UA76106C2 - Formulation containing factor viia and factor xiii, set for intravenous administration, method for increasing generation of fibrin clots, method for treatment or prophylaxis of bleeding episodes, use of factor viia or factor xiii for manufacturing drug for treatment or prophylaxis of bleeding episodes - Google Patents

Abstract

The present invention relates to the use of a factor VIIa and a factor XIII in the treatment or prophylaxis of bleeding episodes.

Description

Description of the invention

The present invention relates to a pharmaceutical composition containing factor Miia and factor XII. The invention also relates to the use of a combination of factor Ma with factor XII for the preparation of a medicament for the treatment of subjects suffering from bleeding attacks or the prevention of such attacks. The invention also relates to a method of treating bleeding attacks in a subject and increasing blood salinity in a subject. The present invention also relates to kits that include these compounds.

Hemostasis is caused by the formation of a complex between tissue factor (TE), exposed to the action of circulating blood 70 due to damage to the vessel wall, and EMIIIa present in the blood circulation in an amount that corresponds to approximately 195 of the total weight of the EMIII protein. This complex is fixed on the cell containing TE and activates EX to EX and GRIX to RiIX on the cell surface. EXa activates prothrombin to thrombin, which activates EMI, EM, EXI, and EX. In addition, the limited amount of thrombin generated in this first step of hemostasis also activates platelets.

After the action of thrombin on platelets, they change shape and open charged phospholipids on their surface. 12 This surface of activated platelets forms the template for further activation of the EC and complete generation of thrombin. Further activation of EX on the surface of activated platelets occurs through the RIXa-EM Sha complex formed on the surface of the activated platelet, and EXa then converts prothrombin to thrombin while still on the surface. Thrombin then converts fibrinogen to fibrin, which is insoluble and which stabilizes the original platelet plug. This process is compartmentalized, that is, limited to the site of TE expression or action, thus minimizing the risk of systemic activation of the coagulation system.

Insoluble fibrin, which forms the plug, is additionally stabilized by EHI-catalyzed cross-linking of fibrin fibers.

EMiia exists in plasma mostly as a single-chain zymogen, which cleaves EXa into its two-chain activated form, EMiia. Recombinant activated factor Miia (Tema) reveals itself as a prohemostatic agent. Administration of heme provides a rapid and highly effective prohemostatic response in subjects with hemophilia, (3 suffering from bleeding that is not helped by products containing coagulation factors due to the formation of antibodies. EMIa can also be successfully used in subjects suffering from from bleeding, with MIII deficiency, or subjects who have a normal coagulation system but have experienced excessive bleeding. No adverse side effects of MIIIa (including thromboembolic events) were observed in these studies.

Additional exogenous administration of EMI increases the production of thrombin on the surface of activated platelets. This occurs in subjects with hemophilia who are deficient in SIN or RMIII, and therefore are deprived of the most efficient pathway for complete thrombin production. Also, in the presence of a reduced number of platelets or platelets with insufficient function, additional EMIIa increases the production of thrombin. Gee)

EX, a fibrin-stabilizing factor, is a transglutaminase that cross-links fibrin monomers, thereby providing a fibrin structure more resistant to dissolution by plasmin and other proteolytic enzymes. in

Factor XII is also known as "fibrin ligase" and "fibrin-stabilizing factor". Activated EHS can form intermolecular gamma-glutamyl-epsilon-lysine cross-links between side chains of fibrin molecules and between other substrates. EHIIA is found in plasma and platelets. This enzyme exists in plasma as a tetrameric zymogen consisting of two alpha-subunits and two beta-subunits of C 70 (denoted a2p2), and in platelets as a zymogen consisting of two alpha-subunits (denoted c ao-dimer).

From" Both zymogens are activated by thrombin and Sak. Calcium is released from platelets and accumulates at the site of damage. Thrombin is cleaved from 1-37 M-terminal amino acid residues (a o-dimer). In the case of abo-zymogen, the beta subunits are separated from the activated alpha subunits. Calcium binds equally well to zymogen and thrombin-modified molecule. After thrombin and calcium activation, cysteine 7 of the active site on the alpha chain is opened and a fully activated enzyme is formed. It was

Ge") subjects with severe thrombocytopenia were found to have low levels of EHIA! in plasma

It is well known that subjects who have experienced excessive bleeding due to surgery or significant trauma and require blood transfusions develop more complications than those who do not experience av! 20 bleeding. However, even moderate bleeding that requires the administration of human blood or blood products (platelets, leukocytes, plasma-derived concentrates for the treatment of coagulation disorders, etc.) can lead to complications associated with the risk of transmission of human viruses (hepatitis, HIV, parvovirus and other, still unknown viruses). Extensive bleeding, which requires blood transfusion in large volumes, can lead to the development of multiple organ failure, including impaired lung and kidney function. With the development of these serious complications in the subject, a series of phenomena associated with many cytokines and

HF) inflammatory reactions, which makes any treatment extremely difficult and often, unfortunately, useless. Therefore, the main goal of both surgical intervention and treatment in case of significant tissue damage is to avoid bleeding or to reduce it to a minimum.

To avoid or minimize such bleeding, it is important to ensure the formation of persistent and firm 60 hemostatic plugs that cannot be easily dissolved by fibrinolytic enzymes. In addition, it is important to ensure the rapid and efficient formation of such plugs and clots.

Japanese patent application Mo2-167234 A relates to an adhesive for biological tissue, which is characterized by containing fibrinogen, prothrombin, blood coagulation factor II, blood coagulation factor IX, blood coagulation factor X, blood coagulation factor XII, antithrombin, proteinase inhibitor and calcium ion . because Japanese patent application Mo59-116213A relates to an aerosol composition for use as a fabric adhesive, which contains a blood coagulant as an active ingredient. The coagulant for blood is chosen from blood coagulation factors I, I, Sh, IM, M, MI, MII, IX, X, Xi, Xii, and XIS, prekallikrein, high-molecular polymer kininogen, and thrombin. Preference is given to combinations of E XII! and thrombin.

MO 93/12813 (2utosSepeiiisv) refers to the use of E XII to reduce perioperative blood loss by a subject undergoing surgery. The composition may also contain aprotinin. EX is administered to the subject in the form of a bolus infusion, as a rule, the day before surgery.

European patent Mo225.160 (Momo MogaizK) relates to EMIIIa compositions and methods of treatment of bleeding-related disorders caused not by damage to the coagulation factor and not by inhibitors of coagulation factor 7/0.

European patent Mo82.182 (Vahieg Tgamepoi! I ar.) relates to the composition of the Miia factor for use in order to counteract the deficiency of coagulating factors or the influence of inhibitors of coagulating factors on the subject's body.

International patent publication Mo MO 93/06855 (Momo MogaizK) concerns the local application of EMIIa.

The work of KiaiKe ei AI., Tygotrosis apa Naetosiavziz, 1999 (Zirri), 095 1, concerns the introduction of additional exogenous EMI and the effect on thrombin production on the surface of activated platelets in dynamic models with imitation of hemophilia A or B conditions.

There is still a need in this field for an improved, reliable and widely used method of increasing coagulation, rapid formation of stable hemostatic plugs and achieving complete hemostasis in 20 subjects, in particular, in subjects with impaired thrombin production. Also needed is a method that would require less EMI to achieve complete hemostasis.

One of the purposes of this invention is to provide compositions that can be effectively used to treat or prevent bleeding attacks and coagulation disorders.

A second object of this invention is to provide compositions in a single dosage form that can be effectively used for the treatment or prevention of bleeding attacks or as procoagulants.

Another object of this invention is to provide compositions, methods of treatment or kits that exhibit i) a synergistic effect.

Another object of the present invention is to provide compositions, methods of treatment or kits that do not exhibit significant side effects, such as a high level of systemic activation of the coagulation system. Other objects of this invention will become clear after reading the description presented.

The authors of this invention have shown that the combination of factor Miia and factor XII can reduce the clotting time of normal human plasma more effectively than either factor Miia or XII taken alone. It has also been "- demonstrated that the combination of factor Miia and factor XIII can increase the stability of the clot more effectively than either factor Ma or XII taken alone. The combination of factor Miia in a concentration at which no further increase in clot stability is observed with factor XII, also in a concentration at which no further increase in clot stability is observed, unexpectedly revealed that in this case a further increase in clot stability is achieved. It was also demonstrated that the combination of the Ma factor and the factor

CP can prolong the time to lysis of an ip migo clot in normal human plasma more effectively than either of the inhibitors of factors Ma or Xi taken alone. Thus, by increasing coagulation, it is possible to treat bleeding subjects more effectively. In addition, patients can be treated with lower concentrations of factor MPa, which reduces the need for relatively high costs associated with traditional treatment with factor Miia alone. ;" In the first aspect, the invention relates to a pharmaceutical composition containing factor Ma and factor XII, and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a pharmaceutical composition containing factor Ma and factor XII as the only active agents and, optionally, a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a pharmaceutical composition prescribed for intravenous use

Me, input that includes factor Miia and factor XII! and, optionally, a pharmaceutically acceptable carrier. - In another aspect, the invention relates to a pharmaceutical composition formulated for intravenous administration, which includes factor Miia and factor XIII as the only active agents and, optionally, a pharmaceutically acceptable carrier.

Ф In one embodiment, the Ma factor is a recombinant Miia factor, and the XII factor is a recombinant XII factor.

In one embodiment of the invention, the Ma factor is a recombinant Miia factor. In another embodiment of this embodiment, the Ma factor is recombinant human Ma factor. In another embodiment, the Ma factor is a variant of the Ma factor.

F) In one embodiment, the variant Miia factor is a variant amino acid sequence that has no more than 20 substituted, deleted, or inserted amino acids compared to the natural Miia factor (ie, a polypeptide having the amino acid sequence described in US Pat. No. 4,784,950). In another embodiment, the variants of factor Ma have no more than 15 substituted, deleted or inserted amino acids; in another embodiment, variants of the Ma factor have no more than 10 substituted, deleted or inserted amino acids; in another embodiment, variants of the Ma factor have no more than 8 substituted, deleted or inserted amino acids; in another embodiment, variants of the Ma factor have no more than 6 substituted, deleted or inserted amino acids; in another embodiment, varieties of the Ma factor have no more than 5 65 substituted, deleted or inserted amino acids; in another embodiment, variants of the Ma factor have no more than 3 substituted, deleted or inserted amino acids compared to the natural Ma factor. In one version of the embodiment, the varieties of the Ma factor are selected from among |G/305M1-EMITA, IS3O05M/M3060/530951-EM Pa,

I-30511-EMPa, (C305711-EMIa, (E374Р|-EMIa, (M1587/M29801-EM Pa, (M1580/E296M/M29891-EmiIIa) and (KZZ7A1-EMPa.

In one embodiment, the XIIII factor is EHIII a2r2. In another version of the embodiment, factor XII is EHIII a2. In another embodiment, the factor XII is activated factor XII (EHSHa). In one embodiment, factor XII is a variant of factor CP. In one embodiment, the Xi factor is a human XII factor. In one version of the embodiment of the XII factor! there is a recombinant factor XII. In one embodiment, factor XII is recombinant human factor XII. In one embodiment, the HII factor is a human α2-dimer.

In one aspect, the composition also comprises a TERI inhibitor. In another aspect, the composition also contains a 7/0 MIII factor. In yet another aspect, the composition also contains the MI factor! and a TERI inhibitor.

In one embodiment, the composition also includes an inhibitor of the fibrinolytic system, for example, aprotinin, /-aminocaproic acid, or tranexamic acid.

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes a) an effective amount of factor Miia and, optionally, a pharmaceutically acceptable carrier in a first dosage unit form; b) an effective amount of factor XII and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; and c) a container for placing the aforementioned first and second dosage unit forms. In one aspect, the kit includes a) an effective amount of factor Miia and, optionally, a pharmaceutically acceptable carrier in a first dosage unit form; b) an effective amount of factor XII and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; c) an effective amount of the TERI inhibitor and, optionally, a pharmaceutically acceptable carrier in the third form of the DdOZO unit; and d) container for placing the aforementioned first, second and third dosage forms. and)

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes a) an effective amount of factor Miia and a TERI inhibitor and, optionally, a pharmaceutically acceptable carrier in a first dosage unit form; (b) an effective amount of factor XII and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; and o c) a container for placing the aforementioned first and second dosage unit forms. "-

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes a) an effective amount of factor Miia and, optionally, a pharmaceutically acceptable carrier in the first form of a) dosage unit; i- b) the effective amount of the KHI factor! and a TERI inhibitor and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; and c) a container for placing the aforementioned first and second dosage unit forms. In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes "a) an effective amount of factor Ma and factor XII! and, optionally, a pharmaceutically acceptable carrier in the first dosage unit form;

And b) an effective amount of the TERI inhibitor and, optionally, a pharmaceutically acceptable carrier in the second form and? dosage unit; and c) a container for placing the aforementioned first and second dosage unit forms.

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes -I a) an effective amount of factor Miia and, optionally, a pharmaceutically acceptable carrier in the first form of a dosage unit;

Me, b) an effective amount of factor XII and, optionally, a pharmaceutically acceptable carrier in the second form - a dosage unit; and c) an effective amount of the MI factor, optionally, a pharmaceutically acceptable carrier in a third dosage form; and

F d) an effective amount of the TERI inhibitor and, optionally, a pharmaceutically acceptable carrier in the fourth dosage form; and e) a container for placing the aforementioned first, second, third and fourth dosage forms.

In another aspect, the kit includes a) an effective amount of factor Ma and factor XII! and, optionally, a pharmaceutically acceptable carrier

F) the first form of the dosage unit; ka b) an effective amount of the MI factor and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; 60 c) an effective amount of the TERI inhibitor and, optionally, a pharmaceutically acceptable carrier in the third dosage unit form; and d) container for placing the aforementioned first, second and third dosage forms.

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes a) an effective amount of a Miia factor and a TERI inhibitor and, optionally, a pharmaceutically acceptable carrier in a first dosage unit form; b) an effective amount of factor XII and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; and c) an effective amount of the MI factor and, optionally, a pharmaceutically acceptable carrier in the third dosage unit form; and d) container for placing the aforementioned first, second and third dosage forms.

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes a) an effective amount of factor Ma and factor MI, optionally, a pharmaceutically acceptable carrier in a first dosage unit form; b) an effective amount of factor XII and, optionally, a pharmaceutically acceptable carrier in the second form of 70 dosage units; and c) an effective amount of the TERI inhibitor, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; and d) container for placing the aforementioned first, second and third dosage forms.

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes a) an effective amount of factor Ma and factor XII! and, optionally, a pharmaceutically acceptable carrier in the first dosage unit form; b) the effective amount of the MI factor! and a TERI inhibitor and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; and c) a container for placing the aforementioned first and second dosage unit forms.

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes a) an effective amount of factor MI and factor HII and, optionally, a pharmaceutically acceptable carrier in a first dosage unit form; b) an effective amount of the TERI inhibitor and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; and c c) an effective amount of the Miia factor and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; and d) container for placing the aforementioned first, second and third dosage forms.

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes a) an effective amount of factor XII! and a TERI inhibitor and, optionally, a pharmaceutically acceptable carrier in the first dosage unit form; b) an effective amount of the MI factor and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; and "- c) an effective amount of the Miia factor and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; and re) d) container for placing the aforementioned first, second and third dosage forms. uh-

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes a) an effective amount of a TERI inhibitor and an MI factor and, optionally, a pharmaceutically acceptable carrier in a first dosage unit form; b) an effective amount of factor Miia and, optionally, a pharmaceutically acceptable carrier in the second form of a "dosage unit; and c) an effective amount of factor XII and, optionally, a pharmaceutically acceptable carrier in the second form. dosage unit; and and?" d) container for placing the aforementioned first, second and third dosage forms.

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes a) an effective amount of a TERI inhibitor and factor XII and, optionally, a pharmaceutically acceptable carrier in a first dosage unit form; b) an effective amount of factor MI and factor Miia and, optionally, a pharmaceutically acceptable carrier in

Me, the second form of dosage unit; and - c) a container for placing the aforementioned first and second dosage unit forms.

In another aspect, the invention relates to a kit for the treatment of bleeding attacks, which includes o a) an effective amount of factor Miia and a TERI inhibitor and, optionally, a pharmaceutically acceptable carrier in

F of the first form of the dosage unit; b) an effective amount of factor MI and factor XII and, optionally, a pharmaceutically acceptable carrier in the second dosage unit form; and c) a container for placing the aforementioned first and second dosage unit forms.

In another aspect, the invention relates to the use of a factor in combination with an XII factor to obtain (F) a medicament for the treatment of bleeding attacks. In another aspect, the invention relates to the use of a factor in combination with an XII factor to provide a medicament for reducing clotting time in a subject. In another aspect, the invention relates to the use of a factor in combination with an XII factor to provide a medicament for prolonging clot lysis time in mammalian plasma.

In another aspect, the invention relates to the use of a factor in combination with an XII factor for the preparation of a medicament for increasing the strength of a clot in mammalian plasma.

In another aspect, the invention relates to the use of a factor in combination with an XIII factor to produce a medicament for enhancing fibrin coagulation in mammalian plasma

In one embodiment, the mammalian plasma is human plasma. In another embodiment, the mammalian plasma is normal plasma; in another embodiment, the plasma is normal human plasma; in one embodiment, the plasma is the plasma of a subject with impaired thrombin production. In one embodiment, the plasma is the plasma of a subject with a reduced concentration of fibrinogen.

In one embodiment, factor Miia and factor XIII prolong the time of lysis of a migo clot in normal human plasma.

In another aspect, the invention relates to a method of enhancing fibrin coagulation in a subject, including administering to the subject an effective amount of the factor in combination with an effective amount of the HIPI factor.

In another aspect, the invention relates to a method of treating bleeding episodes in a subject, including administering to the subject an effective amount of the factor in combination with an effective amount of the HIPI factor.

In another aspect, the invention relates to a method of reducing the clotting time of mammalian plasma, including contacting the plasma with an effective amount of factor Ma in combination with an effective amount of factor XII. In one embodiment, the effective amount of the factor in combination with the effective amount of the factor Chi! administered to a subject in need of such treatment. In another aspect, the invention relates to a method of enhancing fibrin formation in a subject, including administering to the subject an effective amount of the factor in combination with an effective amount of the CP factor.

In one embodiment of the methods of the invention, factor Miia and factor XIII are the only active agents that are administered to the subject. In another embodiment of the invention, the pharmaceutical composition includes factor Miia and factor XIII as the only active agents.

In one embodiment of the invention, factor Ma and factor XIII are administered simultaneously and in the form of a single dose. In another embodiment, factor Miia and factor XII are administered sequentially. In another embodiment, the Miia factor and the XII factor! administered approximately 1 to 2 hours apart, e.g., 30 minutes apart, e.g., 10 minutes apart, e.g., in the form of a kit that includes the factor

Miia in the first dosage unit form and factor XIII in the second dosage unit form. high school

In one embodiment, the effective amount of the Ma factor ranges from 0.05 mg/day to 50 Ω/day (at a subject's weight of 7 Оkg). In one embodiment, the effective amount of factor XII is from 0.005mg/day to i) 500mg/day (at a subject's weight of 7Okg).

In one embodiment of the present invention, the pharmaceutical composition (in the form of a single preparation) consists mainly of factor Miia and factor XII, and, optionally, at least one pharmaceutically acceptable excipient or carrier and/or stabilizer and/or detergent and /or neutral salt and/or antioxidant and/or preservative and/or protease inhibitor. at

In another variant of the embodiment of this invention, the pharmaceutical composition (in the form of a single preparation) "- consists mainly of factor Miia and factor XII, and, optionally, at least one pharmaceutically acceptable filler or carrier and/or stabilizer and/or detergent and /or neutral salt and/or ice)

Зз5 antioxidant and/or preservative and/or protease inhibitor and/or TERI inhibitor. Cha

In another embodiment of the present invention, the pharmaceutical composition (in the form of a single preparation) consists mainly of factor Miia and factor XII, and, optionally, at least one pharmaceutically acceptable excipient or carrier and/or stabilizer and/or detergent and/or neutral salt and/or antioxidant and/or preservative and/or protease inhibitor and/or TERI inhibitor and/or MI factor. "

In another embodiment, the pharmaceutical composition (if in the form of a kit) consists of the first dosage unit form c, which consists mainly of Mig factor and, optionally, at least one pharmaceutically acceptable excipient or carrier and/or stabilizer and/or detergent and/or neutral;" salt and/or antioxidant and/or preservative and/or protease inhibitor, and a second dosage unit form consisting primarily of factor Xi, and optionally at least one pharmaceutically acceptable excipient or carrier and/or stabilizer and/ or a detergent and/or a neutral salt and/or an antioxidant and/or a preservative and/or a protease inhibitor.

In another embodiment, the pharmaceutical composition (if in the form of a kit) consists of a first me) form of a dosage unit, which consists mainly of Mig factor and, optionally, at least one - a pharmaceutically acceptable filler or carrier and/or stabilizer and/ or a detergent and/or a neutral salt and/or an antioxidant and/or a preservative and/or a protease inhibitor and/or a TERI inhibitor, and a second dosage unit form that consists primarily of a HYII factor, and, optionally, at least one

F of a pharmaceutically acceptable filler or carrier and/or stabilizer and/or detergent and/or neutral salt and/or antioxidant and/or preservative and/or protease inhibitor and/or TERI inhibitor and/or factor

M.

In another embodiment, the subject is a human; in another embodiment, the subject has impaired thrombin production; in one embodiment, the subject has a reduced plasma fibrinogen concentration

F) (for example, a subject subjected to multiple transfusions). ka In another aspect, the composition also contains the MI factor. In one embodiment, the MI factor is an activated MI factor! (by the MiPa factor). In another embodiment, the MI factor! is a recombinant factor because Ma. In another embodiment, the MP factor! is a recombinant human MiIia factor.

In another aspect, the composition also includes an inhibitor of the fibrinolytic system, for example, aprotinin, /-aminocaproic acid, or tranexamic acid.

Figure 1 shows that spontaneous clot formation in normal human plasma (NHP) diluted 1/10 in a buffer containing 2O0nM Nerez, 150mM Masi, and 5mM Sasi, pH 7.4 in a 65-well microtitre (total volume with a volume of 250 μl) was reached in approximately 2500-300 Osek. The coagulation of fibrin was traced by an increase in optical density at bOOnm in Zresgat 340. MoiIesshciag Oemisez, Zippu-maye SA. Figure 1 shows

that TONNM of recombinant Miia factor (TEMPa) from Momo MogaizkK A/5 Wadzuaega, Oeptagk reduced the time of clot formation to 1600 seconds (p-2). A further reduction in clot formation time was achieved by adding

ZONNM of factor XIII (EXIII) from Ategisap Oiadpoviisa ips, Sgeepzhisp, ST together with LONM hEMiIa (p-3). The clot formed in the presence of EH was more transparent (had a lower maximum optical density) than in its absence, indicating that the addition of EHIA resulted in a finer fibrin gel structure with thinner fibrils.

Figure 2 shows that additional EX (ZONM) prolongs the time of fibrin lysis of clots formed in the presence of HEMmIIa and tissue plasminogen activator (PA). Clot formation was induced in the presence or in the absence of ZONM XIA by adding from 25 μl of MNR to 225 μl of 200nM Nerez, 150mM Masi, 5mM Sasi", pH 7.4, containing 5ONM hEMiIIia and 0.5nM recombinant (IRA from Momo Mogaizk A/5 Vadzmazga , Oeptagk. Clot formation and subsequent clot lysis caused by RA-mediated activation of plasminogen were observed using Zresigates 340 at bOOnm as an increase in OO sodonm 3 followed by a return of the trajectory to the baseline level. Fig. 2 shows that the time of clot lysis under these conditions is significantly was extended due to the presence of EHIIA.

Figure 3 shows the effect of hEMIA and EHIIA on maximum clot firmness (MCE) and clot resistance to ERA-mediated lysis. Prior to the addition of hEMIia and/or EHIIA, the achieved MCE was 25mm, and the time required to lyse half of the clot was 12.3 minutes (Figure 3). Addition of increased concentrations of EX (0-40 nM) did not change MSE; however, a dose-dependent prolongation of the lysis time of half of the clot was observed, and the optimal one was observed with ZONM EHIY! (time of lysis of half of the clot: 14.3 min, Fig. 3). Similarly, the addition of hEMiia (1NM) as a result provides protection of the clot from mediated

IRA of fibrinolysis (time of lysis of half of the clot; 16.4 min) without any effect on MSE (Fig. 3). However, after the addition of hEMiIa (INM) together with EHNI (ZonNM) an increase in MSE (29 mm) was observed, as well as reliable protection against fibrinolysis (time of lysis of half of the clot; 27.1 min) (Fig. 3). Taken together, these results demonstrate that addition of hEMiIa and EX to plasma synergistically improves clot mechanical strength and resistance to ERA-mediated fibrinolysis.

The present invention provides a composition that includes a combination of factor Miia and factor XII. The invention also i) provides a composition that includes a combination of factor Miia and factor XII! as individual active ingredients.

The composition can exist in the form of a separate composition or in the form of a multi-component set. These compositions are useful as therapeutic and prophylactic procoagulants and fibrin clot stabilization agents, and Ghezo forms rapid fibrin clots in mammals, including primates such as humans.

When mentioning the first, second or third, etc. dosage unit in this description, it is not indicated about the optimal order of administration; this is for convenience only. "-

The present invention also provides a method for the treatment (including prophylactic treatment or prevention) of bleeding episodes in subjects, including humans, caused by, for example, trauma or surgery, ise) or in subjects with blood coagulation factors SIN or EM or platelets missing or damaged. Cha

A combination of factor Ma and factor KPSa has been found to be a useful product that ensures the formation of hard and stable hemostatic plugs that form rapidly.

Complete production of thrombin is necessary for the formation of a solid, stable hemostatic plug. The fibrin structure of such a plug depends on both the amount of thrombin formed and the rate of initial thrombin generation. When the production of thrombin is disturbed, a porous fibrin plug is formed, which has high permeability. Fibrinolytic enzymes, which are normally present on the surface of fibrin, easily dissolve such a fibrin plug. The formation of a stable fibrin plug also depends on the presence of the KhPa factor, which is activated by thrombin and therefore also depends on the complete generation of thrombin. In addition, the above-described thrombin-activated fibrinolytic inhibitor, TARI, requires a fairly large amount of thrombin for its activation. Therefore, with insufficient production of thrombin, TARI cannot be activated, as a result of which a hemostatic plug is formed, which dissolves more easily than normal in the case of normal fibrinolytic activity.

Me, Due to the increase in the production of thrombin, the Miia factor provides the basis for the full activation of the XIA factor, which is the most important for the formation of a fully stabilized hemostatic plug, and therefore, for the maintenance of 5p hemostasis. In cases of reduced number of platelets, thrombocytopenia, acceleration of thrombin production is caused by the introduction of exogenous additional factor MPa. However, total thrombin production does not

Ф is normalized by the Miia factor even in high concentrations.

Combining factor Miia and factor XII, specifically the alpha chain of factor XII! (a2-dimer), ensure full activation of factor ХІІІ, which increases the hemostatic effect of factor MiІІІa.

In addition, in subjects with a reduced concentration of fibrinogen in plasma (subjects exposed to repeated transfusions due to multiple injuries or extensive surgery), full activation of factor XII is not

F) happens. More effective hemostasis is achieved by introducing a combination of factor Miia and factor XII. ka Another way to increase the stability of fibrin hemostatic plugs is to ensure the full presence of the KIP factor (activated KPI factor). in Subjects with thrombocytopenia have impaired thrombin production, as well as impaired stabilization of fibrin plugs, as a result of which hemostatic plugs become prone to premature dissolution. In addition, subjects who have suffered significant trauma or organ damage and have therefore received frequent blood transfusions often have low platelet counts, as well as low levels of fibrinogen, MI factor, and other coagulation proteins. Thrombin production is impaired (or reduced) in these subjects. 65 In addition, the reduced level of fibrinogen negatively affects the activation of factor XII. Therefore, these subjects have insufficient or less effective hemostasis, as a result of which the formed fibrin plugs are easily and prematurely dissolved by proteolytic enzymes; in addition, these enzymes are widely released in cases of significant trauma and organ damage.

To promote the formation of fully stabilized plugs with full ability to maintain hemostasis, the subject is administered a composition according to the invention. This composition is particularly useful for subjects with reduced platelet counts and for subjects with reduced plasma levels of fibrinogen and/or other coagulation proteins.

In the presence of factor XII, a lower concentration of MPa factor may be sufficient to provide the required hemostasis.

As noted above, factor XII exists in plasma as a tetrameric zymogen consisting of two 70 alpha subunits and two beta subunits (denoted a2 p2), but is found in other tissue (eg, platelets) as an a2 dimer. Within the scope of this invention, any of these forms of zymogen or activated factor XII (factor HiIPa) can be used, as well as genetically modified varieties of factor XII, which retain their characteristic ability to cross-link. In one embodiment, factor XII is human factor XIII; in another embodiment, factor XII is a human a2-dimer; in another version of the implementation of the factor Chi! is /5 activated factor of human CSH.

Factor XII and factor Miia, used in this invention, can be isolated from blood or obtained by a recombinant method. It is obvious that the practical implementation of the methods described by us is independent of the origin of the purified XIA factor! and factor Miia, and therefore the present invention is intended to encompass the use of any combination of factor XII and factor Ma suitable for use in the present invention. The preference is given to the human factor Ma and the factor XII. The present invention can also be used genetically modified varieties of the MPa factor and the XII factor, which retain their characteristic activity associated with hemostasis. Fragments of variants of factor Ma or factor XII, or factor Miia-, or factor HIPI-, which retain their characteristic hemostasis-related activity, can also be used in this invention. Hemostasis-related activity of factor Ma is measured, for example, by applying the analysis of activity of factor Miia presented in this description. Hemostasis-related activity of factor XII! measured, for example, using the factor XIII activity assay presented in this description. and)

Examples of variants of the Mii factor that have substantially the same or better biological activity compared to the natural Mii factor are, among others, those described in Danish patent applications MoMo RA 2000 00734,

RA 2000 01360, RA 2000 01361 and RA 2001 00477. Examples, among others, are RMIa, (M1587/M29891-EM Pa, (M1580/E296M/M29801-EMIa and (KZ37A1-EM a. o

In this context, the three-letter or one-letter designations of amino acids are used in their traditional "- meaning, as indicated in the Table. Unless otherwise indicated, the amino acids mentioned by the authors are

And - amino acids. It should be understood that the first letter, for example, in KZ37, represents an amino acid that is naturally present in the indicated position of the natural factor MII, and that, for example, (KZ337A|-EMiIia means a variant of RMII, in which it is represented by a one-letter code K amino acid, which is naturally present in the specified position, is replaced by an amino acid represented by the one-letter code A.

F

- zo o t ov GEN NN tya NO UNN tyuvmio 011 zi1a shchi yuyu (Aspartic acid! dr | 0 60

The term "Ma factor" or "HMIIIa" can be used interchangeably. The term "Miia factor" includes a zymogenic factor

MI (single-chain MI factor). The term "XII factor" or "EXHIM" can be used interchangeably. The term "MI factor! or "RMIM" can be used interchangeably.

It will be clear to a person skilled in the art that substitutions can occur outside the regions that are responsible for the function of the factor MiIa or factor XII molecule! and still provide an active polypeptide as a result. Amino acid residues essential for the activity of the polypeptide of factor Miia or factor XII, and therefore

may not be subject to substitution, are recognized according to procedures known to those skilled in the art, such as site-specific mutagenesis or alanine-scanning mutagenesis |see, e.g., Sippipapat apa UMeiYiz, 1989,

Zsiepse 244:1081-1085). According to the latter method, mutations are carried out in each positively charged residue of the molecule, and the resulting mutant molecules are tested for coagulation, and, accordingly, the ability to cross-link to recognize the amino acid residues that are responsible for the activity of the molecule. Enzyme-substrate interaction sites are also determined by three-dimensional structure analysis, defined by this method as nuclear magnetic resonance analysis, crystallography, or covalent fixation of labeled substrates |see, for example, de Moze et al., 1992, Zsiepse 255: 306-312 ; 1992, pp. 70, 224, 899-904; UModameg eyi ai, 1992, REVZ5Z I eTsegz 309: 59-64.

Introduction of a mutation in a nucleic acid sequence to replace one nucleotide with another can be carried out by site-specific mutagenesis using any methods known to specialists.

A procedure using a supercoiled, double-stranded DNA vector with the desired insert and two synthetic primers containing the desired mutation is particularly appropriate. Oligonucleotide primers, each of which is complementary to the opposite strand of the vector, are unwound during thermocycling with Risch DNA polymerase. After the inclusion of primers, a mutated plasmid is formed, which contains staggered nicks (breaks of one chain in a double-stranded nucleic acid molecule).

After thermocycling, the product is treated with Orli, which is specific for methylated and hemimethylated

DNA, to digest a sample of parental DNA and select synthesized DNA containing the mutation. Other techniques known to those skilled in the art to create, recognize, and isolate varieties may also be used, such as gene transfer or phage detection.

The term "MI factor" or "EMF" includes activated MI factor! (denoted MiIa factor), varieties and truncated forms of the MIP factor, which retain their characteristic coagulant activity. In one embodiment, the MI factor is a human MI factor. high school

The term "TERI inhibitor" refers to compounds that inhibit the anticoagulant activity of TERI (Tissue Factor Pathway Inhibitor). This term includes compounds such as those described in European patent Mo 558529, i)

MO 96/28153 and 05 5,622,988. "TER!" and "ERI" (extrinsic pathway inhibitor) can be used interchangeably.

According to the present invention, an "effective amount" of factor Ma and an "effective amount" of factor XII is defined as an amount of factor Miia and factor XII sufficient to prevent or reduce bleeding or blood loss, for the purpose of treating, alleviating or partially stopping the disease and its complications .

The amount of factor Mia and the amount of factor XII administered according to the present invention may vary from about 1:100 to about 100:1 (μg of factor Mia:/μg of factor XII). "-

In this context, "subjects with impaired thrombin production" means subjects who cannot produce complete thrombin on the surface of activated platelets and includes subjects in whom thrombin is produced in ise) lower amounts than subjects who have a fully functioning, normal coagulation system, including normal amounts and function of coagulation factors, platelets, and fibrinogen, and includes subjects who do not have SIN and/or EM (hemophilia A and B) or subjects with deficiencies RICH and/or EM! or with inhibitors against RICH and/or EM; subjects who lack EHI; subjects with reduced platelet counts or platelets with insufficient function (eg, thrombocytopenia or thrombasthenia "

Glanzmann or subjects with excessive bleeding); as well as subjects who have a reduced level of prothrombin, with EX or MI. . Subjects with reduced plasma fibrinogen concentrations (for example, subjects exposed to repeated transfusions of blood due to multiple injuries or extensive surgery) also suffer from the formation of soft and unstable fibrin plugs that dissolve easily.

The term "complete hemostasis" means the formation at the site of damage of a persistent and solid fibrin clot -I or plug, which effectively stops bleeding and which cannot be easily dissolved by the fibrinolytic system.

The term "factor Miia activity" means the ability to produce thrombin; the term also includes ability

Me, to the production of thrombin on the surface of activated platelets in the absence of tissue factor. - The term "enhancement of the normal hemostatic system" means an increase in the ability to produce thrombin. o The term "bleeding-related disorder" used by the authors means any disorder, congenital, acquired

F or caused, which has a cellular or molecular origin and manifests itself in bleeding. Examples are coagulation factor deficiency (eg, hemophilia A and B or coagulation factor X or MI deficiency), coagulation factor inhibitors, platelet dysfunction, thrombocytopenia, or von

Willebrand (pseudohemophilia).

The term "bleeding attacks" includes uncontrolled and excessive bleeding, which is the main problem that

F) occurs in connection with both surgical intervention and other forms of tissue damage. Uncontrolled and excessive bleeding can occur in subjects with a generally normal coagulation system (however, these subjects develop coagulopathy due to bleeding - dissolution of coagulation proteins, increased fibrinolysis, and decreased platelets due to the soluble effect of bleeding), and subjects subjects who have coagulation disorders or bleeding disorders. Deficiency of coagulation factors (hemophilia A and B, deficiency of coagulation factors XI or MI!) or inhibitors of coagulation factors can cause bleeding disorders. Excessive bleeding also occurs in subjects with a normally functioning coagulation cascade (without coagulation factor deficiency or inhibitors against any of the coagulation factors) and may be 65 Caused by platelet dysfunction, thrombocytopenia, or von Willebrand pseudohemophilia. In such cases, the bleeding can be compared to bleeding caused by hemophilia, because the coagulation system, as in hemophilia, experiences a deficiency or abnormality in the "compounds" responsible for coagulation (such as platelets or von Willebrand factor protein), causing significant bleeding. . In subjects who have sustained significant tissue damage due to surgery or significant trauma, the normal hemostatic mechanism may be impaired due to the need for immediate hemostasis, and they may experience increased bleeding despite a generally normal (pre-injury) hemostatic mechanism. .

Achieving satisfactory hemostasis is also a challenge when bleeding occurs in organs such as the brain, inner ear, and eyes, where surgical hemostasis is limited. The same problem can arise in the process of biopsy from various organs (liver, lungs, tumor tissues, gastrointestinal tract), and also during laparoscopic operations. Common to all these situations are the difficulties associated with ensuring hemostasis by surgical methods (applying sutures, staples, etc.), which also occurs with diffuse bleeding (hemorrhagic gastritis and profuse uterine bleeding). Acute and profuse bleeding can also occur in subjects on anticoagulant therapy in whom hemostasis disorders were caused by the given therapy. Such subjects may require surgical intervention when rapid reversal of the anticoagulant effect is required. For subjects with localized prostate cancer, radical retropubic prostatectomy is usually used. The operation is often complicated by significant, sometimes very large, blood loss. Significant blood loss during prostatectomy is often associated with a complicated anatomical location, with various densely vascularized areas that are difficult to access for surgical hemostasis and can lead to scattered bleeding from a large area. Another situation that can cause problems in the case of unsatisfactory hemostasis is when subjects with a normal hemostatic mechanism undergo anticoagulant therapy to prevent thromboembolism. Such therapy may include the use of heparin, other forms of proteoglycans, warfarin or other forms of vitamin K antagonists, as well as aspirin and other platelet aggregation inhibitors.

In one embodiment of the invention, bleeding is associated with hemophilia. In another embodiment of the disease, bleeding is associated with hemophilia due to acquired inhibitors. In another embodiment, bleeding is associated with thrombocytopenia. In another embodiment, bleeding is associated with pseudohemophilia. In another variant i) embodiment, bleeding is associated with severe tissue damage. In another embodiment, bleeding is associated with severe trauma. In another embodiment, bleeding is associated with surgical intervention. In another embodiment, bleeding is associated with laparoscopic surgery. In another embodiment, He bleeding

Zo is associated with hemorrhagic gastritis. In another embodiment, the bleeding is profuse uterine bleeding. In another embodiment, bleeding occurs in organs with a limited possibility of mechanical hemostasis. In another embodiment, bleeding occurs in the brain, inner ear area, or eyes. In another embodiment, bleeding is associated with the biopsy process. In another embodiment, bleeding is associated with anticoagulant therapy. ise)

The composition according to the invention may also include a TERI inhibitor. Such a composition in its optimal version should be administered to subjects with hemophilia A or B.

The composition according to the invention may also include an MI factor. Such a composition is optimally administered to subjects who do not have inhibitors of the MIP factor.

In this context, the term "treatment" includes both the prevention of possible bleeding, for example, during surgery, and the regulation of bleeding that is already occurring, for example, with hemophilia or with trauma, in order to control or minimize bleeding. Prophylactic administration of factor MiIa and factor XII is therefore covered by the term "treatment". ;" As used by the authors, the term "subject" means any animal, in particular, a mammal such as a human, and may in some cases be used interchangeably with the term "patient". -I Abbreviations

TE tissue factor (o) MI factor MII in its single-chain non-activated form - EMPA factor MI! in its activated form heme recombinant factor MI! in its activated form ге ШХ ЭХ и ий ий and factor ХИЯ in its zymogenic non-activated form 4") ЭХШ factor ХЯІ in its activated form

TEH recombinant EHIII gEhHhSha recombinant. Ehsha huh? alpha- or a-chain of ЕХ or гЭХІ p2 beta- or p-chain of ЭХІІ or гЭЫ!

Ф) ЕХШа?2 dimeric form of EHIIA, which contains two α-chains and EХШаг2р2 tetrameric form of EHIIA, which contains two α- and two Br-chains

E! MI factor in its zymogenic non-activated form 60 TOPICS recombinant EMF!

EMsha factor MI! in its activated form, hEMSh is recombinant. EM Sha

TERI inhibitor of the tissue factor 65 pathway Preparation of compounds

Purified human Miia factor, suitable for use in this invention, is optimally obtained by the method of recombinant DNA, for example, as described in the work Nadep eyi ai!., Rgos.Mak.Asayd.Zsi. OA 83:2412-2416, 1986 or as described in European patent Mo200,421 (7utosepiiisv, Ips.). The recombinant Miia factor can be an authentic Miia factor or a more or less modified Miia factor, provided that such Miia factor has substantially the same biological activity for blood coagulation as the authentic Miia factor (natural Ma factor). Such a modified MiIIa factor is obtained by modifying the nucleic acid sequence encoding the natural MII factor, or by changing the amino acid codons, or by deleting some of the amino acid codons in the nucleic acid encoding the natural MI factor! by known methods, for example, by site-specific mutagenesis. 70 The MII factor is also obtained by the methods described in the works of Vgale apa Ma/egiv, 9U.VioI.Spet. 255 (4): 1242-1247, 1980, and Neadpeg apa Kiviei, U. Siip. ipmevi. 71:1836-1841, 1983. These methods provide the MII factor without appreciable amounts of other blood coagulation factors. Even more purified MII factor is obtained by including additional gel filtration as a final purification step. MI factor! in this case, it is converted to activated factor Miia by known methods, for example, by several different plasma proteins, such as factor 7/5 Hia, Xa or Xa. In an alternative version, as described in the work of Vioegp ei ai. (Kezeagspy Oivzsiosyge, 269

Zerietreg 1986, pp. 564-565), MI factor! activated by passing it through an ion-exchange chromatographic column, such as a Mopo O (RNpagtasia iiipe Spetisai!v) or other similar column.

The XII factor for use according to the present invention is obtained from plasma according to known methods, such as those described in the works of Sooke apa Noibgook (Viospet. .). 141:79-84,1974) and Sygaz apa Gogapa (Meipodz Epgutoi. 45:177-191,1976), to which the authors refer. The dimeric form of the a2 factor of the XIII factor is obtained from the placenta, as described in US patents MoMo 3,904,751; 3,931,399; 4,597,899 and 4,285,933 to which the authors refer. However, preference is given to the use of recombinant factor XII to avoid the use of blood or tissue products associated with the risk of disease transmission. Methods of obtaining recombinant factor XII are known to specialists. See, for example, Joamie ei a), EP 268,772; OSgypdtapp ei ai., sch

AC-A-69896/87; Vivpor ei ai.,, Viospetivhigu 1990, 29:1861-1869; Voaga ef aiYi.,, TygotB. Naetov 1990, 63:235-240; dadadeezzhagap ei ai., Sepe 1990, 86:279-283; and VgoKeg eyai., REV5 Hey. 1989,248:105-110, to which (8) the authors refer in their entirety. In one embodiment, the α2-dimer of factor XII is produced cytoplasmically in the yeast Zasspagotuses segemiviae as described in US Patent Application No. 07/741,263, which is also pending and to which the authors refer in its entirety). Cells are collected and Hezo is subjected to lysis, obtaining a clear lysate. This lysate is fractionated by anion-exchange chromatography at neutral to slightly alkaline pH using a column of derivatized agarose, such as OEAE Ravi-Riom Zernagose fast-flow Sepharose. TM. (RNagtasia) or others. After that, the XII factor is precipitated from the eluate of the -n column by concentrating it and bringing the pH to 5.2-5.5, for example, by diafiltration on a succinate-ammonium buffer. The precipitate is further dissolved and purified using traditional chromatography techniques such as gel filtration and hydrophobic interaction chromatography. uh-

As will be understood by those skilled in the art, the use of factor X and factor Miia proteins syngeneic to the subject is preferred to reduce the risk of triggering an immune response. Obtaining and characteristics of the factor

XII of non-human origin is described in Makatig's work a). (9. Viospet. 78:1247-1266, 1975). The present invention also covers the use of such factor XII proteins! and Miia factor in veterinary procedures. "

Administration and Pharmaceutical Compositions With c For treatment in connection with planned surgical intervention, the MI factor and the HII factor are generally administered within about 24 hours before the intervention and for 7 days or more after it. ;" Administration as a coagulant is carried out in various ways, as described in the successful invention.

A dose of the MI factor! is from about 0.05mg to about 50mg/day, for example from about 1mg to about 200mg/day, or for example from about 1mg to about 175mg/day for a 7kg subject as loading and maintenance doses, depending from the subject's weight, condition and severity.

A dose of the XIA factor! is from about 0.05mg to about 50mg/day, for example from about 1mg to me) about 200mg/day, or for example from about 1mg to about 175mg/day for a 7kg subject as loading and maintenance doses, depending on the subject's weight, condition and severity.

The compositions and kits of the present invention are useful in medicine and veterinary medicine, for example, for the treatment or prevention of subjects suffering from bleeding attacks or coagulation disorders. For

F application according to this invention factor Miia and factor XII! prescribed optionally with a pharmaceutically acceptable carrier. In the optimal version, pharmaceutical compositions are administered parenterally, that is, intravenously, subcutaneously or intramuscularly, or by continuous or pulsatile infusion.

The compositions may also include one or more diluents, emulsifiers, preservatives, buffers, fillers, etc. and may exist in forms such as liquids, powders, emulsions, controlled release

F) release, etc. A person skilled in the art can prescribe the compositions of the invention in a suitable manner and in accordance with generally accepted practice, for example, as described in Ketipdiop's Rpagtaseciis! Zsiepsez, Seppago, ed., Mask Rybijzpipd So., Eaviop, RA, 1990. Compositions for parenteral administration include the MII factor and the CP factor in combination, preferably dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous media are used, such as water, buffered water, 0.490 saline, 0.395 glycine, etc. Varieties of the MI factor! according to the invention can also be formulated into liposomal compositions for administration or targeting to sites of damage. A general description of liposome compositions is contained, for example, in O.5. 4,837,028, 0.5. 4,501,728 and O.5. 4,975,282. 65 A typical pharmaceutical composition for intravenous infusion contains 250 ml of sterile solution

Ringer and 1Omg of factor Mia and/or factor Xi. Actual methods of preparation of compositions for parenteral administration are known or understood by those skilled in the art and are described in more detail, for example, in Ketipdiop's

Rpagtaseciisa! Zsiepsevs, 181st ed., Musk Rybiizpipd Sotrapu, Easiop, RA (1990).

Briefly, pharmaceutical compositions suitable for use according to the present invention are obtained

By mixing factor Miia or factor XIII or factor Miia in combination with factor XIII, preferably in purified form, with suitable adjuvants and a suitable carrier or diluent. Suitable physiologically acceptable carriers or diluents include sterile water and saline. The compositions may contain pharmaceutically acceptable excipients necessary to approximate physiological conditions, such as pH-adjusting agents and buffering agents, tone-adjusting agents, etc., for example, sodium acetate, /o0 sodium lactate, sodium chloride, potassium chloride, calcium chloride and others Suitable adjuvants also include calcium, proteins (e.g., albumins), or other inert peptides (e.g., glycylglycine) or amino acids (e.g., glycine or histidine) to stabilize purified factor Mia and/or factor HIPI. Other physiologically acceptable adjuvants excipients are non-reducing sugars, polyalcohols (eg, sorbitol, mannitol, or glycerol), polysaccharides, such as low molecular weight dextrins, detergents (eg, polysorbate), and antioxidants (eg, bisulfite and ascorbate). Adjuvants typically , present at a concentration of 0.001 to 495 (w/v). The pharmaceutical composition may also contain protease inhibitors, such as aprotinin or tranexamic acid, and preservatives. In addition, the composition may also contain a TERI inhibitor and/or factor

M.

The compositions are sterilized by traditional well-known sterilization methods. Aqueous solutions obtained as a result of sterilization are packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized composition is combined with a sterile aqueous solution before administration.

The concentration of factor Miia, factor XII, or factor in combination with factor XII in these compositions can vary widely, i.e., from less than about 0.595 by weight, usually or at least about 196 by weight, to 15 or 2095 by weight, and is determined , first of all, the volume, viscosity of the liquid, etc. according to the specific selected method of administration.

Preference is given to administration by injection or infusion, better - injections. Thus, factor Ma and (i) factor XII are provided in a form suitable for intravenous administration, such as a composition that is either a dissolved lyophilized powder or a liquid composition that contains both factor Mia and factor XII! in a single dosage form, or a dissolved lyophilized powder, or a liquid composition containing Hezo Mia factor and a dissolved lyophilized powder in a single dosage form, or a liquid composition containing the factor

XII! in a different dosage form. at

Local introduction of factor Miia and factor XIII, for example, local application, is carried out, for example, "- by spraying, perfusion, double balloon catheters, dilator introduced into vascular grafts, hydrogels used to cover balloon catheters, or other traditional means) c5 applied methods. For ambulatory patients who require daily maintenance administration, factor Ia and factor XIII can be administered by continuous infusion using, for example, a portable pump system. In any case, the pharmaceutical composition should provide for the amount of the factor

Miga and factor XII, sufficient for effective treatment of the subject.

The combination of factor Miia and factor XIII shows a synergistic effect in the miigo analysis of clot firmness and fibrinolysis time. In addition, the combination of the Ma factor and the XII factor shows a synergistic effect in the formation of stable fibrin clots, increasing the lysis time of half of the clot, increasing the strength of the clot and resistance to fibrinolysis. ;" Compositions containing factor MII and factor XII are administered for prophylactic and/or therapeutic treatment. When used therapeutically, the composition is administered to a subject who is already suffering from the disease as described above, in an amount sufficient to cure, alleviate, or partially suspend the disease and its -I complications. The amount sufficient for this purpose is defined as an "effective amount" or "therapeutically effective amount. As will become clear to the skilled person, the effective amount depends on the severity of the disease or

Me. damage, as well as the mass and general condition of the subject. It should be taken into account that the materials of this invention are usually used in case of serious illness or damage, that is, when there is or may be a threat to life.

In these cases, in order to reduce side effects and under the conditions of the general lack of immunogenicity of the MiIa factor and the XII factor in humans, the doctor may consider it possible and desirable to introduce a significant excess amount of these

F compositions.

When used prophylactically, compositions containing factor Ma and factor XII are administered to a subject who is susceptible or otherwise at risk of disease or injury to increase their ability to coagulate. This amount is defined as the "prophylactically effective dose."

Single or repeated administration of the compositions is carried out in doses and according to the scheme determined by the doctor (F). The compositions can be administered one or more times a day or a week. The effective amount of such a pharmaceutical composition is the amount that provides a clinically significant effect against bleeding attacks. This amount depends in part on the specific condition being treated, the age, weight and general health of the subject, as well as other factors that are apparent to those skilled in the art.

The composition, as a rule, is administered in a single dose before the expected bleeding or at the beginning of the bleeding. However, it can also be entered | in several doses, ideally at intervals of 2-4-6-12 hours, depending on the dose and the condition of the subject.

The composition can exist in the form of a separate preparation, which contains both factor MiIa and | factor XII in 65 suitable concentrations. The composition can also exist in the form of a kit consisting of a first dosage unit form that includes factor Miia and a second dosage unit form that includes factor XIII and, optionally, one or more other dosage unit forms that include factor MI! and/or inhibitor

TERI. In this case, the Ma factor and the XII factor are administered sequentially, in the optimal version - with an interval of approximately 1-2 hours between them, for example, within 30 minutes between them, better - within 10 minutes, even better - within 5 minutes between them . Either of these two dosage unit forms may be administered first.

Since the present invention relates to the prevention or treatment of bleeding attacks or to the provision of coagulation by treatment with a combination of active ingredients that can be administered separately, the invention also relates to the combination of separate pharmaceutical compositions in the form of a kit. The kit includes at least two separate pharmaceutical compositions. It also includes packaging for individual formulations, such as a 7/0 septum vial or a septum foil bag. As a rule, the kit includes instructions for the introduction of individual components. The kit form is particularly advantageous when it is preferred to administer the individual components in different dosage forms, with different intervals between doses, or when the physician considers it desirable to titrate the individual components of the combination.

Analyzes

Test for the activity of factor Ma:

A suitable assay for Miia factor activity testing, and therefore the selection of suitable Miia factor variants, is carried out as a simple preliminary IP migo test:

IP migo hydrolytic analysis

Natural (wild-type) factor Ma and a variant of factor Miia (hereinafter both referred to as "factor Miia") 2o Can be assayed for specific activity. They can also be assayed in parallel for direct comparison of their specific activity. The analysis is carried out in a microtiter tablet (Mahibogr, Mips, Denmark). The chromogenic substrate O-Pe-Pho-Ago-p-nitroanilide (5-2288, SPpgotodepih, Sweden), final concentration of MM, is added to the Miia factor (final concentration of ТО0О0НМ) in 5 ОМММ Nerez, pH 7.4, containing 0.1 M Masi, 5mm Sasi" and mg/ml bovine serum albumin. Optical density at 405 nm is continuously measured in a ZresigaMach 340 plate reader (Moiesschag Oemisez, USA). From the value of the optical density detected during the 20-minute incubation, the value of the optical density in i) a clean well that does not contain the enzyme is subtracted, and the difference is used to calculate the ratio of the activity indicators of the variety and the natural factor Miia:

The ratio - (Addition of the type of Miia factor)X Addition of the wild-type Miia factor). On the basis of this equation, variants of the Miia factor with activity approximately equal to or higher than the natural Miia factor can be identified, for example, varieties in which the ratio between the activity of the variant and the activity of the natural Miia factor, as shown in Fig. 1, is approximately 1.0, but not more. "-

The activity of factor Miia or varieties of factor Miia is also measured using a physiological substrate, such as factor X, in the optimal version - at a concentration of 100-1000nM, and the produced factor Xa ise) measured after adding a suitable chromogenic substrate (for example, 5-2765). In addition, the analysis of its activity can be carried out at physiological temperature.

The ability of factor Miia or variants of factor Ma to generate thrombin can also be measured by an assay that includes all relevant coagulation factors, inhibitors at physiological concentrations (minus factor Miia to mimic hemophilia A conditions), and activated platelets (as described on page 543 of

Mopgoe her a. (1997) Vgyii. U. Naetayi. 99, 542-547, to which the authors refer). with c Test for the activity of the KIP factor!: . A suitable analysis with a test for factor XII transglutaminase activity, and therefore the selection of suitable and? varieties of factor XII, are carried out as a simple ip migo test, as described, for example, in the work of Meiyoadz oi Epgutoiodu, vol. 45 (1976), Rgoieomis Epgutevs, ch. V, p. 177-191 (Ed. I ogapa, |).

The present invention is further illustrated by the following examples, which, however, should not be construed as limiting the scope of protection. The features described above and the examples below may, either individually or in any combination, be essential to an understanding of the invention in its various forms. me) Examples - Example 1.

The XII factor enhances fibrin salinity caused by the Miia factor. o Normal human plasma (NHP) was diluted 1/10 in a buffer containing 20 nM HERE5, 150 mM Masi, 5 mM

F Sasi", pH 7.4, in a microtiter well (total volume 25 Ωcl) and fibrin salinity was monitored by an increase in optical density at bOOnm in Zresgat 340 (Moiessag Oemisez, Zippumaie SA).

Spontaneous formation of a clot was achieved in approximately 2500-300 seconds. Fig. 1 shows that 1OnM of recombinant Miia factor (TEMIPa) (Momo MogaizK A/5 Wadzuaega, Denmark) reduced the time of clot formation to 1600 seconds (p-2). A further reduction in clot formation time was achieved when CP factor ZONM was added

F) (EX) (Ategisap Oiadpoviisa ips, Sgeepzhisp, ST) together with TONM gEMiIIa (p-3). The clot formed in the presence of EHIA was clearer (had a lower maximum optical density) than in its absence, indicating that the addition of EHIA provides a finer fibrin gel structure with thinner fibrils. in Example 2.

The presence of additional factor Xi during clot formation caused by factor Ma provides increased resistance to fibrinolytic breakdown.

A fibrin clot that consists of thin fibers is mechanically stronger and more difficult to disintegrate than a clot that contains the same amount of fibrin but consists of thick fibers or fewer b5 cross-linked fibers. The results of the experiment, shown in Fig. 2, indicate that additional EH (ZONM) prolongs the time of fibrin lysis of clots formed in the presence of EMIP and tissue plasminogen activator (EPA, Ategisap Oiadpoisis). Clot formation was initiated in the presence or absence of ZONM

EHIIA by adding 25 μl of MNR to 225 μl of 20NM NEREB, 150mM Masi, 5mm Sasi", pH 7.4, containing 50OHM

HEMIa and 0.5 nM of recombinant I-RA. Clot formation and subsequent clot lysis are caused by mediated

ERA by plasminogen activation was observed with the help of Zresigat 340 at bOOnm as an increase in ОЯ vornm. With the subsequent return of the trajectory to the base level. Figure 2 shows that the time of clot lysis under these conditions was significantly extended due to the presence of EHIPIII.

Example 3.

Factor MPa in combination with factor XII increases the maximum hardness of the clot and resistance of the clot to 7/0 fibrinolysis.

Thromboblastographic measurements were performed on normal human plasma to which bnM of recombinant tissue-type plasminogen activator (-RA, Ategisap Oiadpowiisa) was added, and to analyze the effect of adding 1NM hEMiia (Momo MogaizkK A/5, Wadzuaega, Denmark), alone or in combination with different concentrations factor XII (EH, Naeptaioiodis Thesppoiodieg, NOSHP-0O160, oi M1212). Salinity was induced by adding 7/5 Innovin (Ippomip) (final concentration of 2000-fold dilution, OSade Vepygipd Zh 526945) and calcium (final concentration 15 mM) in 20 mM NERE5 buffer, 150 mM mass, pH 74.

Thrombelastographic measurements were used to analyze the effect of heMIP and EHIIA on maximum clot hardness (CME), as well as clot resistance to Y-RA mediated lysis. Before the addition of hEMIIa and/or EHIIA, the achieved MCE was 25 mm, and the time required to lyse half of the clot was 12.3 minutes (Figure 3). Addition of increased concentrations of EHIIA (0-4 ONM) did not change MSE; however, a dose-dependent prolongation of half-clot lysis time was observed, and optimal data were observed with ZONM

EX (time of lysis of half of the clot: 14.3 min, Fig. 3). Likewise, the addition of hEMiia (INM) provides protection of the clot from mediated I-RA fibrinolysis (lysis time of half of the clot 16.4 min) without any effect on MSE (Fig. 3). However, after adding hEMiIIa (1NM) together with EHI! (ZONM) observed an increase in MSE (29mm), as well as a reliable protection against fibrinolysis (time of lysis of half of the clot 27.uh) (Fig. 3). Together, these results demonstrate that the addition of hEMiIa and EHIIA to plasma synergistically improves the mechanical strength of the clot and i) resistance to I-RA-mediated fibrinolysis.

Claims (21)

Formula of the invention at 30 o 1. Pharmaceutical composition for intravenous administration, which contains factor MiIIa and factor XII. "- 2. The composition according to claim 1, which differs in that the MIIa factor is a variant of the MI factor. 3. The composition according to claim 1, which differs in that the Ma factor is a human Ma factor. is), 35 4. The composition according to claim 1 or clause 3, which differs in that the Ma factor and the ХІІІ factor are human recombinant factor МІІІa and recombinant factor ХІІ! a person 5. The composition according to any of claims 1-4, which is characterized by the fact that factor XII is a2-dimer of factor HIPI. 6. The composition according to any of claims 1-5, which is characterized by the fact that the HII factor is an activated factor " XII. w-v c 7. The composition according to any of claims 1-6, which is characterized by the fact that it additionally contains a TERI inhibitor. 8. The composition according to any of claims 1-7, which is characterized by the fact that it additionally contains the MIP factor. :with" 9. A kit for intravenous administration, which contains a drug for the treatment of episodic bleeding, containing a) an effective amount of factor Miia and a pharmaceutically acceptable carrier in a first unit dosage form; - p) an effective amount of factor XIII and a pharmaceutically acceptable carrier in the second single dosage form, and c) a container for placing the specified first and second dosage forms in it. (22) 10. The kit according to claim 9, which is characterized by the fact that it additionally contains an effective amount of a TERI inhibitor and a pharmaceutically acceptable carrier, in a third unit dosage form. 11. The kit according to claim 9, which is characterized by the fact that the first single dosage form or the second single dosage form additionally contains a TERI inhibitor. F 12. The kit according to any of claims 9-11, which is characterized by the fact that it additionally contains the MI factor! or manufactured in the form of a separate unit dosage form, or as part of a unit dosage form that also contains one or more compounds selected from the group consisting of factor Miia, factor XII! or a TERI inhibitor. 13. Use of factor MPa in combination with factor XIiY for the manufacture of a medicinal product for (F) treatment of episodic bleeding in a subject, and the medicinal product is intended for intravenous GI administration. 14. Application according to claim 13 for the manufacture of a medicinal product for reducing the time of clot formation in a subject. 15. Application according to claim 13 for the manufacture of a medicinal product for prolonging the lysis time of a clot in normal plasma of a mammal. 16. Application according to claim 13 for the manufacture of a medicinal product for increasing the strength of a clot in normal mammalian plasma. 65 17. Application according to claim 13 for the manufacture of a medicinal product to enhance the formation of a fibrin clot in normal human plasma. 18. A method of enhancing the formation of a fibrin clot in a subject, which includes administering to the subject an effective amount of the Miia factor in combination with an effective amount of the CP factor. 19. A method of treating episodic bleeding in a subject, which includes administering to the subject an effective amount of the Miia factor in combination with an effective amount of the HIPI factor. 20. The method according to claim 18 or claim 19, which differs in that the Ma factor and the XII factor are administered in the form of a single dosage form. 21. The method according to claim 18 or claim 19, which differs in that factor Ma and factor XII! administered sequentially. 6) (Se) «c) «- (Se) and - - with . and? -I (o) - r SHY 4) name) 60 b5