding is less pronounced at higher levels of this factor in the blood, so that abnormal bleeding does not usually occur when the level of factor VIII is about 35-40% of normal is x levels and above. The usual correlation between the contents of this factor and symptoms of hemophilia As shown below.

The severity of hemophilia, depending on the factor VIII:
SeverityThe factor level (% of normal level)Manifestations
Heavy0-1Seemingly spontaneous bleeding. Heavy

the bleeding.
Average1-5A few bruises. Hemarthrosis, mainly traumatic nature.
Weak5-30Bleeding post-traumatic, surgical,

postdental origin. Several episodes.

Usually treatment of hemophilia And is in substitution restore the missing protein obtained from plasma or recombinant factor VIII. The products of the factor VIII used for intravenous infusion or injection) if necessary for the treatment of acute bleeding. Different types of bleeding are divided into the following categories:

1. Hemarthrosis (bleeding into joints)

2. Bleeding, representing a threat to life and limb (bleeding in the retroperitoneal space, bleeding CNS bleeding in zapoteco the space, muscle bleeding with compartmental syndrome and massive bleeding of the gastrointestinal tract)

3. Prevention of bleeding in connection with surgery (orthopedics, separate procedures, surgery due to an accident).

Experience shows that if, before the completion of the healing process the content of factor VIII is set at the level of 30-40% of its normal level, usually supported normal hemostasis. However, of great importance and also other factors. Move damaged parts, as when hemarthrosis, coughing or standing up from the bed and movement of the patient after abdominal surgery can cause bleeding. Physiotherapy or other impacts may require higher levels, whereas immobilization weak damage can be controlled relatively low levels of this factor. The approximate levels of this factor in the case of specific target organs in the situation shown below:

The standard treatment of hemarthrosis (category 1):

Usually the goal is to achieve the initial concentration of factor VIII in the blood plasma level of at least 30-40% of the normal level, and then, in the next 2-3 days, maintaining a level of at least 10-20% of the normal level.

Treatment of bleeding representing the ASO is the ability for life and limb (category 2):

Usually the goal is to achieve an initial concentration of factor VIII in the blood plasma level of at least 50%, and then, during the next one week, at the level of at least 20%.

Prevention of bleeding in connection with surgery (category 3):

Typically, the goal is to achieve a concentration of factor VIII in plasma on day surgical intervention at the level of at least 60-100%, then, in the next 2-7 days, at the level of at least 30-40%, and then, within one to two weeks, at the level of at least 10-20%.

If treatment is to follow the instructions above regarding the number of injections of factor VIII in connection with these types of bleeding can say the following.

Considering the fact that the average half-life of factor VIII in plasma is 10 to 12 hours, usually in clinical practice to use the average the following number of injections of factor VIII that accompany each bleeding episode:

Hemarthrosis (bleeding into joints): Home treatment of minor hemarthrosis: 1-3 injections; treatment in hospital, more extensive hemarthrosis: 6-14 injection.

Bleeding, representing a threat to life and limb: 10-20 injection.

Prevention of bleeding in connection with surgery: 30-40 injection.

Currently, in the treatment of the AI hemophilia in a clinical setting using factor VIIa to stop bleeding in patients which are inhibitors of factors VIII or IX (which prevent substitution therapy). Typically, however, clinicians tend not to use factor VIIa as a primary treatment in cases of hemophilia patients without inhibitors (which can be used, respectively, factors VIII or IX), since it is assumed that the short half-life of factor VIIa compared with that of factor VIII (2.5 hours compared to 10-12 hours) will require more frequent injections of factor VIIa to maintain a certain level of hemostasis.

European Patent No. 225160 (Novo Nordisk) refers to the compositions of factor VIIa and treatments related to bleeding disorders, which are not defects of coagulation factors or inhibitors of coagulation factors.

European Patent No. 82182 (Baxter Travenol Lab.) refers to the composition of factor VIIa used to arouse the patient opposition to deficits or the effects of inhibitors of blood coagulation factors.

Publication Lusher et al., Haemophilia, 1998, 4, pp. 790-798, associated with the introduction of recombinant factor VIIa in the treatment of joint, muscle hemorrhage and mucosal hemorrhage in patients with hemophilia A and B, have or not have inhibitors.

Publication Kjalke et al., Thrombosis and Haemostasis, 1999 (Suppl), 095 1, associated with the introduction of extra-exogenous factor VIIa and effect the formation of thrombin on the surface of activated platelets on the model system, simulating conditions of hemophilias A and B.

U.S. patent No. 5891843 (Measurement) refers to the composition of factor VIIa in combination with a second ingredient, with FEIB-activity, such as activated prothrombin complex or FEIBA preparation.

Today, many products of factor VIII used to treat hemophilia, contain obtained by recombinant factor VIII. However, these products can also be isolated from blood plasma of pigs or humans. These refined products often contain smaller amounts of other coagulation factors or other components of blood plasma. Typically, these incremental plasma components is undesirable (because there is the risk of introducing a virus or other contaminants), and the replacement of these products with the recombinant protein (e.g., factor VIIa) is regarded as an improvement of the properties of the composition and treatment as an advantage for the patient.

Today in subjects with a reduced level of factor VIII (for example, in patients with hemophilia a) and survivors of episodic bleeding, stop the bleeding usually only possible after treatment in the form of multiple injections or infusions of factor VIII. Moreover, you need a certain number of injections to maintain hemostasis up until the injury that caused bleeding, completely SAI the et.

Victims of trauma who are suffering from excessive bleeding, usually treat large volumes infusium fluids, such as liquids for intravenous infirmerie by injection of colloidal products, albumin, including concentrates of red blood cells, etc. Extensive bleeding requiring massive blood transfusion can lead to the failure of many organs, including the weakening of the function of the lungs and kidneys.

Faster stop bleeding in such entities could create a very big advantage. Thus, it would reduce the number of injections needed to stop the bleeding and maintain haemostasis, and/or to reduce the number of clotting protein used to stop bleeding and maintain haemostasis.

There is still a need for improved therapy of subjects experienced episodic bleeding, including entities that have cases of bleeding associated with low levels of factor VIII clotting. There is also a need for improved, reliable and widely applicable methods of increasing clotting, boost or ensure the formation of a stable hemostatic clots, improve facilities used method of treatment for patients or achieving a complete or satisfactory hemost is for patients, in particular subjects, whose weak formulation of thrombin. There is a need for ways to reduce the amount of factor VIIa or a number of factor VIII required for achieving a complete or satisfactory hemostasis. There is also a need for ways to reduce the total number of protein - coagulation factor required for achieving a complete or satisfactory hemostasis, and in ways, enabling to shorten the time required to stop bleeding.

SUMMARY of INVENTION

One object according to the invention is the provision of compositions which can effectively be used in the treatment or prevention of cases of bleeding or clotting diseases.

The second object according to the invention is the provision of compositions of unit dosage forms, which can effectively be used in the treatment or prevention of cases of bleeding or as a proco-agulant. Another object according to the invention is to provide compositions, methods of treatment or sets, providing a synergistic effect.

Another object according to the invention is to provide compositions, methods of treatment or sets that do not cause significant adverse effects, such as high level the system ü activation of the blood coagulation system.

Other objects according to the invention will become apparent in the course of reading this description.

In the first aspect, the invention relates to a pharmaceutical composition comprising a preparation of factor VII or a related factor VII polypeptide, and obtaining factor VIII or related to the factor VIII polypeptide.

In the second aspect of this invention relates to a set of components for the treatment of bleeding, including: a) an effective amount of a preparation of factor VII or a related factor VII polypeptide and a pharmaceutically acceptable carrier in a first unit dosage form; b) an effective amount of a preparation of factor VIII or related to the factor VIII polypeptide and a pharmaceutically acceptable carrier in a second unit dosage form; and c) a container for the contents of these first and second dosage forms.

In another aspect this invention relates to the use of a preparation of factor VII or a related factor VII polypeptide in combination with a preparation of factor VIII or related to the factor VIII polypeptide for the manufacture of a medicinal product for the treatment of the patient cases, bleeding.

In another aspect this invention relates to the use of a preparation of factor VII or a related factor VII polypeptide in to the munali with a preparation of factor VIII or related to the factor VIII polypeptide for the manufacture of a medicinal product to reduce the time of the formation of a clot.

In another aspect this invention relates to the use of a preparation of factor VII or a related factor VII polypeptide in combination with a preparation of factor VIII or related to the factor VIII polypeptide for the manufacture of a medicinal product to increase the time of fibrinolysis (the time of the dissolution of a blood clot).

In another aspect this invention relates to the use of a preparation of factor VII or a related factor VII polypeptide in combination with a preparation of factor VIII or related to the factor VIII polypeptide for the manufacture of a medicinal product to increase the strength of the clot.

In another aspect this invention relates to a method for the treatment of episodic bleeding in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to treat bleedings.

In another aspect, the invention relates to a method for reducing the time of the formation of a clot in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII is whether a related factor VIII polypeptide, where the first and second amount together are effective to reduce the time of the formation of a clot.

In another aspect, the invention relates to a method for enhancing hemostasis in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to enhance haemostasis.

In another aspect, the invention relates to a method for reducing the number of injections protein clotting factors needed to stop bleeding and establish hemostasis in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to stop bleeding and establish hemostasis.

In another aspect, the invention relates to a method for reducing the number of input protein clotting factors needed to stop bleeding and establish hemostasis in a subject comprising administration to the subject if necessary the value of the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to stop bleeding and establish hemostasis.

In another aspect, the invention relates to a method for increasing the time of fibrinolysis (the time of dissolution of the clot) in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to increase the time of fibrinolysis.

In another aspect, the invention relates to a method for increasing clot strength in a subject, comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to increase clot strength.

In another aspect, the invention relates to a method for enhancing the formation of a fibrin clot in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or relationship is the main factor VIII polypeptide, where the first and second amount together are effective to enhance formation of a fibrin clot.

In another aspect this invention relates to a set of components for the treatment of bleeding, including: a) an effective amount of a preparation of factor VII or a related factor VII polypeptide and an effective amount of a preparation of factor VIII or related to the factor VIII polypeptide and a pharmaceutically acceptable carrier in unit dosage form; and (b) the container for the contents of the specified unit dosage forms.

In one series of aspects according to the invention, the factor VII or related to the factor VII polypeptide is a related factor VII polypeptide. In another aspect, the factor VII or related to the factor VII polypeptide is a factor VII polypeptide. In one series of aspects according to the invention specified related to the factor VII polypeptide is a variant amino acid sequence of factor VII. In one aspect the ratio between the activity related to the factor VII polypeptide and the activity of native human factor VIIa (factor VIIa wild type) is at least about 1.25 when tested by the method of hydrolysis in vitro, as described in materials of this application.

In one series is oplasty according to the invention, the factor VII or related to the factor VII polypeptide is a factor VII polypeptide. In one aspect of the factor VII is a human factor VII. In one aspect of the factor VII is a factor VII ox, pig, dog, horse, mouse, or salmon. In another aspect, the factor VII polypeptide is a recombinant factor VII. In another aspect, the factor VII polypeptide is obtained from a plasma factor VII. In another aspect, the factor VII polypeptide is obtained from a plasma factor VII human. In another aspect, the factor VII polypeptide is a recombinant factor VII human. In one series of aspects according to the invention, the factor VII or related to the factor VII polypeptide is presented in its activated form. In another aspect according to the invention, the factor VII polypeptide is a recombinant factor VIIa person.

In one series of embodiments according to the invention, the factor VIII or related to the factor VIII polypeptide is a related factor VIII polypeptide. In one aspect related to the factor VIII polypeptide is a variant amino acid sequences of factor VIII. In one aspect the ratio between the activity related to the factor VIII polypeptide and the activity of native human factor VIII (factor VIII wild type) is at least about 1.25 when tested by the method of "chrome is Nickel analysis", as described in the materials of this application. In one aspect of factor VIII or related to the factor VIII polypeptide is a polypeptide factor VIII. In one aspect of the factor VIII is a human factor VIII. In one aspect of the factor VIII is a factor VIII ox, pig, dog, horse, mouse, or salmon. In another aspect of the factor VIII polypeptide is a recombinant factor VIII. In another aspect of the factor VIII polypeptide is obtained from a plasma factor VIII polypeptide. In another aspect of the factor VIII polypeptide is obtained from a plasma polypeptide of human factor VIII. In another aspect of the factor VIII polypeptide is a recombinant human factor VIII. In one series of aspects according to the invention, the factor VIII or related to the factor VIII polypeptide is presented in its activated form. In one embodiment related to the factor VIII polypeptide is a fragment of factor VIII. In one embodiment related to the factor VIII polypeptide is a hybrid polypeptide factor VIII pig/human.

In one embodiment the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide is represented in the mass ratio between about 100:1 and about 1:100 factor VII to factor VIII.

In the following aspect, the polypeptides of factor VII or a related factor VII polypeptides have an increased independent of tissue factor activity compared to native human factor VIIa coagulation. The following aspect of the enhanced act shall want to make was not accompanied by changes in substrate specificity. In another aspect according to the invention the binding of factor VII or a related factor VII polypeptide with a tissue factor should not be weakened and the factor VII or a related factor VII polypeptides must have at least the activity of factor VIIa wild type when binding to tissue factor.

In one preferred embodiment the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide is a recombinant human factor VIIa and recombinant human factor VIII.

In one embodiment shortens the time of the formation of a clot in the blood of a mammal. In another embodiment in the blood of the mammal is enhanced hemostasis. In another embodiment in the blood of a mammal extends the time of fibrinolysis. In another embodiment increases the strength of the clot in the blood of a mammal. In another embodiment increases the formation of fibrin clot in the blood of a mammal. In one embodiment of the blood of the mammal is human blood. In another embodiment of the blood of the mammal is a normal blood; in another embodiment the blood of the mammal is blood, with normal levels of proteins, coagulation factors; in another embodiment the blood of the mammal is blood, having a normal level of factor VIII; in another embodiment krovavaya normal human blood; in one embodiment of the blood is blood obtained from the subject with impaired production of thrombin. In one embodiment of the blood is blood obtained from the subject having a deficiency of one or more coagulation factors; in another embodiment, the blood is blood obtained from the subject, which are inhibitors against one or more clotting factors. In one embodiment, the blood is blood obtained from the subject, which reduced fibrinogen concentration. In one embodiment, the blood is the blood of a person with deficiency of factor VIII.

In one of the embodiments of the present invention, the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide are only used hemostatic agents. In another embodiment the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide are only used active haemostatic agents. In another embodiment the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide are only used by the coagulation factors. In one embodiment of this invention, the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide are only used in the diversified active agents. Here the use of "only" in relation to the agents or factors refers to situations in which the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide, taken together, are only used hemostatic agents, active haemostatic agents, or coagulation factors contained in the pharmaceutical composition or kit, or are the only haemostatic agents, active haemostatic agents, or coagulation factors, administered to the patient during a particular course of treatment, such as in the treatment of episodic bleeding. Needless to say that such situations include cases where other applicable haemostatic agents, or coagulation factors not represented either in sufficient numbers or with sufficient activity so that significantly affect one or more parameters of coagulation.

In another embodiment the pharmaceutical composition is formulated for intravenous administration. In one embodiment of the present invention the composition further comprises a pharmaceutically acceptable excipient.

In one embodiment of the present invention the composition is presented in the form of a single drug dose, where a single drug dose contains both a factor in the collapse. In one embodiment of the present invention, the composition is represented as a set of parts (components) containing a preparation of factor VII or a related factor VII polypeptide as a first unit dosage form and a preparation of factor VIII or related to the factor VIII polypeptide as a second unit dosage forms containing the container in which is placed above the first and second unit dosage forms. In one embodiment of the applicable composition or the kit optionally contain instructions for use of the composition or of the individual components.

In one embodiment of this invention, the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide is introduced in the form of a single dosage form. In one embodiment of this invention, the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide is entered as the first single dosage form containing the preparation of factor VII or a related factor VII polypeptide and the second single dosage form containing the drug factor VIII or related to the factor VIII polypeptide.

In one embodiment of this invention, the factor VII or a related factor VII and the factor VIII or related to the factor VIII polypeptide is introduced at the same time. In another embodiment the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide are introduced sequentially. In one embodiment the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide are inserted one after another at intervals of not more than 15 minutes, preferably 10, more preferred 5, more preferred 2 minutes. In one embodiment of this invention, the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide are introduced one after the other at intervals up to 2 hours, preferably from 1 to 2 hours, more preferably up to 1 hour, more preferably from 30 minutes to 1 hour, more preferably up to 30 minutes, more preferably from 15 to 30 minutes.

In one embodiment an effective amount of factor VII or a related factor VII polypeptide is a number from about 0.05 mg/day to about 500 mg/day (for a subject weighing 70 kilograms). In one embodiment an effective amount of a preparation of factor VIII or related to the factor VIII polypeptide is from about 0.01 mg/day to about 500 mg/day (for a subject weighing 70 kilograms).

In one embodiment the factor VII or related to the factor VII polypeptide and the factor VIII or related the initial factor VIII polypeptide is represented in the mass ratio between about 100:1 and about 1:100 factor VII to factor VIII.

In one embodiment of the present invention a pharmaceutical composition is provided in a single dosage form and consists essentially of a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide and one or more components selected from a list of pharmaceutically acceptable excipients or carriers, stabilizers, detergents, neutral salts, antioxidants, preservatives and proteinase inhibitors.

In another embodiment the subject is a human; in another embodiment the subject has a disorder of output thrombin; in one embodiment the subject has reduced fibrinogen concentration in the plasma (for example, the subject is repeatedly exposed transfusion); in one embodiment the subject has reduced the concentration of factor VIII in plasma.

In one embodiment the pharmaceutical composition is presented in the form of a drug for treatment of humans.

In another aspect of the invention involves the use of a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide to obtain drugs for the treatment of bleeding in a subject suffering from syndrome sensitivity to factor VIII.

In another aspect of the invention involves the use of the Reparata factor VII or related to the factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide to obtain drugs for the treatment of bleeding in a subject, which has a reduced level of factor VIII.

In another aspect the invention relates to a method to enhance haemostasis in a subject suffering from syndrome sensitivity to factor VIII, as compared with when the subject is treated with factor VIII as the only protein of the coagulation factor, and this method includes the introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to enhance haemostasis.

In another aspect the invention relates to a method to enhance haemostasis in a subject, which has a reduced level of factor VIII, as compared with when the subject is treated with factor VIII as the only protein of the coagulation factor, and this method includes the introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to enhance haemostasis.

In another aspect the invention relates to a method to enhance formation of thrombin in a subject, and this method includes the SEB is an introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to enhance formation of thrombin.

In another aspect the invention relates to a method to enhance formation of thrombin in a subject suffering from syndrome sensitivity to factor VIII, as compared with when the subject is treated with factor VIII as the only protein of the coagulation factor, and this method includes the introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to enhance formation of thrombin.

In another aspect the invention relates to a method to enhance formation of thrombin in a subject, which has a reduced level of factor VIII, as compared with when the subject is treated with factor VIII as the only protein of the coagulation factor, and this method includes the introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to enhance formation of thrombin.

In other aspects the e invention relates to a method of reducing the number of injections of the protein of coagulation factor required to achieve hemostasis in a subject suffering from syndrome sensitivity to factor VIII, in comparison with the number of injections required, when factor VIII is administered to the subject as the only protein of the coagulation factor, and this method includes the introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective in reducing the number of injections of the protein of the coagulation factor.

In another aspect the invention relates to a method of reducing the number of injections of the protein of the coagulation factor necessary for achieving hemostasis in a subject, which has a reduced level of factor VIII, in comparison with the number of injections required, when factor VIII is administered to the subject as the only protein of the coagulation factor, and this method includes the introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective in reducing the introduction the protein deposits - factor out.

In another aspect the invention relates to a method of reducing the amount of input protein coagulation factor required to achieve hemostasis in a subject suffering from syndrome sensitivity to factor VIII compared to the number of input protein coagulation factor required when factor VIII is administered to the subject as the only protein of the coagulation factor, and this method includes the introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to reducing the amount of input protein coagulation factor.

In another aspect the invention relates to a method of reducing the amount of input protein coagulation factor required to achieve hemostasis in a subject, which has a reduced level of factor VIII compared to the number of input protein coagulation factor required when factor VIII is administered to the subject as the only protein of the coagulation factor, and this method includes the introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polyp is putida and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective in reducing the number of input protein coagulation factor.

In another aspect the invention relates to a method of treating bleedings in a subject suffering from syndrome sensitivity to factor VIII, and this method includes the introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective in the treatment of bleeding.

In another aspect the invention relates to a method of treating bleedings in a subject, which has a reduced level of factor VIII, and this method includes the introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective in the treatment of bleeding.

In one embodiment the subject has a reduced level of factor VIII. In another embodiment, the subject suffers from a syndrome sensitivity to factor VIII. In one embodiment syndrome sensitivity to factor VIII is hemophilia A.

In od the second embodiment of a reduced level of factor VIII is 90% or lower than normal level, in another embodiment, the level of factor VIII is 80% or below, in another embodiment is 50% or below, in another embodiment 40% or below, in another embodiment is 30% or below, in another embodiment is 20% or below, in another embodiment is 10% or below, in another embodiment is 5% or below, in another embodiment is 2% or below. These terms can be used interchangeably. In the preferred embodiment the level of factor VIII is below 30% of normal level.

In another aspect the invention relates to a method of treating bleedings in a subject suffering from syndrome sensitivity to factor VIII, and the method includes the introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective in the treatment of bleeding.

In one embodiment the factor VII is a recombinant human factor VIIa (rFVIIa). In another embodiment the factor rFVIIa is a product NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark).

In one embodiment the factor VIII is a recombinant human factor VIII (rFVIII). In another embodiment the factor VIII is a product ReFacto (AHP/Genetics Institute), Alphanate (Alpha), Biocate (Aventis) Monoclate-P (Aventis), Helixate (Aventis), Recombinate (Baxter), Hemophil M (Baxter), Kogenate (Bayer), Nordiate (HemaSure), FACTEUR VIII-LFB (French Laboratory fractionation and biotechnologies (LFB)), Hyate:C (Spaywood) and Kogenate SP (Bayer).

In one embodiment the pharmaceutical composition is formulated for intravenous administration. In one embodiment, the composition additionally includes fibrinolytic system, including, but not limited to, Aprotinin, ε-aminocaproic acid or tranexamic acid.

LIST of DRAWINGS

Figure 1: figure 1 shows the effect of accelerating collapse under the action of rFVIIa in FVIII-deficient plasma in the absence and in the presence of FVIII.

Figure 2: figure 2 shows the effect of accelerating collapse under the action of rFVIIa in normal plasma in the absence and in the presence of FVIII.

DETAILED description of the INVENTION

In subjects who lose a lot of blood in connection with the operation or serious injury and which, therefore, need for blood transfusion, develop more serious complications than those who usually bleeding does not occur. However, moderate bleeding can also lead to complications if this requires the introduction of human blood or blood components (platelets, leukocytes obtained from plasma concentrates for the treatment of coagulation defects, and so on), because it is associated with the risk of transmitting the human is x viruses (for example, virus hepatitis, HIV, parvovirus or other unknown today, viruses and non-viral pathogens of nature. Extensive bleeding requiring massive blood transfusions can lead to failure of multiple organs, including the failure function of the lungs and kidneys. As soon as the subject starts to develop such serious complications, a cascade of events involving a number of cytokines and inflammatory reactions that exclusively makes any treatment, often, unfortunately, ineffective. Consequently, the main task in surgery and in the treatment of serious tissue damage associated with in order to avoid or minimize bleeding. To avoid or minimize such unwanted bleeding, it is important to ensure the formation of a stable and solid hemostatic plugs, which are not very susceptible to dissolution under the action of fibrinolytic enzymes. Moreover, it is very important to ensure the rapid and efficient formation of such tubes or clots.

In subjects with thrombocytopenia (when reduced amount or activity of platelets) also attenuated the generation of thrombin, and in addition, they have the defect of fibrin stabilizing tubes, in which a hemostatic plug is subjected to premature is agradeciy. In addition, in subjects who have undergone extensive injury or damage to the body and which have been subjected to frequent blood transfusions are often reduced number of platelets, as well as reduced levels of fibrinogen, factor VIII and other clotting proteins in the blood. Such entities are usually weakened (or decreased) production of thrombin. In these subjects, therefore, defective or less effective hemostasis, leading to the formation of fibrin plugs that are easily before they ripen dissolve under the action of proteolytic enzymes, in addition to these enzymes in situations of extensive injury or lesion of the body, are released in large quantities.

The patient underwent extensive blood loss, becomes clinically unstable. In these patients there is a risk of atrial fibrillation, which can lead to fatal stopping of cardiac activity; the weakening of renal function or extravasation of fluid from the vessels into the tissue of the lungs (the so-called "wet lungs, or respiratory distress syndrome in adults (ARDS)).

Bleeding in the tissues can also lead to bruising. The size of the hematoma (in particular, intercranial and spinal) are closely associated with the degree of loss of neurological function, difficulties rehabilitation and/or the severity and degree of permanent impairment of neurological function after rehabilitation. The most severe consequences of hematomas can be traced in those cases when they are localized in the brain, and in this case, they can even cause the death of the patient. The so-called compartmental syndrome is a clinical condition caused severe internal bleeding in an extremity. In the hands and feet muscles and bones externally surrounded by an almost inelastic collagen layer called fascia. Bleeding into the space surrounded by fascia, increases the pressure in this compartment, and, consequently, increases the pressure on the nervous, vascular and muscle tissue, causing as a result, if no urgent action is taken, extensive tissue necrosis. In the formation of necrosis necrotic tissue is largely due to the healing process will be transferred to the connective tissue, which is shortened (reduced) compared to the original muscle tissue. Such contractures lead to the fact that the subject is forced to limit the mobility of the affected joints, which in turn again leads to the need for surgical correction. Severe bruising can then lead to the formation of pseudo-cysts, which may resemble benign tumors, in which such cysts like tumors erogenous damaged muscle or bone tissue. And again there is a necessity to the operation for the removal of such pseudo-cyst.

In addition, the formation of hematomas increases the incidence of infections in the subject. The same result can lead and infusion of blood components such as erythrocytes. Infusion of red blood cells lead to the risk of antibody formation in the subject. When a subject having antibodies against blood components, very difficult transfusion in this patient, as it becomes increasingly difficult to match blood types.

Thus, the main objectives in the treatment of bleeding are reduced to achieve hemostasis within the minimum possible period of time, thus minimizing blood loss.

Thus, the present invention provides a beneficial composition, application and methods of treatment for elimination of bleeding episodes in subjects in case of need of such treatment. Such compositions, applications and methods can be associated with beneficial effects such as reduction of blood loss before achieving hemostasis, reducing blood loss during operations, maintenance of blood pressure at an acceptable level until you have reached a state of hemostasis, faster stabilization of blood pressure, shortening the recovery time of the patient being treated, the shortening of the time of rehabilitation of the patient being treated, ukrashenie education bruising or haematoma formation of smaller size, including hematoma in the brain, reduced formation of pseudo-cysts, reduced formation of muscle contractures, faster stopping bleeding, reducing the number of injections needed to stop bleeding and achieve hemostasis, reducing clotting protein used to stop bleeding and achieve hemostasis.

The introduction of a preparation of factor VII or a related factor VII polypeptide, such as factor VIIa, in combination with a preparation of factor VIII or related to the factor VIII polypeptide provides a shortening of the clotting time of blood compared to the clotting time, when either factor VIIa or factor VIII are assigned separately.

The introduction of a preparation of factor VII or a related factor VII polypeptide, such as factor VIIa, in combination with a preparation of factor VIII or related to the factor VIII polypeptide also provides a decrease in the total number of clotting factor used to stop bleeding and achieve hemostasis in a subject in case of need of such treatment compared to the amount of protein applied when either factor VIIa or factor VIII are assigned separately.

The introduction of a preparation of factor VII or a related factor VII polypeptide, such as factor VIIa, in combination with a preparation of factor VIII or related to factor VIII polypeptid is and also provides a reduction in time stop the bleeding and reduce the number of injections to achieve haemostasis compared to the situation when either factor VIIa or factor VIII are assigned separately. The introduction of a preparation of factor VII or a related factor VII polypeptide, e.g., factor VIIa, in combination with a preparation of factor VIII or related to the factor VIII polypeptide also provides a reduction in the number of injection to ensure complete haemostasis compared to the situation when either factor VIII or factor VIIa are entered separately.

Patients with hemophilia A, the present invention provides beneficial effects in simultaneous or sequential introduction of doses of factor VIII or related to the factor VIII polypeptide and a preparation of factor VII or a related factor VII polypeptide. As the substitution of coagulation factor VIII, and a preparation of factor VII or a related factor VII polypeptide induce hemostasis in these groups of patients, but have different biochemical mechanisms of action. Simultaneous introduction improves hemostatic effect.

The present invention provides a pharmaceutical composition comprising a combination of a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide. The composition may be in the form of a single composition or in the form of a multi-component set (set of parts). The composition of solenoidality applicable as a therapeutic and preventive proco-agulant in mammals, including primates and humans. In addition, the present invention provides a method of treatment (including prophylactic treatment or prevention) of bleeding episodes in a subject, including humans.

Wherever in this description neither mentioned first, or second, or third, etc. the introduction of drug doses, does not mean the preferred order of introduction, but rather referred to for the sake of convenience of presentation.

The combination of a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide is a beneficial product, that reduce the clotting time and the rapid formation of hemostatic plugs. The authors of this invention will be shown that combination of factor VIIa and factor VIII can more effectively reduce the clotting time of normal human plasma than factor VIIa or factor VIII separately. Thus, shortening the clotting time, you can achieve a more effective treatment of bleeding in subjects. Moreover, patients can be treated with a lower total amounts of factor VII and factor VIII or related to the factor VIII polypeptide.

The factor VII polypeptides:

In the practical use of the present invention can use any factor VII polypeptide, to which were effective in preventing or treating bleeding. This includes the factor VII polypeptides derived from blood or plasma or obtained by recombinant means.

The present invention encompasses polypeptides of factor VII, such as those that have an amino acid sequence described in U.S. patent No. 4784950 (human factor VII (wild-type). In some embodiments the factor VII polypeptide is human factor VIIa, as described, for example, in U.S. patent No. 4784950 (factor VII (wild-type). In one series of embodiments, the factor VII polypeptides include polypeptides that exhibit at least about 10%, preferably at least about 30%, more preferably at least about 50% and most preferably at least about 70% of the specific biological activity of human factor VIIa. In one series of embodiments the factor VII polypeptides include polypeptides that exhibit at least about 90%, preferably at least about 100%, preferably at least about 120%, more preferably at least about 140%, and most preferably at least about 160% of the specific biological activity of human factor VIIa. In one series of embodiments the factor VII polypeptide is clucalc the polypeptides, which exhibit at least about 70%, preferably at least about 80%, more preferably at least about 90% and most preferably at least about 95% identity with the sequence of factor VII wild type, as described in U.S. patent No. 4784950.

In the present description, the term "factor VII polypeptide" encompasses, without limitation, factor VII polypeptides, as well as related factor VII polypeptides. The term " factor VII" means, without limitation, polypeptides having the amino acid sequence 1-406 of human factor VII wild-type (as described in U.S. patent No. 4784950), and factor VII wild type obtained in other species, such as factor VII, ox, pig, dog, mouse, and salmon, and the factor VII derived from blood or plasma, or obtained by recombinant. It additionally includes natural allelic variants of factor VII that may exist and occur from one or another of the individual. The degree and localization of glycosylation or other posttranslational modifications may vary depending on the choice of the host cell and the nature of the environment of the host cells. The term "Factor VII" also refers to the coverage of factor VII polypeptides in their unsplit form (FMR is e zymogen), as well as those polypeptides that have undergone proteolytic processing that resulted in their respective bioactive forms, which may be described as factor VIIa. Typically, the factor VII is cleaved between residues 152 and 153, producing the factor VIIa.

"Related to factor VII polypeptides" include, but are not limited to, factor VII polypeptides that are either chemically modified compared to human factor VII and/or contain one or more changes in amino acid sequence compared to human factor VII (i.e. variants of factor VII), and/or contain truncated amino acid sequences were compared to human factor VII (i.e. fragments of factor VII). Such related factor VII polypeptides can exhibit properties similar to the properties of human factor VII, including stability, phospholipid binding, altered specific activity, and the like. The term "related to factor VII polypeptides" is intended to refer to such polypeptides in their unsplit form (form zymogen), as well as those polypeptides that have undergone proteolytic processing, and as a result their respective bioactive forms, which may be designated as a related factor is VIIa polypeptides" or "activated related to factor VII polypeptides".

Here, the term "related to factor VII polypeptides" encompasses, without limitation, polypeptides exhibiting essentially the same or improved biological activity compared to human factor VII wild type, as well as polypeptides, in which the biological activity of factor VIIa substantially modified or reduced relative to the activity of human factor VIIa wild type. These polypeptides include, without limitation, factor VII or factor VIIa, which has been chemically modified, and variants of factor VII, amino acid sequences which have made specific changes that modify or disrupt the bioactivity of the polypeptide.

In addition, it encompasses polypeptides with a slightly modified amino acid sequence, for example, polypeptides having a modified N-end including N-terminal amino acid deletions or additives, and/or polypeptides that have been chemically modified compared to human factor VIIa.

Related to factor VII polypeptides, including variants of factor VII, regardless, does it essentially the same or improved biological activity than the factor VII wild-type, or, alternatively, exhibiting substantially modified or reduced biological activity is compared with factor VII wild-type, include, but are not limited to, polypeptides having the amino acid sequence that differs from the sequence of factor VII wild-type insertion, deletion or replacement of one or more amino acids.

Related to factor VII polypeptides, including variants include polypeptides that exhibit at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, or at least about 130% of the specific activity of factor VIIa wild type, which was produced by the same cell type when tested in one or more tests for coagulation, the analysis of proteolysis or analysis of TF binding, as described above.

Related to factor VII polypeptides, including variants, having essentially the same or improved biological activity compared to the factor VIIa wild type, include polypeptides that exhibit at measures is approximately 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120%, and most preferably at least about 130% of the specific activity of factor VIIa wild type, which was produced by the same cell type when tested in one or more tests for coagulation, the analysis of proteolysis or analysis of TF binding, as described above.

Related to factor VII polypeptides, including variants, having substantially reduced biological activity compared to the factor VIIa wild type are those polypeptides which exhibit less than about 25%, preferably less than about 10%, more preferably less than about 5% and most preferably less than about 1% of the specific activity of factor VIIa wild type, which was produced by the same cell type when tested in one or more tests for coagulation, the analysis of proteolysis or analysis of TF binding, as described above. Variants of factor VII, which has essentially a modified biological activity compared with factor VII wild tee is a, include, but are not limited to, variants of factor VII, which show a TF-independent proteolytic activity of factor X, and such variants that bind TF, but not split factor X.

In some embodiments the factor VII polypeptides are related to the factor VII polypeptides, in particular variants where the ratio between the activity of the polypeptide of this factor VII and the activity of native human factor VIIa (wild-type FVIIa) is at least about 1.25 when tested by the method of hydrolysis in vitro (see Analysis, below); in other embodiments, this ratio is at least approximately 2.0; in other additional embodiments, this ratio is at least approximately to 4.0. In some embodiments according to the invention, the factor VII polypeptides are related to the factor VII polypeptides, in particular variants where the ratio between the activity of the polypeptide of this factor VII and the activity of native human factor VIIa (wild-type FVIIa) is at least about 1.25 when tested by the method of proteolysis in Vitro (see Analysis, below); in other embodiments the ratio is at least approximately 2.0; in further embodiments, the ratio is at least approximately 4,0; the further embodiments the ratio is at least approximately 8,0.

In some embodiments the factor VII polypeptide is human factor VII, as described, for example, in U.S. patent No. 4784950 (factor VII (wild-type). In some embodiments the factor VII polypeptide is human factor VIIa. In one series of embodiments the factor VII polypeptides are related to the factor VII polypeptides that exhibit at least about 10%, preferably at least about 30%, more preferably at least about 50% and most preferably at least about 70% of the specific biological activity of human factor VIIa. In some embodiments the factor VII polypeptides have an amino acid sequence that differs from the sequence of factor VII wild-type inclusion, deletion or replacement of one or more amino acids.

Non-limiting examples of factor VII, which has essentially the same or improved biological activity as the factor VII wild type, include S52A-FVII, S60A-FVII (lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998); L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII and S336G-FVII; FVIIa variants exhibiting increased p is osteoliticescuu stability, as described in U.S. patent No. 5580560; factor VIIa, which proteoliticeski cleaved between residues 290 and 291 or between residues 315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48: 501-505, 1995); and oxidized forms of factor VIIa (Kornfelt et al., Arch. Biochem. Biophys. 363: 43-54, 1999). Non-limiting examples of factor VII, which has significantly reduced or modified biological activity compared with factor VII wild type, include R152E-FVIIa (Wildgoose et al., Biochem 29: 3413-3420, 1990), S344A-FVIIa (Kazama et al., J. Biol. Chem. 270: 66-72, 1995), FFR-FVIIa (Holst et al., Eur. J. Vasc. Endovasc. Surg. 15: 515-520, 1998) and factor VIIa, lacking Gla domain (Nicolaisen et al., FEBS Letts. 317: 245-249, 1993). Non-limiting examples of chemically modified factor VII polypeptides and variants of the sequences described, for example, in U.S. patent No. 5997864.

The biological activity of factor VIIa in blood coagulation associated with its ability (i) to contact with tissue factor (TF) and (ii) to catalyze the proteolytic cleavage of factor IX or factor X with the production of activated factor IX or X (factor IXa or Xa, respectively).

For the purposes of the present invention, the biological activity of factor VII polypeptides ("biological activity of factor VII") can be quantified by measuring the ability of the drug to start the clotting process using plasma deficient in factor VII, and thromboplastin, is described, for example, in U.S. patent No. 5997864. In this assay, the biological activity is expressed as the decrease in clotting time compared with the control sample and is converted into units of factor VII by comparison with the combined standard human serum, containing 1 unit/ml activity of factor VII. Alternative biological activity of factor VIIa can be quantified by:

(i) measuring the ability of factor VIIa or a related factor VIIa polypeptide to produce activated factor X (factor Xa) in the system containing tissue factor (TF), embedded in a lipid membrane and factor X. (Persson et al., J. Biol. Chem. 272: 19919-19924, 1997);

(ii) measuring the hydrolysis of factor X in the aqueous system ("analysis of proteolysis in Vitro", see below);

(iii) measuring the physical binding of factor VIIa or a related factor VIIa polypeptide with a tissue factor using a unit-based surface plasmon resonance (Persson, FEBS Letts. 413: 359-363, 1997);

(iv) measuring hydrolysis of a synthetic substrate by the action of factor VIIa and/or related to the factor VIIa polypeptide (analysis of hydrolysis in Vitro", see below);

(v) measuring generation of thrombin in a TF-independent in vitro system.

The term "biological activity of factor VII"or "activity of factor VII", refers to the ability to generate thrombin; this Ter is in also implies the generation of thrombin on the surface of activated platelets in the absence of tissue factor.

Preparation of factor VIIa, which can be used according to the invention, is not limited to, the drug produced by the company NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark).

The polypeptides of factor VIII:

The present invention includes polypeptides of factor VIII, such as, for example, polypeptides having the amino acid sequence described, for example, in the publication'toole et al., Nature 1984; 312: 342-347 (human factor VIII wild type).

In fact, in connection with the present invention may be any polypeptide factor VIII, which is effective in preventing or treating bleeding. This includes polypeptides of factor VIII derived from blood or plasma, or obtained by recombinant.

Here, "factor VIII polypeptide" refers, without limitation, factor VIII, and is also related to the factor VIII polypeptide. The term "factor VIII" includes, without limitation, polypeptides having the amino acid sequence described in the publication'toole et al., Nature 1984 (see above) (human factor VIII wild-type), and factor VIII wild type obtained in other species, such as factor VIII, bull, pig, dog, mouse, and salmon. In addition, it includes natural allelic variants of factor VIII that may exist and occur from and who and other individuals. The degree and localization of glycosylation or other posttranslational modifications can vary depending on the choice of the host cell and the nature of the environment of the host cells. The term "Factor VIII" also refers to the coverage of the polypeptides of factor VIII in their unsplit form (form zymogen), as well as those polypeptides that have undergone proteolytic processing, with education as a result of their respective bioactive forms, which may be characterized as a factor VIIIa.

"Related to factor VIII polypeptides" include, but are not limited to, factor VIII polypeptides that are either chemically modified compared to human factor VIII, and/or contain one or more changes in amino acid sequence compared to human factor VIII (i.e. variants of factor VIII), and/or contain truncated amino acid sequences were compared to human factor VIII (i.e., the fragments of factor VIII). Such related factor VIII polypeptides can exhibit properties similar to the properties of human factor VIII, including stability, phospholipid binding, altered specific activity, and the like. The term "related to factor VIII polypeptides" is intended to refer to such polypeptides in their unsplit form(form zymogen), as well as those polypeptides that have undergone proteolytic processing, and as a result their respective bioactive forms, which may be designated as "related to factor VIIIa polypeptides" or "activated related factor VIII polypeptides".

Here, the term "related to the factor VIII polypeptide" encompasses, without limitation, polypeptides exhibiting essentially the same or improved biological activity compared to human factor VIII wild type, as well as polypeptides, in which the biological activity of factor VIII is substantially modified or reduced relative to the activity of human factor VIII of the wild type. These polypeptides include, without limitation, factor VIII or factor VIIIa, which has been chemically modified, and variants of factor VIII in the amino acid sequence of which were made specific changes that modify or disrupt the bioactivity of the polypeptide.

In addition, it encompasses polypeptides with a slightly modified amino acid sequence, for example, polypeptides having a modified N-end including N-terminal amino acid deletions or additives, and/or polypeptides that have been chemically modified compared to human factor VIII.

Ro is a significant factor VIII polypeptides, including variants of factor VIII, regardless, does it essentially the same or improved biological activity than the factor VIII wild-type, or alternative exhibits substantially modified or reduced biological activity compared to factor VIII include, without limitation, polypeptides having the amino acid sequence that differs from the sequence of factor VIII wild-type insertion, deletion or replacement of one or more amino acids.

A related factor VIII polypeptides, including variants include polypeptides that exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, and at least about 130% of the specific activity of factor VIII wild type, which was produced by the same cell type when tested in the analysis of the activity of factor VIII, as described in this application.

A related factor VIII polypeptides, including variants, have their essentially the same or improved biological activity compared to factor VIII wild-type, include polypeptides that exhibit at least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120%, and most preferably at least about 130% of the specific activity of human factor VIII wild type, which was produced in the same cell type when tested in one or more analyses of the specific activity of factor VIII, as described above. For purposes associated with the present invention, the biological activity of factor VIII can be quantitatively determined, as described below in the present description ("tests").

A related factor VIII polypeptides, including variants, having substantially reduced biological activity compared to factor VIII wild type are those polypeptides which exhibit less than about 25%, preferably less than about 10%, more preferably less than about 5% and most preferably less than about 1% of the specific activity of factor VIII wild type, which was produced in the cells of the same the IPA when tested in one or more analyses of the specific activity of factor VIII, as explained above.

Non-limiting examples of factor VIII polypeptides include obtained from plasma of human factor VIII, as described, for example, Fulcher et al., Proc. Acad. Nat. Sci. USA 1982, 79: 1648-1652; and Rotblat et al., Biochemistry 1985, 24: 4294-4300; and obtained from plasma pig factor VIII, as described, for example, Fass et al., Blood 1982, 59:594-600 and Knutson et al., Blood 1982, 59:615-624.

In some embodiments, the polypeptides of factor VIII are related to the factor VIII polypeptide, and the ratio between the activity of the polypeptide of the factor VIII and the activity of native human factor VIII (factor VIII wild type) is at least about 1.25 when tested by the method of "chromogenic analysis" (see below); in other embodiments, this ratio is at least approximately 2.0; in other additional embodiments, this ratio is at least approximately 4,0.

Non-limiting examples of variants of the sequence of factor VIII are described, for example, Lollar et al., Blood, 2000; 95(2): 564-568 (hybrid FVIII polypeptides pig/human) and Lollar et al., Blood, 2001; 97(1): 169-174.

Commercially available products of factor VIII (so-called substitution products) obtained from the joint normal plasma or obtained by genetic engineering methods lines of mammalian cells. Substitution products often classificar the t in accordance with their ultimate purity, defined as a specific activity (international units of activity of coagulation factor per mg protein, IU/mg). Intermediate products have a relatively low specific activity(<50 IU/mg), because they also contain extraneous plasma proteins such as fibrinogen, fibronectin and other coagulant proteins. A high degree of purity (>50 IU/mg) and ultra-high purity (>3000 IU/mg) correspond to a very low content of impurities other plasma proteins or their absence, in addition to albumin, which is added as a stabilizer.

Non-limiting examples of commercially available products of factor VIII concentrates and preparations), which can be used according to the invention are, for example, without limitation, ReFacto (devoid of In-domain rFVIII) from AHP/Genetics Institute, Alphanate (FVIII) Alpha, Bioclate (rFVIII) company Aventis, Monoclate-P (factor VIII:C) company Aventis, Helixate (rFVIII) company Aventis, Recombinate (rFVIII) firm Baxter, Hemophil M (FVIII) firm Baxter, Kogenate (rFVIII) Bayer, Nordiate (FVIII firms HemaSure, FACTEUR VIII-LFB (FVIII-based human plasma) from the French Laboratory fractionation and biotechnologies (LFB), Hyate:C (FVIII pigs) company Spaywood, and Kogenate SP (rFVIII in the composition to sucrose) from Bayer.

Non-limiting examples of products with high activity and high activity are Alphanate (Alpha) (low), ReFacto (AHP/Genetics Institute) Kogenate SF (Bayer), Kogenate (Bayer), Helixate (Aventis), Recombinate (Baxter), Monoclate-P (Aventis), Hemophil M (Baxter) (all ultra-high activity).

Definitions:

In the text of this description three-letter or one-letter symbols of amino acids are used in their usual meaning, as indicated in table 1. Unless explicitly, under specified here refers to amino acids L-amino acids. It should be understood that the first letter, for example, K337 means amino acid, natural at a specified position of the factor VII wild type, and that, for example, K337A-FVIIa means FVII variant where the amino acid is presented in one-letter code as K and natural in that position and replaced by the amino acid presented in one-letter code as A.

Table 1

Abbreviation of amino acids:
Amino acidThree-letter codeSingle-letter code
GlycineGlyG
ProlineProP
AlanineAlaA
ValineValV
LeucineLeuL
IsoleucineIleI
Methionine MetM
CysteineCysC
PhenylalaninePheF
TyrosineTyrY
TryptophanTrpW
HistidineHisH
LysineLysK
ArginineArgR
GlutamineGlnQ
AsparagineAsnN
Glutamic acidGluE
Aspartic acidAspD

The term "factor VII", "Factor VII" or "FVII" can be used interchangeably. The term "factor VIIa", "Factor VIIa" or "FVIIa" can be used interchangeably. The term "factor VIII", "Factor VIII " or "FVIII " can be used interchangeably.

In this context, the term "subjects with impaired thrombin generation" refers to entities that have not generated a full release of thrombin on the surface of activated platelets; it includes subjects who do not produce thrombin below the level that is achieved in individuals with fully functioning n malnoy the hemostatic system, including the normal amount and the normal function of coagulation factors, platelets and fibrinogen, and includes, without limitation, entities that do not have factor VIII clotting; subjects with a reduced number of platelets or with platelet functional defect (e.g., thrombocytopenia or thrombasthenia of Glanzmann, or subjects with excessive bleeding); subjects who have reduced levels of prothrombin, FX or FVII; subjects, who have reduced levels of several coagulation factors (for example, due to excessive bleeding in trauma or extensive surgery); and subjects, have reduced the concentration of fibrinogen in the blood plasma (for example, subjects who underwent repeated transfusion).

The term "enhancing the system of hemostasis" refers to the increased ability to generate thrombin. The term "enhancing hemostasis" refers to the situation when measured thrombin generation in a sample containing a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide, prolonged compared with the individual thrombin generation in a control sample containing only the factor VII or related to the factor VII polypeptide or the factor VIII or Rhodes is ment factor VIII polypeptide, when testing the same method of measuring thrombin generation. The production of thrombin can be estimated as described in the method of analysis generation of thrombin in the present description (see "assays").

The time parameters of clot dissolution (fibrinolysis), the strength of the clot, the formation of fibrin clots and clotting time are the clinical parameters used to study the status of the hemostatic system in patients. The blood samples at appropriate intervals taken from patients and study one or more parameters using, for example, thromboelastography, as described, for example, in the publications Meh et al., Blood Coagulation &Fibrinolysis 2001; 12: 627-637; Vig et al., Hematology, Vol. 6 (3) pp. 205-213 (2001); Vig et al., Blood coagulation &fibrinolysis, Vol. 12 (7) pp. 555-561 (2001) Oct; Glidden et al., Clinical and applied thrombosis/hemostasis, Vol. 6 (4) pp. 226-233 (2000) Oct; McKenzie et al., Cardiology, Vol. 92 (4) pp. 240-247 (1999) Apr; or Davis et al., Journal of the American Society of Nephrology, Vol. 6 (4) pp. 1250-1255 (1995).

The term "prolonging the time of dissolution of the clot" covers situations where the designated time of dissolution of the clot in the sample containing a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide, prolonged compared with an individual time of dissolution of the clot in the control sample containing only the factor VII or a related factor VI polypeptide or the factor VIII or related to the factor VIII polypeptide, when testing the same method of analysis of the dissolution of the clot. The time of dissolution of the clot can be estimated as described above.

The term "increasing clot strength" refers to the situation when determining the strength of the clot, such as mechanical strength, in samples containing a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide, is increased in comparison with an individual time of dissolution of the clot in the control sample containing only the factor VII or related to the factor VII polypeptide or the factor VIII or related to the factor VIII polypeptide, when testing the same method for assessing the strength of the clot. The strength of the clot can be estimated, as described, for example, Carr et al, 1991. (Carr ME, Zekert SL. Measurement of platelet-mediated force development during plasma clot formation. AM J MED SCI 1991; 302: 13-8), or, as described above, by using thromboelastography.

The term "enhancing education fibrin clot" refers to situations when the measured rate or extent of the formation of a clot in a test sample containing a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide, is increased in comparison with individual speed or degree of formation of a clot in a test sample, the soda is containing only the factor VII or related to the factor VII polypeptide or the factor VIII or related to the factor VIII polypeptide, when testing the same method for assessing coagulation. The formation of a fibrin clot can be estimated as described above.

The term "reducing clotting time" refers to the situations when the measured time of the formation of a clot (clotting time) in a test sample containing a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide, is increased in comparison with an individual time in the control sample containing only the factor VII or related to the factor VII polypeptide or the factor VIII or related to the factor VIII polypeptide, when testing the same method for assessing coagulation. The clotting time can be assessed using standard assays PT og aPTT, which are known to experts in this field.

Used herein, the term "disorder associated with bleeding reflects any congenital, acquired or induced defect cellular or molecular nature, which manifests itself in the form of occasional bleeding. Examples of disorders associated with bleeding, include, but are not limited to, deficiencies of coagulation factors (for example, deficiency of factors VIII, IX, XI or VII), inhibitors of coagulation factors, the defective platelet function (e.g., disease of Glanzmann and sink the m Bernard-Soulier syndrome), thrombocytopenia, disease von Willebrand's disease and coagulopathy, such as coagulopathy caused by dilution of the protein coagulation, increased fibrinolysis and decreased number of platelets as a result of bleeding and/or transfusions (e.g., patients, or patients with trauma exposed to repeated transfusions).

Bleeding is transudate blood outside of any component of the circulation system. The term "bleeding episodes" refers to unwanted, uncontrollable and often excessive bleeding in connection with surgery, trauma, or other forms of tissue damage, as well as undesirable bleeding in subjects with disorders associated with bleeding. Occasional bleeding may occur in subjects with baseline normal blood clotting system, but which moved the (temporary) coagulopathy, as well as subjects who have inherited or acquired clotting disorders or disorders associated with bleeding. In subjects with disorders of platelet function, bleeding can remind bleeding caused by hemophilia, because in these cases, as in hemophilia, in the hemostatic system are missing the main "connection" coagulation or they have abnormal nature (e.g., platelets or protein factor von Willebrand's disease). Subjects undergoing extensive tissue damage, for example, in connection with the operation or extensive trauma, the normal mechanism of hemostasis can be suppressed as a result of the continued relevance of immediate hemostasis, and they in spite of initial normal mechanism of hemostasis (prior injury or prior surgical exposure) may develop excessive bleeding. These entities, which are then often subjected to multiple transfusions, developing (temporarily) coagulopathy as a result of bleeding and/or transfusions (i.e. dilution coagulating proteins, increased fibrinolysis and a low platelet count in the result of bleeding and/or blood transfusions). Bleeding can also occur in organs such as the brain, the area of the inner ear, as well as in the eye; these are areas in which the possibility of surgical hemostasis is limited, and therefore, satisfactory hemostasis in these cases is problematic. Such problems can occur during sampling biopsies of various organs (liver, lung, tumor tissue, gastrointestinal tract), as well as in laparoscopic surgery and the radical zagalabatay prostatectomy. Common in all these situations is the difficulty in providing hemostasis using the surgeon who ical techniques (stitches, clamps etc.), in particular, when the bleeding is diffuse (e.g., acute hemorrhagic gastritis and profuse uterine bleeding). Bleeding can also occur under the action of anticoagulation therapy in subjects who have defective hemostasis induced by therapy; such bleeding often acute and extensive. Anticoagulant therapy is often prescribed to prevent thromboembolic disease. Such therapy may include heparin, other forms of proteoglycans, warfarin or other forms of vitamin K antagonists, and aspirin and other platelet aggregation inhibitors, such as antibodies or other inhibitors of the activity of GP IIb/IIIa. Under the episodes of bleeding also means, not limited to, uncontrolled and excessive bleeding in connection with surgery or trauma in subjects with acute hemarthrosis (bleeding into joints), chronic the hemophilic arthropathy, hematoma (e.g., muscle, retroperitoneal, sublingual and retropharyngeal), bleeding in other tissues, hematuria (bleeding from the renal tract), cerebral haemorrhage, the subjects during surgery (for example, hepatectomy), tooth extraction and gastrointestinal bleeding (such as bleeding of the upper section as docno-intestinal tract). Occasional bleeding may be associated with inhibitors of factor VIII; haemophilia A; haemophilia a with inhibitors hemophilia B; deficiency of factor VII; deficiency of factor XI; thrombocytopenia; deficiency of factor von Willebrand's disease (a disease von Willebrand's disease); severe tissue damage; severe trauma; surgery; laparoscopic surgery; haemorrhagic gastritis; the selection of biopsy material; anticoagulant therapy; bleeding of the upper section of the gastrointestinal tract (UGI); or stem cell transplantation. Occasional bleeding may be extensive uterine bleeding; bleeding that occurs in the bodies, in respect of which limited the possibility of mechanical hemostasis; bleeding that occurs in the brain; produced in the region of the inner ear; or arising in the eyes. The terms "bleeding episodes" and "bleeding" can accordingly be used interchangeably.

In this context, the term "treatment" includes preventing any bleeding, such as bleeding in surgical interventions and regulation of an already existing bleeding, such as in trauma, in order to inhibit or minimize bleeding. The above "possible bleeding may be bleeding, the buildings permanently, it is incurred which can be expected in the specific tissue or specific body or it may be unexpected bleeding. Prophylactic purpose of the preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide is thus included in the term "treatment".

Here, the term "subject" refers to any animal, particularly mammals, such as man, and this term may optionally be used along with the term "patient" are used interchangeably.

The factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide, as defined in the present description, may be introduced simultaneously or sequentially. These factors can be represented in the form of a single dosage form, where a single dosage form contains both coagulation factor, or set of parts containing preparation of factor VII or a related factor VII polypeptide as a first dosage form and a preparation of factor VIII or related to the factor VIII polypeptide as a second dosage forms. The second dosage form may be represented as a product with high, medium or low activity of factor VIII. Predpochtitelnye are highly active products. Most preferred are combinatie high-level products. In the present description reference to the first, second or third etc. dosage form refers not to the preferred order of their introduction, but rather made out of convenience.

By "simultaneous" dosing of a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide is meant as an introduction proteins coagulation factors in a single dosage form or the introduction of the first protein of the coagulation factor, and then the second protein of the coagulation factor to the interval between injections is not more than 15 minutes, preferably 10, more preferred 5, more preferred 2 minutes. Any of the factors can be entered in the first place.

By "consistent" dose refers to the introduction of the first protein of the coagulation factor, and then the second protein - factora with an interval between doses up to 2 hours, preferably from 1 to 2 hours, more preferably up to 1 hour, more preferably from 30 minutes to 1 hour, more preferably up to 30 minutes, more preferably from 15 to 30 minutes. Any of these two unit dosage forms, or proteins, coagulation factors, can be entered in the first place. Preferably, both the product was is injected using the same devices for intravenous administration.

Under the "level of factor VIII" refers to the level of activity of coagulation factor VIII in a patient compared to its level in healthy subjects. This level is indicated as a percentage of the normal level.

Under the "reduced level of factor VIII" refers to the reduction of the present amount or activity of factor VIII in the blood stream compared with the average level of factor VIII in a population of subjects that do not have deficiency of factor VIII clotting inhibitors or factor VIII clotting. On the basis of data obtained during the purification of this factor isolated from human blood plasma, the concentration of factor VIII in normal adult is approximately 100 to 200 ng/ml plasma (mean value), which is equivalent to approximately 0.1 μm; this is equivalent to 0.6 to 1.6 units/ml

In normal healthy individuals the activity of factor VIII and antigen levels vary between 60 and 160% of that of the normal United plasma. Clinically, the level of circulating factor VIII can be measured either by assessing coagulation, or by immunological analysis. Procoagulant activity of factor VIII is determined based on the ability of the plasma of the patient to correct the clotting time of plasma deficient in factor VIII (for example, in the analysis of the APTT (activated partial thromboplastin the new time), see below; see also "analysis" of the present description).

Unit of factor VIII is defined as the amount of factor VIII in one milliliter of normal (pooled) plasma (corresponding to 100%increase the level of factor VIII).

Unit of factor VII is defined as the amount of factor VII, in 1 ml of normal pooled) plasma, corresponding to approximately 0.5 μg of protein. After activation, 50 units corresponding to approximately 1 μg of protein.

Under the "deficit" means reducing the amount or activity of, for example, factor VIII in plasma compared with those in normal healthy individuals. This term may optionally be used along with the term "reduced level of factor VIII" interchangeably.

The term "APTT or aPTT" refers to activated partial thromboplastin time (described, for example, in the publication Proctor RR, Rapaport SI: The partial thromboplastin time with kaolin; a simple screening test for first-stage plasma clotting factor deficiencies. Am J Clin Pathol 36: 212, 1961).

The term "syndrome sensitivity to factor VIII" refers to a syndrome in which the introduction to the subject, if necessary, exogenous factor VIII can prevent, cure or improve the condition when any symptoms, conditions or diseases, suspected, or present, caused by this syndrome. In their numbers is included, not limited to the above, syndromes caused by a reduced level of factor VIII, such as disorders associated with bleeding, such as the following, not limited to, hemophilia A, or syndromes caused by inhibitors of factor VIII.

The term "syndrome sensitivity factor VII" refers to a syndrome in which the introduction to the subject, if necessary, exogenous factor VII, preferably of factor VIIa, can prevent, cure or improve the condition when any symptoms, conditions or diseases, suspected, or present, caused by this syndrome. These include, but are not limited to the mentioned above, syndromes caused by a reduced level of clotting factors VIII, IX, XI or VII, inhibitors of coagulation factors, defective platelet function (e.g., when trombastenii of Glanzmann and syndrome Bernard-Soulier syndrome), thrombocytopenia, disease von Willebrand's disease and coagulopathy, such as caused by dilution of clotting proteins, increased fibrinolysis and decreased platelet counts due to bleeding and /or blood transfusions (for example, in subjects exposed to repeated transfusions due to surgery or trauma).

"Half-life" means the time required for plasma concentration of factor VII or a related factor VII in which peptide or factor VIII or related to the factor VIII polypeptide has decreased by half compared with concrete (initial) value.

Under the "primary hemostasis" refers to the original generation of thrombin under the action of FXa and TF:factor VIIa, subsequent activation of platelets and the formation of loose tubes from the activated attached platelets that have not yet stabilized fibrin, and, finally, (stable) cross linked fibrin. If the formed tube is not stabilized by fibrin in the second stage of the hemostatic process (supported hemostasis), it is easily subjected to dissolution under the action of the fibrinolytic system.

Under "secondary hemostasis", or "supported hemostasis"means secondary, complete, and the main emission (thrombin burst), or generation, thrombin, taking place on the surface of activated platelets and catalyzed by factor VIIIa and factor VIIIa, subsequent fibrin formation and stabilization of the initial platelet plug. Stabilization of the tube under the action of fibrin leads to complete hemostasis.

Under "complete hemostasis" refers to the formation of a stable and solid fibrin clot, or tube, in the site of damage, which effectively stops the bleeding and which is not subject to dissolution under the action of the fibrinolytic system. In this context, the term hemostasis will be used to denote the complete hemostasis, as described the above.

In this description, the term "drug" of coagulation factor, such as factor VIII, means a preparation in which factor VIII is a predominant factor. In one aspect of the coagulation factor is represented in the product in quantities of more than 20% (in/in) of total protein, more preferably 30%, more preferably 40%, more preferably 50%, more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%.

In the preferred embodiment, the coagulation factor present in an amount of more than 50% (V/V) of the total number of protein - coagulation factor, more preferably 80%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99%.

The total amount of protein in this preparation may be measured using a key known methods, for example, by measuring the optical density. The amount of protein factor VIII clotting can be measured by known methods such as standard ELISA Elisa method. In General, such analysis is carried out by contacting the solution containing the protein of factor VIII with anti-FVIII-antibody immobilized on the elisa plate, subsequent to the clocking immobilized antibody complex factor VIII with secondary anti-FVIII-antibody bearing marker, the amount of which is determined in the third stage. The amount of each of the factors can be estimated in a similar manner using the appropriate antibodies. The total number of protein - coagulation factor in the product is determined by adding the amounts of individual proteins coagulation factors. In one of the embodiments of the drug contains the selected coagulation factor. In another embodiment, the drug-free coagulation factor II coagulation factor IIa.

In this context, the term "isolated" refers to the coagulation factors such as factor VIII or related factor VIII polypeptides, which are isolated from the cells in which they were synthesized, or from the environment in which they exist in nature (e.g., plasma or blood). The selection of polypeptides from cells from which they originated, can be achieved using methods known in this field, including, but not limited to, removal of culture medium containing the desired product from the culture of attached cells; the method of centrifugation or filtration to remove unattached cells; and the like. The selection of polypeptides from the medium in which they are formed in nature, can be achieved by several methods known in this field, including, without limiting them, affinity chromatography, such voltage is emer, as for the column with the antibody against factor VII or with antibody against factor VIII, respectively; chromatography using hydrophobic interactions; ion exchange chromatography; chromatography excluded size; electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., precipitation with ammonium sulfate), or extraction and the like,

Notations:

TFtissue factor
FVIIfactor VII in its single-stranded, non-activated form
FVIIafactor VII in its activated form
rFVIIarecombinant factor VII in its activated form
factor VIIIfactor VIII in his imagenow, inactive form
factor VIIIafactor VIII in its activated form
factor VIIIrecombinant factor VIII
factor VIIIarecombinant factor VIIIa

Getting connections:

Human purified factor VIIa, suitable for use in the present invention, preferably obtained using recombinant DNA technologies, for example, as described agen et al., Proc. Natl. Acad. Sci. USA 83: 2412-2416, 1986, or as described in European patent No. 200421 (ZymoGenetics, Inc.).

Factor VII can be produced by methods described by Broze and Majerus, J. Biol. Chem. 255 (4): 1242-1247, 1980 and Hedner and Kisiel, J. Clin. lnvest. 71: 1836-1841, 1983. These methods allow to obtain the factor VII without appreciable amounts of other blood clotting factors. Even more purified preparation of factor VII can be obtained if, in addition to hold the gel filtration as the final purification stages. Then factor VII converted into activated factor VIIa by known techniques, for example, using several different plasma proteins, such as factor XIIa, IXa or Xa. Alternatively, as described Bjoern et al. (Research Disclosure, 269 September 1986, pp. 564-565), factor VII may be activated by passing it through an ion-exchange chromatographic column, such as Mono Q® (Pharmacia fine Chemicals) or similar.

Related to factor VII polypeptides can be produced by modification of factor VII wild-type or using recombinant technology. Related to factor VII polypeptides with altered amino acid sequence when compared with factor VII wild-type can be obtained by modifying the nucleic acid sequence that encodes a factor VII wild type, or by changing the codons corresponding to amino acids, or by uralenergostory of codons, corresponding to amino acids, nucleic acid encoding the factor VII, by known methods, for example by site-specific mutagenesis.

For professionals it will be obvious that substitutions can be made outside the regions critical factor VIIa or factor VIII molecule, so that the activity of the polypeptide is preserved. Amino acid residues essential for the activity of factor VII or a related factor VII polypeptide or the factor VIII or related to the factor VIII polypeptide, and therefore preferably not subject to substitution, may be identified according to procedures known in the field, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique mutations are introduced at every positively charged residue in the molecule, and the resulting mutant molecules are tested for coagulant in accordance with the cross-binding activity to identify amino acid residues that are critical for activity of the molecule. Sites of enzyme-substrate interaction can also be determined by analyzing the three-dimensional structure determined by methods such as nuclear magnetic resonance, crystallography or photoaffinity tagging (see, for example, de Vos et al., 192, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

Introduction mutations in the sequence of the nucleic acid with the purpose of replacement of one nucleotide with another nucleotide can be achieved by site-directed mutagenesis using any of the known in this connection methods. In particular, applicable technology, which uses the vector superscreen, double-stranded DNA with the desired insert and two synthetic primers containing the desired mutation. Oligonucleotide primers, each complementary to opposite chain vector is extended (increased) in the cyclization process temperature using DNA polymerase Pfu. When embedding primers produced a mutated plasmid containing zigzag located single-strand breaks. Subsequent temperature cycle, the product is treated with Dpnl that specific methylated and paleometeorology DNA for cleavage of the parental DNA template and selection synthesized DNA containing the mutation. You can also use other procedures known in the art, to create, identify, and highlight options, such as the shuffling of genes or the technology of phage display.

Separation of polypeptides from cells from which they originated, mo the em can be achieved by using one of the methods known in this field, including, without limitation, the Department of culture medium containing the desired product from the culture of the attached cells; centrifugation or filtration to remove unattached cells; and so on

The factor VII or related to the factor VII polypeptide may be - optional - extra refined. Purification may be achieved using any known in the field method, including, without limitation listed, affinity chromatography, such as affinity chromatography on a column with an antibody against factor VII (see, for example, Wakabayashi et al., J. Biol. Chem. 261: 11097, 1986; and Thim et al., Biochem. 27: 7785, 1988); hydrophobic chromatography; ion exchange chromatography; chromatography excluded size; electrophoretic approaches (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., precipitation with ammonium sulfate), or extraction and the like, In General, see Scopes, Protein Purification, Springer-Verlag, New York, 1982; and Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989. After cleaning, the preparation preferably contains less than about 10% (by weight), more preferably less than about 5%, and most preferably less than about 1% of the polypeptides, which was not a factor VII or non-related factor VII is polypeptide of host cells.

The factor VII or a related factor VII polypeptides may be activated by proteolytic cleavage with factor XIIa or other proteases having trypsin-like specificity, such as factor IXa, kallickrein, factor Xa and thrombin. See, for example, Osterud et al., Biochem. 11: 2853 (1972); Thomas, U.S. patent No. 4456591; Hedner et al., J. Clin. Invest. 71: 1836 (1983). An alternative factor VII or a related factor VII polypeptides may be activated as a result of their transmission through a column of ion-exchange chromatography, such as Mono Q® (Pharmacia) or the like. The resulting activated factor VII or related to the factor VII polypeptide can then be compiled (in composition), and can be entered (the patient), as described below.

For use in the present invention, the factor VIII can be isolated from plasma in accordance with known techniques, such as techniques described, for example, in the publications Fulcher et al., Proc. Acad. Nat. Sci. USA 1982, 79: 1648-1652; and Rotblat et al., Biochemistry 1985, 24: 4294-4300. However, preferably the use of recombinant factor VIII in order to avoid the use of products derived from blood or other tissues, as it is associated with the risk of transmitting diseases. Purified human factor VIII suitable for use in the present invention, preferably obtained using the technology of recombinant DNA, for example, as described in U.S. patent No. 4757006 and 4965199.

A related factor VIII polypeptides can be obtained by modifying factor VIII wild-type or using recombinant technology. A related factor VIII polypeptides with altered amino acid sequence compared to factor VIII wild-type can be obtained by modifying the nucleic acid sequence that encodes a factor VIII wild type, either by altering the amino acid codons or by removal of some of the amino acid codons in the nucleic acid that encodes a natural factor VIII, using known techniques such as site-directed mutagenesis, as described in more detail above. Separation of polypeptides from cells from which they originated, can be achieved using any of the methods known in this field, including, without limitation, the Department of culture medium containing the desired product from the culture of the attached cells; centrifugation or filtration to remove unattached cells; and the like Factor VIII or related to the factor VIII polypeptide can be - optional - extra refined. Purification may be achieved using any known in the field method, including, without limitation listed, affinity chromatography, t is positive, for example, as affinity chromatography on a column with an antibody against factor VIII; hydrophobic chromatography; ion exchange chromatography; chromatography excluded size; electrophoretic methods (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., precipitation with ammonium sulfate), or extraction and the like, as described more fully above. After cleaning, the preparation preferably contains less than about 10% (by weight), more preferably less than about 5%, and most preferably less than about 1% of the polypeptides, which was not a factor VIII or non-related factor VIII polypeptides from host cells. The resulting activated factor VIII or related to the factor VIII polypeptide can then be compiled (in composition), and can be entered (the patient), as described below.

Professionals in this field should be obvious that the preferred is the use of factor VIII polypeptides and related to the factor VIII polypeptide, syngeneic to the subject, in order to reduce the risk of inducing an immune response. The method of preparation and the properties of factor VIII non-human nature are described, for example, Fass et al., Blood 1982, 59: 594-600. The present invention also relates to use of such polypeptides factor VIII and floor of the peptides of factor VII in veterinary practice.

Pharmaceutical compositions and methods of use

The preparations according to the invention can be used to treat any symptoms of sensitivity to factor VIII, such as disorders associated with bleeding, including, but not limited to, disorders caused by deficiencies of coagulation factors (for example, hemophilia A), or caused by inhibitors (low or medium titer) of factor VIII.

The preparations according to the invention can be used to treat any symptoms of sensitivity to factor VII, such as disorders associated with bleeding, including, without limitation, syndromes caused by a reduced level of clotting factors VIII, IX, XI or VII, the presence of inhibitors of coagulation factors, defective platelet function (e.g., when trombastenii of Glanzmann and syndrome Bernard-Soulier syndrome), thrombocytopenia, disease von Willebrand's disease and coagulopathy, such as coagulopathy caused by dilution of clotting proteins, increased fibrinolysis and reduce the number of platelets as a result of bleeding and/or transfusions (for example, many times transfusion subjects subjected to surgery or trauma).

Pharmaceutical compositions containing a preparation of factor VII or a related factor II polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide according to the invention, primarily intended for parenteral administration for prophylactic and/or therapeutic applications. Preferably the pharmaceutical composition is injected parenterally, for example intravenously, subcutaneously or intramuscularly; however, intravenous administration is preferred. They can also be entered by continuous or pulsating infusion.

Pharmaceutical compositions or formulations according to the invention contain the drug preparation of factor VII or a related factor VII polypeptide or drug preparation of factor VIII or related to the factor VIII polypeptide or drug preparation of factor VII or a related factor VII polypeptide in combination with a preparation of a preparation of factor VIII or related to the factor VIII polypeptide in combination with (preferably when breeding in it) pharmaceutically acceptable carrier, preferably water carrier or diluent. Can be used in a variety of aqueous media such as water, buffered water, and 0.4% saline, 0.3% glycine and the like, the Preparations according to the invention can be prepared using non-aqueous media, such as drugs in the form of a gel or liposomal preparations for delivery or targeting to specific sites of damage. Liposomal drugs in General are described, e.g. the, in U.S. patent No. 4837028, 4501728 and 4975282. The composition can be sterilized by commonly used well-known sterilization technologies. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, and the dried product can be combined with a sterile aqueous solution prior to introduction.

The compositions may contain pharmaceutically acceptable excipients or adjuvants, including, without limitation, stabilizers, pH and tabularasa agents and/or agents, regulatory toychest solution, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

In addition, the composition can include one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. can be obtained in such forms as the liquid form, powders, emulsions, form a controlled release etc. Specialist in this field, you can prepare compositions according to the invention accordingly and in accordance with acceptable practice, such as described inRemington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, PA, 1990. Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 50 ml of sterile ringer's solution and 10 mg of the drug.

Compositions containing preparations according to the invention can be put into preventive and/or therapeutic purposes. With therapeutic use of the composition is introduced to a subject already suffering from a disease, as described above, in a quantity sufficient to cure, alleviate, or to partially stop the clinical manifestations of the disease and its complications. Adequate to achieve this amount is defined as "therapeutically effective amount". Effective for each goal amount will depend on the severity of the disease or damage, and the weight and General condition of the subject. Of course, determining the appropriate dosage can be achieved through conventional experimentation, by creating a matrix of values (parameters) and testing different points (indicators) in this matrix.

Local delivery of drugs according to the invention, such as, for example, local application, may be implemented, for example, by spray, perfusion, double balloon catheters, stent embedded in vascular implants or stents, hydrogels used for coating balloon catheters, or other well known methods. In any case, the pharmaceutical compositions should provide a quantity of the drug sufficient for effective treatment (ecologicheskogo) status.

The concentration of factor VII or a related factor VII polypeptide, factor VIII or related to the factor VIII polypeptide or the factor VII or related to the factor VII polypeptide in combination with factor VIII or related to factor VIII by the polypeptide in the compositions can vary widely, i.e. from less than about 0.5% by weight, typically at 1% or at least about 1% by weight, up to 15 or 20% by weight and the first place they will be selected based on the volume of fluid, viscosity, etc. in accordance with specifically selected by way of introduction. Introduction by injection or infusion, in particular, injection is preferred. Thus, the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide is obtained in a form suitable for intravenous administration, such as the drug that is either dissolved liofilizirovannam powder or liquid composition containing as a factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide in a single dosage form, or containing a dissolved lyophilized powder or a liquid composition comprising factor VII or a related factor VII polypeptide, in the same dosage form and dissolved lyophilized powder or a liquid composition containing factor VIII alertthingy factor VIII polypeptide, in another dosage form.

It should be obvious that the amount of factor VII or a related factor VII polypeptide and the amount of factor VIII or related to the factor VIII polypeptide together comprise the total effective amount for the treatment of bleeding episodes.

It should be borne in mind that the materials according to the invention can generally be used in case of serious disease or condition damage, such as situation, life threatening or potentially life-threatening situation. In such cases, reasons minimization of extraneous substances and usually the lack of immunogenicity of factor VIIa or factor VIII in humans possible, as may be necessary appointment your doctor significantly excessive amounts of these compositions.

For prophylactic use of a composition containing a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide, is administered to a subject susceptible to or otherwise - with the risk of illness or injury, to improve their own coagulating ability of the subject. This number is defined as a "prophylactically effective dose." It should be understood that the amount of factor VII or a related factor VII polypeptide and the number of factor VIII or related to the factor VIII polypeptide together comprise the total effective amount to prevent episodic bleeding.

Single or repeated administration of the compositions can be carried out in the levels and nature of the dosage selected by the attending physician. Such compositions can be administered one or more times per day or per week. An effective amount of such pharmaceutical compositions is an amount that provides a clinically significant effect against occasional bleeding. Such amount will be partly depend on the specific condition to be treated, the age, weight and General health of the subject, and other factors evident to the experts in this field.

The composition according to the invention is normally administered in a single dose to the expected bleeding or early bleeding. However, it can also be entered and re (multiple doses), preferably at intervals of 2-4-6-12 hours depending on the input dose and condition of the subject.

For treatment in connection with deliberate interventions, the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide is typically administered in intervals of approximately 24 hours before implementation of the intervention and for 7 days or more after him. Introduction as a coagulant can be produced in various ways, as described in this application.

Composers who s can be represented as a single drug (single dosage form), containing the drug preparation of factor VII or a related factor VII polypeptide and a preparation of a preparation of factor VIII or related to the factor VIII polypeptide, in appropriate concentrations. The composition may be presented in the form of a kit of parts comprising a first dosage form containing the drug preparation of factor VII or a related factor VII polypeptide and the second dosage form containing the drug preparation of factor VIII or related to the factor VIII polypeptide. In this case, the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide should be administered sequentially, preferably within 15 minutes, one after the other, for example, within 10 minutes, one after the other or preferably within 5 minutes, or more preferably within 2 minutes, one after the other. Any of the two dosage forms can be entered in the first place.

This set includes at least two separate pharmaceutical compositions. The set includes a device is a container for content of separate compositions, such as distributed vials or distributed in foil packages. Typically, the kit also contains instructions for use of the individual components of the set. The composition in the form set is especially preferred is sustained fashion then when the individual components are preferably introduced into various dosage forms, are different dosage intervals, or when making the appointment the doctor considers it necessary to attirbute individual components of this combination.

The number of input according to this invention, the factor VII or related to the factor VII polypeptide and the amount of factor VIII or related to the factor VIII polypeptide can vary, ranging from a ratio of one to the other is approximately between 1:100 and about 100:1 (V/V). The ratio of factor VII to factor VIII may, therefore, be approximately 1:100, or 1:90, or 1:80, or 1:70 or 1:60, or 1:50 or 1:40 or 1:30, or 1:20 or 1:10 or 1:5 or 1:2 or 1:1 or 2:1 or 5:1 or 10:1 or 20:1 or 30:1 or 40:1 or 50:1 or 60:1, 70:1, 80:1, 90:1, or 100:1; or between about 1:90 and 1:1, or between about 1:80 and 1:2, or approximately between 1:70 and 1:5, or between about 1:60 and 1:10, or between about 1:50 and 1:25, or roughly between 1:40 and 1:30, or between about 90:1 and 1:1, or between about 80:1 and 2:1, or between about 70:1 and 5:1, or approximately between 60:1 and 10:1, or between about 50:1 and 25:1, or between about 40:1 and 30:1.

The dose of factor VII or a related factor VII polypeptide varies from dose corresponding CA is approximately from 0.05 mg to 500 mg/day factor VII wild-type, for example, from about 1 mg to 200 mg/day, or, for example, from about 5 mg to 175 mg/day for a subject weighing 70 kg as loading and maintenance doses, depending on the weight of the subject, its condition and the severity of the condition.

The dose of factor VIII or related to the factor VIII polypeptide varies, ranging from a dose corresponding to about 0.05 mg to about 500 mg/day factor VIII wild-type, for example, from about 1 mg to approximately 200 mg/day, or, for example, from about 1 mg to 175 mg/day for a subject weighing 70 kg as loading and maintenance doses, depending on the weight of the subject, its condition and the severity of the condition.

In the treatment of subjects with low levels of factor VIII preferred dose is presented below:

With the high dosage of factor VIII or related to the factor VIII polypeptide to the level of its activity in plasma was achieved up to 10% of normal activity in plasma factor VIII

preferred levels of factor VII or a related factor VII polypeptide comprise from 15 to 300 micrograms/kg body weight;

more preferred levels of factor VII or a related factor VII polypeptide: 30-250 micrograms/kg body weight;

the most preferred levels of factor VII or a related factor VII polypeptide: 60-180 micrograms/the g body weight.

With the high dosage of factor VIII or related to the factor VIII polypeptide to the level of its activity in plasma was achieved up to 30% of normal activity in plasma factor VIII

preferred levels of factor VII or a related factor VII polypeptide comprise from 15 to 300 micrograms/kg of body weight,

more preferred levels of factor VII or a related factor VII polypeptide: 30-250 micrograms/kg body weight;

the most preferred levels of factor VII or a related factor VII polypeptide: 60-180 micrograms/kg of body weight.

With the high dosage of factor VIII or related to the factor VIII polypeptide to the level of its activity in plasma was achieved up to 50% of the normal level of activity in plasma factor VIII

preferred levels of factor VII or a related factor VII polypeptide comprise from 15 to 300 micrograms/kg body weight;

more preferred levels of factor VII or a related factor VII polypeptide: 30-250 micrograms/kg body weight;

the most preferred levels of factor VII or a related factor VII polypeptide: 60-180 micrograms/kg of body weight.

With the high dosage of factor VIII or related to the factor VIII polypeptide to the level of its activity in plasma was achieved up to 80% of the normal level of activity in plasma factor VIII

preferred ur the attention of factor VII or a related factor VII polypeptide comprise 5 are 300 micrograms/kg of body weight;

more preferred levels of factor VII or a related factor VII polypeptide: 10-180 micrograms/kg body weight;

even more preferred levels of factor VII or a related factor VII polypeptide: 30-120 micrograms/kg body weight;

the most preferred levels of factor VII or a related factor VII polypeptide: 60 to 120 micrograms/kg of body weight.

With the high dosage of factor VIII or related to the factor VIII polypeptide to the level of its activity in plasma was achieved up to 100% of the normal level of activity in plasma factor VIII

preferred levels of factor VII or a related factor VII polypeptide comprise 5 are 300 micrograms/kg body weight;

more preferred levels of factor VII or a related factor VII polypeptide: 10-180 micrograms/kg body weight;

the most preferred levels of factor VII or a related factor VII polypeptide: 60 to 120 micrograms/kg of body weight.

The dosage can be estimated based on the premise that 1 unit of FVIII substitution per kg body weight increases the activity in the plasma of approximately 0.02 units per ml (2%). Monitoring of the level of factor VIII in the blood of a patient is carried out, producing appropriate time intervals testing of blood samples of the patient in relation to the activity of factor VIII (see description above).

Tests:

Tested the e activity of factor VIIa:

To implement the appropriate analysis to test the activity of factor VIIa, and thus, selection of appropriate variants of factor VIIa may be taken by simple preliminary testing in vitro.

Analysis of hydrolysis in vitro

Native factor VIIa (wild-type) and factor VIIa (hereafter equally referred to as "factor VIIa") can be tested in respect of their specific activities. They can also be tested in parallel to directly compare their specific activities. The analysis is performed in microtiter tablet (MaxiSorp, Nunc, Denmark). Chromogenic substrate D-Ile-Pro-Arg-para-nitroanilide (S-2288, Chromogenix, Sweden), final concentration 1 mm, is added to the factor VIIa (final concentration 100 nm) in 50 mm Hepes buffer, pH of 7.4, containing 0.1 M NaCl, 5 mM CaCl2and 1 mg/ml bovine serum albumin. The absorption at 405 nm continuously measured in tablet SpectraMax™ 340, supplied by the reader (Molecular Devices, USA). Absorption, developing during the 20-minute incubation, after subtraction of the absorption index in the control well containing no enzyme, is used to calculate the ratios between the activities of variant and wild-type VIIa:

Ratio = (A405nmvariants of factor VIIa)/(A405nmfactor VIIa wild type).

The outcome is from this we can identify variants of factor VIIa activity, comparable to the activity of native factor VIIa or above it, such as the cases in which the ratio between the activity of the variant and the activity of native factor VII (FVII wild type) is approximately greater than 1.0.

The activity of factor VIIa or variants of factor VIIa can also be measured using a physiological substrate, such as factor X, in the respective concentrations of 100-1000 nm, where the generated factor Xa was measured after adding the appropriate chromogenic substrate (e.g., S-2765). In addition, the testing activity can be carried out at physiological temperature.

Analysis of proteolysis in vitro

Native factor VIIa (wild-type) and factor VIIa (hereafter equally referred to as "factor VIIa") are tested in parallel, in order to directly compare their specific activities. The analysis is performed in microtiter tablet (MaxiSorp, Nunc, Denmark). Factor VIIa (10 nm) and factor X (0.8 μm) in 100 μl of 50 mm Hepes buffer, pH of 7.4, containing 0.1 M NaCl, 5 mm CaCl2and 1 mg/ml bovine serum albumin, incubated for 15 minutes Then cleavage of factor X is stopped by adding 50 μl of 50 mm Hepes buffer, pH of 7.4, containing 0.1 M NaCl, 20 mm EDTA and 1 mg/ml bovine serum albumin. The number of generated factor Xa is measured by adding the chromogenic subst the ATA Z-D-Arg-Gly-Arg-para-nitroanilide (S-2765, Chromogenix, Sweden), final concentration 0.5 mm. The absorption at 405 nm continuously measured in tablet SpectraMax™ 340, supplied by the reader (Molecular Devices, USA). Absorption, developing within a 10-minute incubation, after subtraction of the absorption index in the control well, not containing FVIIa, is used to calculate the ratio between the proteolytic activities of variant and wild-type VIIa:

Ratio = (A405nmvariants of factor VIIa)/(A405nmfactor VIIa wild type).

From this we can identify variants of factor VIIa activity comparable to the activity of native factor VIIa or above it, such as the cases in which the ratio between the activity of the variant and the activity of native factor VII (FVII wild type) is approximately greater than 1.0.

Analysis of thrombin generation:

The ability of factor VII or a related factor VII polypeptides (e.g., variants) or factor VIII or related to the factor VIII polypeptide (e.g., variants) to generate thrombin can be measured through an analysis that includes all relevant coagulation factors and inhibitors at physiological concentrations and activated platelets (as described on page 543 in the publication Monroe et al. (1997) Brit. J. Haematol. 99, 542-547, which is incorporated into this op is a description by reference).

Testing of the activity of factor VIII:

To implement the appropriate analysis to test the activity of factor VIII and, thus, providing an opportunity for selection of suitable variants of factor VIII can be made simple in vitro tests, such as described by Kirkwood TBL, Rizza CR, Snape N.J., Rhymes I.L., Austen DEG. Identification of Sources of Interlaboratory Variation in Factor VIII Assay. B.J.Haematol. 1981; 37; 559-68; or Ressels et al., British Journal of Haematology, Vol. 76 (Suppl.1), pp. 16 (1990)). Wagenvoord et al., 1989, Haemostasis, 19 (4): 196-204) ("chromogenic analysis").

The biological activity of factor VIII can also be assessed by measuring the ability of the drug to correct the clotting time in plasma, deficient for the content of factor VIII, for example, as described in Nilsson et al., 1959. (Nilsson IM, Blombaeck M, Thilen A, von Francken I., Carriers of haemophilia A - A laboratory study, Acta Med Scan 1959; 165: 357). In this assay, the biological activity is expressed in units/ml plasma (1 unit corresponds to the amount of FVIII present in normal United plasma).

Aspects of the present invention:

In one aspect the present invention relates to a pharmaceutical composition comprising the polypeptide FVII and FVIII polypeptide as the only active clotting factors. In one aspect of the FVII polypeptide is a recombinant human FVIIa. In one aspect of the FVIII polypeptide is recombin ntny human FVIII. In one aspect, the polypeptide FVII and FVIII polypeptide is mixed (combined). In one aspect, the polypeptide FVII and FVIII polypeptide represented in different packages. In one aspect the composition is intended for the treatment of humans.

In another aspect, the present invention relates to a kit for the treatment of episodic bleeding, containing:

a) an effective amount of a FVII polypeptide and optionally a pharmaceutically acceptable carrier in a first unit dosage form;

b) an effective amount of FVIII polypeptide and optionally a pharmaceutically acceptable carrier comprising a second unit dosage form; and

c) the device is a container for the contents of these first and second dosage forms.

In one of the embodiments of the FVII polypeptide is a recombinant human FVIIa. In one of the embodiments of the FVIII polypeptide is a recombinant human FVIII. In one of the embodiments of the kit is intended for the treatment of humans. In another aspect of the invention involves the use of a FVII polypeptide and FVIII polypeptide to obtain drugs for the treatment of bleeding in a subject suffering from syndrome sensitivity factor FVIII. In another aspect of the invention involves the use of a FVII polypeptide and polypeptid is and FVIII to obtain drugs for the treatment of bleeding in a subject, having a reduced level of FVIII. In one aspect, the drug is intended for treatment of bleeding episodes in patients with hemophilia A. In one aspect, the drug includes a mixture of polypeptide factor FVII and polypeptide factor FVIII. In one aspect, the drug is obtained in the form of a first dosage form containing FVII polypeptide, and the second dosage forms containing the FVIII polypeptide. In one aspect of the FVII polypeptide is a recombinant human FVIIa. In one aspect of the FVIII polypeptide is a recombinant human FVIII.

In another aspect, the present invention relates to a method of improving hemostasis in a subject suffering from syndrome sensitivity factor FVIII, compared with a situation where the subject is administered FVIII as the only coagulating protein, where the method includes introduction to the subject, if necessary, an effective amount of a FVII polypeptide and an effective amount of FVIII polypeptide.

In another aspect, the present invention relates to a method of improving hemostasis in a subject having a reduced level of FVIII compared to a situation where the subject is administered FVIII as the only coagulating protein, where the method includes the introduction of the su is the target, if necessary, an effective amount of a FVII polypeptide and an effective amount of FVIII polypeptide.

In another aspect, the present invention relates to a method of increasing the production of thrombin in a subject suffering from syndrome sensitivity factor FVIII, compared with a situation where the subject is administered FVIII as the only coagulating protein, where the method includes introduction to the subject, if necessary, an effective amount of a FVII polypeptide and an effective amount of FVIII polypeptide.

In another aspect, the present invention relates to a method of increasing the production of thrombin in a subject having a reduced level of factor FVIII, compared with a situation where the subject is administered FVIII as the only coagulating protein, where the method includes introduction to the subject, if necessary, an effective amount of a FVII polypeptide and an effective amount of FVIII polypeptide.

In another aspect, the present invention relates to a method of reducing the number of injections of the protein of the coagulation factor required to achieve hemostasis in a subject suffering from syndrome sensitivity factor FVIII compared to the number of injections required, if the subject was administered FVIII as the only protein of the coagulation factor, where the method includes introduction to the subject, if necessary, an effective amount of a FVII polypeptide and effectivnogo number of FVIII polypeptide.

In another aspect, the present invention relates to a method of reducing the number of injections of the protein of the coagulation factor required to achieve hemostasis in a subject having a reduced level of factor FVIII compared to the number of injections required, if the subject was administered FVIII as the only protein of the coagulation factor, where the method includes introduction to the subject, if necessary, an effective amount of a FVII polypeptide and an effective amount of FVIII polypeptide.

In another aspect, the present invention relates to a method of reducing the number of input protein coagulation factor required to achieve hemostasis in a subject suffering from syndrome sensitivity factor FVIII compared to the number of input protein coagulation factor required if the subject was administered FVIII as the only protein of the coagulation factor, where the method includes introduction to the subject, if necessary, an effective amount of a FVII polypeptide and an effective amount of FVIII polypeptide.

In another aspect, the present invention relates to a method of reducing the number of input protein coagulation factor required to achieve hemostasis in a subject having a reduced level of factor FVIII compared with the number Vladimov the protein factora required if the subject was administered FVIII as the only protein of the coagulation factor, where the method includes introduction to the subject, if necessary, an effective amount of a FVII polypeptide and an effective amount of FVIII polypeptide.

In another aspect, the present invention relates to a method of treating bleedings in a subject suffering from syndrome sensitivity factor FVIII, where the method includes introduction to the subject, if necessary, an effective amount of a FVII polypeptide and an effective amount of FVIII polypeptide.

In another aspect, the present invention relates to a method of treating bleedings in a subject having a reduced level of factor FVIII, where the method includes introduction to the subject, if necessary, an effective amount of a FVII polypeptide and an effective amount of FVIII polypeptide.

In another embodiment of these methods, the FVII polypeptide is a recombinant human factor FVIIa.

In one embodiment, the FVIII polypeptide is a recombinant human factor FVIII. In one embodiment of the specified subject suffering from hemophilia A.

The present invention optionally further illustrated in the following examples, which, however, should not be viewed as the exhaust gas is unicefusa scope of protection of this invention. The signs described in the foregoing description and in the examples that follow, can both separately and in combination with each other to serve as a material for realizing the present invention in its various embodiments.

EXAMPLES

Example 1: In vivo treatment of intracranial bleeding in patients with hemophilia

In the treatment without inhibitor patient with hemophilia And suffering from intracranial bleeding, commercially available FVIII product to achieve hemostasis had, as a rule, do 10 to 20 injections or infusions of factor FVIII. Infusion of FVIII is designed to achieve the concentration of FVIII in the plasma component initially at least 80%, and then, after one week, another 50% of the normal concentration in plasma.

Such a patient was administered a single dose of 90-180 μg/kg body weight of the drug NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark) and simultaneously administered FVIII product, or has introduced a single dose of 90-180 μg/kg body weight of the drug NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark), and FVIII product was introduced after some time, for example after 5 minutes. Both products were injected with through the same device for an intravenous injection. The patient has reduced the time required to stop bleeding, and decreased the number of injections required to maintain hemostasis. This scheme Leche is tion leads to a decrease in the total number of protein - factor coagulation, used to stop bleeding and geostationary.

Example 2: In vivo treatment of a patient with hemophilia a patient with bleeding in compartmental syndrome

The patient is without inhibitor patient with hemophilia And suffering with compartmental syndrome from bleeding in the right upper limb as a result of external trauma. When this patient was treated with commercially available FVIII product, it was necessary, to achieve hemostasis, to do, usually from 20 to 40 injections or infusions of factor FVIII, most often in connection with the need for surgery in an accident. Infusion of FVIII is designed to achieve the concentration of FVIII in the plasma component initially at least 80 to 100% of the normal concentration in plasma, and then, after 1-2 weeks, which is 50%.

Such a patient was administered a single dose of 90-180 μg/kg body weight of the drug NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark) and simultaneously administered FVIII product, or was administered one dose of 90-180 μg/kg body weight of the drug NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark), and FVIII product was introduced after some time, for example after 5 minutes. Both products were injected with through the same device for an intravenous injection. The patient had decreased the time required to stop bleeding, and reduced quantities of the injection, necessary to maintain hemostasis. This treatment leads to a decrease in the total number of protein - coagulation factor used to stop bleeding and geostationary.

In this case, it may be appropriate introduction immediately before the operation is repeated dose factor VIIa in combination with factor VIII.

Example 3: In vivo treatment of a patient with hemophilia bleeding of the upper section of the gastrointestinal tract

The patient is without inhibitor patient with hemophilia And suffering from bleeding in the upper section of the gastrointestinal tract secondary to the use of NSAID (nonsteroidal anti-inflammatory drug). When treating such a patient is commercially available FVIII product to achieve hemostasis have, as a rule, do 20 to 40 injections or infusions of factor FVIII, most often in connection with an accident in connection with the scope.

Infusion of FVIII is designed to achieve the concentration of FVIII in the plasma component initially at least 80% to 100% of the normal concentration in plasma, and then another 50% after 5-10 days.

Such a patient was administered a single dose of 90-180 μg/kg body weight of the drug NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark) and simultaneously administered FVIII product, or was administered one dose of 90-180 μg/kg body weight ol the parathas NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark), and FVIII product was introduced after some time, for example after 5 minutes. Both products were injected with through the same device for an intravenous injection. The patient had decreased the time required to stop bleeding, and was able to reduce the number of injections required to maintain hemostasis. This treatment leads to a decrease in the total number of protein - coagulation factor used to stop bleeding and geostationary.

In this case, it may be appropriate introduction immediately before the operation is repeated dose factor VIIa in combination with factor VIII.

Example 4: In vivo treatment of the injured multiple transfusions a patient with diffuse bleeding

The patient suffers from diffuse bleeding as a result of external trauma. Before the state of diffuse bleeding, the patient was injected large quantities of liquids for intravenous injection, infusium colloidal products, albumin and concentrates of red blood cells. This clinical condition that occurs when multiple transfusions, characterized by low platelet counts and low concentration of fibrinogen and factor VIII.

Treatment should include transfusion of platelets, fresh frozen plasma and FVIII products. Infusion of FVIII p is odnaznachno to achieve plasma concentrations, component of at least 80% of the normal level, and then should continue until such time as the state of diffuse bleeding completely resolved.

Such a patient was administered a single dose of 90-180 μg/kg body weight of the drug NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark) and simultaneously administered FVIII product, or has introduced a single dose of 90-180 μg/kg body weight of the drug NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark), and FVIII product was introduced after some time, for example after 5 minutes. Both products were injected with through the same device for an intravenous injection. The patient had decreased the time required to stop multifocal bleeding, and was able to reduce the number of injections required to maintain hemostasis. This treatment leads to a decrease in the total number of protein - coagulation factor used to stop bleeding and hemostaseology, and to increase survival.

In this case, it may be appropriate introduction immediately before the operation is repeated dose factor VIIa in combination with factor VIII.

Example 5: a Study of the coagulation status of the blood without inhibitor patient with hemophilia And

The patient is without inhibitor patient with hemophilia A, who suffers from bleeding, such as intracranial bleeding.

In the treatment of such item is the patient should therefore be commercially available FVIII product had usually do 10 to 20 injections or infusions of factor VIII to achieve hemostasis. Infusion of FVIII is designed to achieve the concentration of FVIII in the plasma component initially at least 80% of the normal concentration in plasma, and then, after one week of 50%.

Such a patient was administered a single dose of 90-180 μg/kg body weight of the drug NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark) and simultaneously administered FVIII product, or was administered one dose of 90-180 μg/kg body weight of the drug NovoSeven® (Novo Nordisk A/S, Bagsvaerd, Denmark), and FVIII product was introduced after some time, for example after 5 minutes. Both products were injected with through the same device for an intravenous injection. Ten minutes after administration of the last two coagulating proteins the patient made a complete analysis of coagulation using thromboelastographic method, which is a standardized method of analysis, clinically relevant coagulation status (see, for example, Meh et al., BLOOD COAGULATION &FIBRINOLYSIS 2001; 12: 627-637). On the basis of data obtained through standard readers, as a result of this analysis, it was found increased formation of fibrin clot, increasing the strength of the bunch, and lengthening the time of dissolution of the clot. Such measurements during subsequent blood tests showed the nalitch the e variability in these parameters, depending on the time after injection products of factor VII and factor VIII.

Example 6: a Decrease in clotting time under the effect of combinations of factors VIIa and factor VIII

METHODS:

Analysis of the bunch: the Specific clotting activity of recombinant human coagulation factor VIIa (rFVIIa) in the presence or absence of various concentrations of purified from human plasma factor VIII (FVIII) was determined using a one-stage analysis as described above (Persson et al., J Biol Chem 276: 29195-9, 2001). Briefly, aliquots (55 μl) rFVIIa (0.2 to 3 mg/ml, Novo Nordisk stock) in 50 mm pipes, 100 mm NaCl, 2 mm EDTA, 1% BSA, pH of 7.2, was mixed with an equal volume of buffer containing 50 mm CaCl2and phosphatidylcholine/phosphatidylserine vesicles (total concentration of phospholipids 100 μm; 80% phosphatidylcholine/20% phosphatidylserine), and clotting was initiated by adding 55 μl of normal human plasma (Novo Nordisk pooled plasma standard) or FVIII-deficient plasma (Helena Helena Labs Labs #5793), to which are added various concentrations of FVIII (10, 50, and 80% of the concentration in plasma, Haematologic Technologies). The clotting reaction was carried out for 400 seconds in coagulometer for research ACL 300 (Instrumentation Laboratory, Milan, Italy) using the standard program APTT.

The RESULTS:

Analysis of the clot: rFVIIa and FVIII, separately and in combination with each other, added to FVIII-deficient and normal human plasma and determined the time of swertia the Oia. To add rFVIIa/FVIII clotting time of both plasmas were more than 400 seconds. The effect of reducing clotting time under the action of rFVIIa in the presence and in the absence of FVIII in FVIII-deficient and normal human plasma is shown in figures 1 and 2, respectively.

CONCLUSION:

The results show that the combination of rFVIIa and FVIII can cause the effect of shortening the clotting time FVIII-deficient plasma to a greater extent than when the proteins were added separately.

1. A pharmaceutical composition comprising a preparation of factor VII or a related factor VII polypeptide and a preparation of factor VIII or related to the factor VIII polypeptide, under related to each of the factors a polypeptide refers to a chemically modified factor and/or a variant amino acid sequence containing not more than 20 amino acid substitutions, deletions or insertions, compared to the corresponding factor wild type, and/or a fragment of the corresponding factor.

2. The composition according to claim 1, where the factor VII or related to the factor VII polypeptide is a related factor VII polypeptide, such as a variant of the amino acid sequence of factor VII.

3. The composition according to claim 1 or 2, where the ratio between the activity of the specified related to the factor VII polypeptide and the activity of native the th human factor VIIa (wild-type FVIIa) is at least about 1.25 when tested using analysis of hydrolysis In Vitro", as described in the present description.

4. The composition according to claim 1, where the factor VII or related to the factor VII polypeptide is a factor VII polypeptide, such as recombinant human factor VII.

5. Composition according to any one of claims 1 to 4, where the factor VII or related to the factor VII polypeptide is presented in its activated form.

6. The composition according to claim 5, where the specified factor VII polypeptide is a recombinant human factor VIIa.

7. Composition according to any one of claims 1 to 6, where the factor VIII or related to the factor VIII polypeptide is a related factor VIII polypeptide, such as a variant of the amino acid sequence of factor VIII.

8. The composition according to claim 1, where the ratio between the activity of the specified related to the factor VIII polypeptide and the activity of native human factor VIII (FVIII wild type) is at least about 1.25 when tested using chromogenic analysis, as described in the present description.

9. Composition according to any one of claims 1 to 6, where the factor VIII or related to the factor VIII polypeptide is a factor VIII polypeptide, such as human factor VIII or recombinant human factor VIII.

10. Composition according to any one of paragraphs. 1-9, where the factor VII or related to the factor VII polypeptide ukazanny factor VIII or related to the factor VIII polypeptide are present in a mass ratio between about 100:1 and 1:100 factor VII to factor VIII.

11. Set for treatment of bleeding episodes containing (a) an effective amount of a preparation of factor VII or a related factor VII polypeptide and a pharmaceutically acceptable carrier in a first dosage form; b) an effective amount of a preparation of factor VIII or related to the factor VIII polypeptide and a pharmaceutically acceptable carrier in a second dosage form; and C) a container for the contents of these first and second dosage forms.

12. Set according to claim 11, where the factor VII or related to the factor VII polypeptide is a related factor VII polypeptide, such as a variant of the amino acid sequence of factor VII.

13. Set in § § 11 or 12, where the ratio between the activity of the specified related to the factor VII polypeptide and the activity of native human factor VIIa (wild-type FVIIa) is at least about 1.25 when tested using analysis of hydrolysis In Vitro, as described in the present description.

14. Set according to claim 11, where the factor VII or related to the factor VII polypeptide is a factor VII polypeptide, such as human factor VII or recombinant human factor VII.

15. Set according to any one of § § 11-14, where the factor VII or related to the factor VII polypeptide is presented in his aktivirovannoi form.

16. Set on 15, where specified, the factor VII polypeptide is a recombinant human factor VIIa.

17. Set according to any one of § § 11-16, where the factor VIII or related to the factor VIII polypeptide is a related factor VIII polypeptide, such as a variant of the amino acid sequence of factor VIII.

18. Set on 17, where the ratio between the activity of the specified related to the factor VIII polypeptide and the activity of native human factor VIII (FVIII wild type) is at least about 1.25 when tested using chromogenic analysis, as described in the present description.

19. Set according to any one of § § 11-16, where the factor VIII or related to the factor VIII polypeptide is a factor VIII polypeptide, such as human factor VIII or recombinant human factor VIII.

20. Set according to any one of § § 11-19, where the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide is represented in the mass ratio between about 100:1 and 1:100 factor VII to factor VIII.

21. The use of a composition according to any one of claims 1 to 10 for the manufacture of a medicinal product for the treatment of the subject of bleeding episodes.

22. The use of a composition according to any one of claims 1 to 10 for the manufacture of a medicinal product to reduce the possible clotting time.

23. The use of a composition according to any one of claims 1 to 10 for the manufacture of a medicinal product to increase the time of dissolution of the clot.

24. The use of a composition according to any one of claims 1 to 10 for the manufacture of a medicinal product to increase the strength of the clot.

25. The use of a composition according to any one of claims 1 to 10 for the manufacture of a medicinal product to enhance the formation of a fibrin clot.

26. The use according to any one of p-25, where the subject is suffering from a syndrome of sensitivity to factor VIII and/or has a reduced level of factor VIII.

27. Application on p. 26, where the subject suffers from hemophilia A.

28. The use according to any one of p-27, where the medicinal product is presented in a single dosage form.

29. The use according to any one of p-27, where the medicinal product is obtained in the form of a first dosage form containing preparation of factor VII or a related factor VII polypeptide and the second dosage form containing the drug factor VIII or related to the factor VIII polypeptide.

30. A method of treating bleeding episodes in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are the Xia effective for the treatment of bleeding.

31. A method of reducing clotting time in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to reduce clotting time.

32. A way to enhance haemostasis in a subject, comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to enhance haemostasis.

33. The way to reduce the number of injections of the protein clotting factor needed to stop bleeding and establish hemostasis in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to stop bleeding and establish hemostasis.

34. The way to reduce the amount of typing for protein-factora, neobhodimosti stop bleeding and establish hemostasis in a subject, includes introduction to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to stop bleeding and establish hemostasis.

35. Method of increasing the time of dissolution of a clot in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to increase the time of dissolution of the clot.

36. Method of increasing the strength of a clot in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor VII polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to increase clot strength.

37. Method of strengthening education fibrin clot in a subject comprising administration to the subject, if necessary, the first amount of a preparation of factor VII or a related factor II polypeptide and a second amount of a preparation of factor VIII or related to the factor VIII polypeptide, where the first and second amount together are effective to enhance formation of a fibrin clot.

38. The method according to any of PP-37, where the subject is suffering from a syndrome of sensitivity to factor VIII and/or has before treatment reduced the level of factor VIII.

39. The method according to § 38, where the subject suffers from hemophilia A.

40. The method according to any of p-39, where the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide is administered as part of a single dosage form.

41. The method according to any of p-39, where the factor VII or related to the factor VII polypeptide and the factor VIII or related to the factor VIII polypeptide is administered in the form of a first dosage form containing preparation of factor VII or a related factor VII polypeptide and the second dosage form containing the drug factor VIII or related to the factor VIII polypeptide.

42. The method according to paragraph 41, where the first dosage form and the second dosage form is entered at intervals of time not more than 15 minutes.

43. Set for treatment of bleeding episodes comprising (d) an effective amount of factor VII or a related factor VII polypeptide and an effective amount of factor VIII or related to the factor VIII polypeptide and a pharmaceutically acceptable carrier in a single dosage form; and (e) a container for content in asanoi a single dosage form.



 

Same patents:

Improved separation // 2311183

FIELD: biochemical methods.

SUBSTANCE: invention provides a method for binding and separating at least one component from a body fluid. Body fluid, such as whole blood, is passed through integral separating matrix without displacing components according their sizes from the matrix, the latter having porous structure with pore size within a range of 5 to 500 μm and active surface between 0.5 and 10 m2. During passage, at least one component of the body fluid binds to at least one functional group in the matrix. Matrix is prepared by one of following methods: caking, molding, or foaming. Use of bile acid residue immobilized of a carrier is proposed to eliminate a component from aqueous solution containing it. Invention further provides device for selective binding of at least one component from body fluid comprising case with inlet and outlet and at least one separation matrix, said matrix being rigid and integral during passage of the body fluid therethrough.

EFFECT: achieved selectivity in separation of at least one component from a body fluid.

32 cl, 4 dwg, 2 tbl, 35 ex

FIELD: medicine, peptides.

SUBSTANCE: invention relates to osteogenic growth oligopeptides used as stimulators of hemopoiesis. Invention proposes using an oligopeptide of molecular mass in the range from 200 to 1000 Da, comprising one of the following sequence: Tyr-Gly-Phe-Gly-Gly, Met-Tyr-Gly-Phe-Gly-Gly used in preparing a pharmaceutical composition and enhancing mobilization of hemopoietic stem cells from many differentiation line into peripheral blood, in particular, CD34-positive hemopoietic stem cells. Advantage of the invention involves expanding field in using oligopeptides used in stimulation of hemopoiesis.

EFFECT: enhanced and valuable properties of oligopeptides.

34 cl, 2 tbl, 7 dwg, 4 ex

FIELD: animal science.

SUBSTANCE: the suggested preparation contains disodium or dipotassium salt of ethylenediamine-N,N1succinic acid, iron (III), manganese (II), zinc (II), copper (II), cobalt (II), iodine (I), selenium (IV) and water at the following ratio of components, weight%: disodium or dipotassium salt of ethylenediamine-N,N1disuccinic acid 25.1-57.5; iron (III) 0.5-5.5; manganese (II) 0.25-4.9; zinc (II) 0.05-2.0; copper (II) 0.10-0.55, cobalt (II) 0.05-0.3, iodine (I) 0.01-0.08, selenium (IV) 0.01-0.06, water - the rest. The preparation suggested enables to increase vitamin full value of feedstuffs.

EFFECT: higher efficiency.

4 ex, 4 tbl

FIELD: medicine, endocrinology.

SUBSTANCE: one should intravenously inject perfluoran 12 h before operation at the dosage of 5 mg/kg body weight along with inhaling oxygen-enriched air mixture. In the course of operation perfluoran should be injected intraarterially at the dosage of 100 ml. Such a scheme for introducing perfluoran provides maximal manifestation of its gas-transport function that enables to carry out efficient therapy due to considerable decrease of tissue hypoxia.

EFFECT: higher efficiency of therapy.

1 ex

FIELD: medicine, experimental medicine.

SUBSTANCE: for the purpose to stop hemorrhages it is necessary to introduce fibrin-monomer for a rabbit at the dosages ranged 0.5-5 mg/kg animal body weight. In case of intravenous injection at the dosages ranged 0.5-5 mg/kg animal body weight fibrin-monomer is of immediate hemostatic action and has no influence upon basic hemostatic parameters.

EFFECT: higher efficiency.

5 ex

FIELD: medicine.

SUBSTANCE: method involves applying ultrasonic functional debitometry in pre-operative period. Lower extremity microcirculation bed capacity is determined. Clopidogrel preparation is administered at a dose of 75 mg a day. Then, ultrasonic functional debitometry is repeatedly applied under dynamic conditions. Reliable microcirculation bed capacity being the case, patient preparation to reconstruictive reparative vascular surgical interventions.

EFFECT: enhanced effectiveness of microcirculation bed preparation; improved rheological blood properties.

3 tbl

FIELD: organic chemistry.

SUBSTANCE: invention relates to new amide-type carboxamide derivatives of formula [1] , wherein X represents -N= or -CH= group; R1 represents halogen atom, lower alkyl and a like; R1 represents -CO-R21-R22 (meanings of R21 and R22 are as defined in claim 1); Y1 and Y2 are independently halogen atom, lower alkyl, lower alcoxy group, and a like; ring A represents phenyl and a like; or pharmaceutically acceptable salts thereof. Said derivatives are useful as FXa inhibitors. Also disclosed are pharmaceutical composition based on abovementioned compounds and uses thereof.

EFFECT: new amide-type carboxamide derivatives.

7 cl, 105 ex

FIELD: organic chemistry, drugs.

SUBSTANCE: invention relates to imidazo(or triazolo)pyrimidine derivatives of general formula I wherein Y represents CH or N; R1, R2 and R3 are as defined in the description, and pharmaceutically acceptable salt thereof. Such compounds inhibit SYK tyrosine kinase and are useful in medicine to suppress immediate allergic reaction and late inflammation reaction. Described is method for production of abovementioned compounds by interaction of compound of formula II wherein Y, R2 and R3 are as defined above; L is leaving group, with compound of formula III: HR1, wherein R1 is as defined above. Also disclosed is pharmaceutical agent inhibiting SYK tyrosine kinase activity.

EFFECT: new inhibitors of SYK tyrosine kinase activity.

7 cl, 17 tbl, 17 ex

FIELD: organic chemistry, pharmaceuticals.

SUBSTANCE: Described are derivatives of general formula I (all symbols are as described in specification), pharmaceutically acceptable salts thereof or cyclodextrin clathrates. Such compounds hardly bind of EP2 subtype of PGE receptor and are useful in prophylaxis of immune diseases, allergy, death of neuronal cells, liver or kidney insufficiency, etc.

EFFECT: new agent for prophylaxis of various diseases.

18 cl, 388 ex, 68 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: invention relates to application of erythropoetin and analogs thereof (darbepoetin alpha, modified erythropoetin, glycolizied erythropoetin conjugated with polyethylene glycol) to treating disturbances of iron distribution in diabetes. Disclosed are appropriate method for treatment and drug. It is demonstrated that patients suffering from diabetes have high susceptibility to disturbances of iron distribution at normal iron concentration in organism. Subdermal application of erythropoetin in dose of 150 U/kg suppresses this disturbance.

EFFECT: new method for erythropoetin application.

17 cl, 2 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method involves carrying out tonometry, patient examination in slit lamp light, determining filtration cushion size and relief, carrying out test with sterile air introduced under conjunctiva in operation zone in combination with glycocorticosteroid preparation. Filtration cushion height increasing and air entering the anterior chamber, diuretic drugs are prescribed and preparations inhibiting intraocular fluid secretion. Filtration cushion becoming flat, glycocorticosteroid preparations, enzyme preparations are introduced and antimetabolite is introduced under conjunctiva outside of operation field. Filtration cushion relief being variable and air passes into the anterior chamber, anti-inflammatory therapy, anti-proliferative treatment course, glycocorticosteroid and enzyme preparations are prescribed. Filtration cushion becoming flat, and air does not enter the anterior chamber, gonioscopy is carried out and surgical intervention zones are revised in the cases of open internal fistula and antibiotic of cytostatic activity is to be prescribed. Internal fistula being blocked, laser surgical intervention is carried out. Hypertension being persistent, anti-glaucoma intervention is carried out in another eyeball segment with antibiotic of cytostatic activity being applied.

EFFECT: enhanced effectiveness of treatment selection.

FIELD: medicine, surgery.

SUBSTANCE: on the 1st d after operation a patient should be injected with mexidol intravenously by drops at the dosage of 300 mg, during the next 2 d mexidol should be injected intramuscularly twice daily at the dosage of 200 mg. Moreover, during 10 d after operation a patient should be perorally introduced with ADP-37 preparation a the dosage of 6 capsules daily and during 5 d one should affect the wound with impulse magnetic field at about 0.5-1.0 Tl intensity through Dalcex-trypsin filter cloth impregnated with 0.01%-serotonin solution, once daily. The innovation enables to accelerate the terms of wound healing up to 6-7 d and avoid therapy-induced complications.

EFFECT: higher efficiency of therapy.

1 ex

FIELD: medicine, surgery.

SUBSTANCE: the present innovation deals with treating osseous gunshot fractures due to fixing osseous fragments with the help of the apparatus of extrafocal osteosynthesis followed by their reposition and surgical treatment of the wound. After surgical treatment on the 1st d after operation a patient should be intravenously injected with mexidol by drops at the dosage of 300 mg once daily, and during the next 2 d - intramuscularly twice daily at the dosage of 200 mg, moreover during 10 d after surgical treatment a patient should be perorally introduced with ADP-37 at the dosage of 6 capsules daily and during 5 d it is necessary to affect the wound with impulse magnetic filed of 0.5-1.0 Tl intensity through Dalcex-trypsin filter cloth impregnated with 0.01%-serotonin solution in physiological solution once daily. The innovation enables to accelerate the terms for wounds healing, avoid complications due to complex pathogenetic approach during therapy conducted.

EFFECT: higher efficiency of therapy.

2 ex

FIELD: medicine, gastroenterology.

SUBSTANCE: invention relates to selection of tactics in treatment of acute viral hepatitis. Method involves determination of the relative content of fractions of lysophosphatidylcholine (LPHCH), sphingomyelin (SM) and phosphatidylcholine (PHCH) in blood phospholipid spectrum. Then coefficient is calculated by the formula: PHCH2/SM x LPHCH. Semi-bed regimen, diet № 5, drinking liquid up to 2 l and vitamins are prescribed at coefficient value 6-13. Enzyme preparations and infusion therapy in volume up to 1200 ml of liquid with 5 ml of 5% ascorbic acid solution are prescribed at coefficient value 15-27. Enterosorbents, massive disintoxicating therapy, intravenous infusion of amino acid mixtures and glucocorticosteroids in the doses 40 mg/24 h and 120 mg/24 h orally that are equivalent to prednisolon dose are prescribed at coefficient value 30-70.

EFFECT: improved method of treatment.

3 ex

FIELD: medicine.

SUBSTANCE: method involves placing bone implant in 2-5% tripsin solution at temperature 30-40°С for 6-8 h. Then, bone implant washing is carried out in turn with physiological solution and 2-5% tripsin solution at the same temperature. Alternation frequency rate depends on morphologically determined final bone implant cleaning degree. Final washing is carried out with physiological salt solution.

EFFECT: enhanced effectiveness of treatment; improved plastic bone implant properties, full bone marrow removal; accelerated implant preparation to clinical use or morphological research.

2 cl, 1 tbl

FIELD: medical engineering.

SUBSTANCE: method involves Chondroxide ointment is mixed with Indometacin ointment and Essaven gel in proportion of 1:1:1. 50 mg of a Caripazyme enzyme preparation, dissolved in 5 ml of the physiological salt solution is added. Then, the produced suspension put on injured regions. Ultraphonophoresis is applied daily for 5 min on each region. The total treatment course is 15-20 procedures long.

EFFECT: improved anesthetic action, anti-inflammatory and chondroprotective action; improved microcirculation in injured region; accelerated rehabilitation period.

4 dwg, 1 tbl

FIELD: medicine, pharmacology, biochemistry, enzymes.

SUBSTANCE: invention relates to an enzyme-containing medicinal agent wherein enzymes are chosen from a group consisting of hydrolases, lipases, amylases, glycosidases, phospholipases, phosphodiesterases, phosphatases prepared from infusoria. Also, invention relates to a method for using indicated enzymes in improving digestion or in treatment of digestion disorders. Invention enhances resistance to stomach acid media and preparing from safety microorganism.

EFFECT: valuable medicinal properties of enzymes.

7 cl, 2 tbl, 7 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: method involves setting draining means like silicon tube or umbilical catheter No5 into anterior horns of lateral ventricles, tube diameter being equal to 2,2 mm. Their fixation is carried out with counteraperture. 2-З hours later after patient condition having been stabilized, one of draining tubes is connected to the inflow system designed as bottle having measuring scale, connected to infusion pump. The second draining tube is connected to outflow system, representing the second a bottle having measuring scale. Continuously active physiological saline infusion containing antibacterial and fibrinolytic preparations is carried out into ventricular system at a rate of 1-2 ml/h until full liquor sanitation is achieved.

EFFECT: simplified method; accelerated surgical intervention process.

1 dwg

FIELD: medicine, traumatology, orthopedics.

SUBSTANCE: it is necessary to irradiate affected area with low-intensity He-Ne laser at wave length of 0.63 mcm and density of radiation power being 10 mW/sq. cm, impact exposure - 10 min. Quantity of seances corresponds to 10. Before irradiation one should apply a single-layer filter cloth impregnated with 1%-carypazim solution onto the skin of affected area. The innovation enables to quickly remove pain syndrome, reconstruct the volume of movements in affected area and achieve prolonged remission of the disease mentioned.

EFFECT: higher efficiency of therapy.

2 dwg, 2 ex, 1 tbl

FIELD: medicine, hepatology, virology.

SUBSTANCE: invention relates to a method for treatment of chronic viral hepatitis C of genotype-2 with moderate activity. Method is carried out by every day parenteral administration of solutions containing a mixture of amino acids, nucleosides and enzyme. In the first day 0.2 ml of 0.01% solution of a solution containing a mixture of amino acids: lysine, arginine, asparagines, glutamic acid taken in the equal amounts is administrated; in the next day 0.2 ml of 0.02% solution containing a mixture of nucleosides: adenosine, inosine, amino acids, guanosine, and enzyme lysozyme also taken in equal amounts. Administration of solution is alternated every day during carrying out the treatment course and each 2 months break in administration of amino acids is carried out for 28-32 days. After 9-11 days of administration of proposed solutions method involves additional course of parenteral administration of interferon-α 2b in the dose 3 ml IU, 3 times per a week for 6 months. The total duration of treatment course is 6 months, 9 days - 6 months, 11 days. Method provides clinical remission and elimination of hepatitis virus and normalization of biochemical indices for 6 months after termination of treatment.

EFFECT: improved method of treatment.

2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to methods for preparing biologically active substances from donor blood. Method for enriching donor blood plasma cryoprecipitate with factor VIII involves defrosting freshly frozen donor blood plasma at +1-4°C to obtain cryofractions from plasma cryosupernatant and cryoprecipitate. After formation of the donor blood freshly frozen plasma dry calcium chloride is added in the amount from 0.001 to 0.004 mole/l followed by addition of sodium citrate in the amount from 0.05 to 0.10 mole/l and stirring at +1-4°C for 10-15 min. Then prepared cryoprecipitate deposit is separated by centrifugation. For preparing cryoprecipitate freshly frozen plasma is used prepared by usual method with using conventional preserving agents. Using the method provides significant increasing the content of factor VIII in cryoprecipitate being without appreciable co-precipitation of inert proteins.

EFFECT: improved enriching method.

2 tbl, 3 ex

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