DK156699B - Use of factor VIIa for producing a therapeutic composition for treating bleeding disturbances - Google Patents

Description

1 DK 156699 B

The present disclosure relates to the use of factor VIIa in the manufacture of a pharmaceutical composition for the treatment of patients suffering from bleeding disorders, such as platelet defects including thrombocytopenia, von Willebrand's disease, and others typically present in associated with severe tissue damage. According to the present invention, factor VIIa can also be used to prepare a composition for the treatment of gastrointestinal bleeding and oral oral disorders, even in cases where no baseline haemostatic disorders have been diagnosed.

Uncontrolled and violent bleeding is a major problem in both surgery and various types of tissue damage. Bleeding disorders may be due to coagulation factor defects or coagulation factor inhibitors (hemophilia A and B). However, bleeding disorders can also be observed in patients who do not suffer from haemophilia A or B, e.g. in patients with von Willebrand's disease. Patients with von Willebrand's disease have defective primary hemostasis as they lack or have an abnormal von Willebrand 20 factor. Bleeding disorders are also seen in patients with a normally functioning blood clotting cascade and may be due to defective platelet function, thrombocytopenia or even unknown factors.

Blood coagulation is mainly induced by the conversion of the soluble plasma protein fibrinogen to insoluble fibrin catalyzed by the enzyme thrombin. The blood components that participate in the coagulation cascade are proenzymes or zymogens, enzymatically inactive proteins which are converted to proteolytic enzymes under the action of an activator which is itself an activated coagulation factor. Coagulation factors that have undergone such transformation are commonly referred to as "activated factors" and are denoted by the addition of a lowercase letter "a" (e.g. Vlla).

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There are two separate systems that can promote blood coagulation. These systems are referred to as the internal and external coagulation systems. In the internal system, only factors found in plasma are used. In internal system 5, the activation of factor IX to factor IXa is a reaction catalyzed by factor X1a and calcium ions. Factor IXa then participates in the activation of Factor X to Factor Xa in the presence of Factor Villa, phospholipid and calcium ions. The external system also involves plasma factors and components found in tissue extracts. Factor VII, one of the aforementioned proenzymes, participates in the external blood coagulation system by (after its conversion to factor VIIa) converting factor X to factor Xa in the presence of tissue factor and calcium ions. Thereby, factor Xa converts prothrombin to throm-15 bin in the presence of factor Va, calcium ions and phospholipid.

Since the activation of Factor X to Factor Xa is an event common to both the internal and external systems, Factor VIIa can be used to treat patients with defects in or inhibitors of Factor VIII (U.S. Patent No. 4,382,083) and factor VIIa have been shown to be able to "bypass" the initial phase of the coagulation cascade of haemophilia A patients with antibodies against factor VIIIC, Hedner and Kisiel, J.Clin.Invest., 71: 1836 - 1841 , 1983.

Thrombocytopenia, defined as "a reduced number of circulating platelets", is a common clinical problem associated with various disorders and complicated conditions where various factors contribute to the low platelet count. Decreased platelet counts will result in an increased bleeding tendency which appears as mucosal bleeding from e.g. the nasal-oral or gastrointestinal tract, as well as ulceration, ulceration and injection sites. Thrombocytopenic bleeding can be extensive and cause serious problems both during and after surgery. Even 35 minor surgical procedures such as tooth extractions may result

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severe bleeding. Furthermore, spontaneous intracranial hemorrhages may occur at extremely low platelet counts (less than 10 x 109/1).

A low number of circulating plates may be the result of: (1) a production defect, (2) an abnormal distribution, (3) a dilution loss (massive blood transfusion), or (4) an abnormal destruction.

A defective production of bone marrow plates may be the result of various conditions including effects of toxins such as radiation, cytostatic drugs, certain drugs, etc., tumor formation (metastatic cancer and leukemia) or degenerative processes of unknown origin (often associated with anemia or other bleeding disorders). An abnormal distribution of platelets is seen in association with hematological disorders (leukemia, myeloma, lymphoma), liver disease, tumors, etc. In these situations, the platelets may be accumulated in an enlarged spleen or liver, thereby avoiding circulating blood. Massive blood transfusion without special supply of fresh platelets will result in a diminished concentration of platelets in the circulating blood and is thought to be the cause of thrombocytopenic hemorrhages which can occur in such situations. An abnormal platelet destruction may be the result of: (1) increased consumption in cardiac transplants or in traumatized tissues or (2) an immune response seen in drug-induced thrombocytopenia, idiopathic thrombocytopenic purpura (ITP), autoimmune disease, hematoma disorders (leukemia, lymphoma) etc.

Platelets are important for primary hemostasis by causing the formation of a primary hemostatic plug, which is then enhanced by the activation of the coagulation cascade and the formation of fibrin. Platelets usually provide coagulation factors, including Factor V, Factor V1, and fibrin as well as phospholipids, which are necessary in initiating local hemostasis.

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In patients with thrombocytopenia, the normal coagulation cascade is disabled due to a lack of initiation of the primary stages of the coagulation cascade. The treatment of these patients has significant difficulties. Patients with thrombocytopenia are now most commonly treated by donor platelet concentrate administration. Such concentrates consist of mixed platelets from 5-6 donors. Most recipients of repeat platelet transfusions develop antibodies to platelet antigens, resulting in a poor or complete failure of additional platelet transfusions. There are no treatment options available for these patients yet.

Defective platelet function is quite common in both congenital disorders (Glanzmann's thrombasthenia, other congenital forms of thrombasthenia, platelet aggregation defects) and as a complication of a variety of diseases such as leukemia, dysproteinemia (eg myeloma), autoimmune disorders (eg, autoimmune disorders) -toid arthritis, systemic lupus erythematosus etc.) and uremia. Patients with defective platelet function may develop mucosal bleeding most frequently as described above in connection with patients with thrombocytopenia. For surgery, these patients also need treatment to avoid severe bleeding. Currently, antifibrinolytic treatment (tranexamic acid, epsilon-aminocaproic acid) is used alone or in conjunction with desmopressin (DDVAP), a vasopressin analog. However, desmopressin also has cardiovascular effects that cause vasoconstriction. This renders the drug unsuitable for treating patients suspected of having cardiovascular problems.

30 Patients with von Willebrand's disease have a defective primary hemostasis because they lack or have an abnormal von Willebrand factor. Therefore, patients with von Willebrand's disease have mucosal bleeding both from the nasal-oral and gastrointestinal tract. The patients who have the most serious forms of von Willebrand's disease also suffer

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bleeding from the joints. In patients with von Willebrand's disease, one factor may be beneficial, which is capable of causing hemostasis by circumventing the initial hemostatic steps.

As a result, there is a need for an improved method for treating patients with defective platelet dysfunction, as well as for treating patients suffering from thrombocytopenia and von Willebrand's disease, which method does not have the undesirable side effects and drawbacks that are characteristic of previous treatments. The present invention meets this need and further provides other related benefits, including the treatment of gastrointestinal and nasal-oral bleeding, even in cases where specific haemostatic disorders have not been diagnosed.

The present invention thus relates to the use of factor VIIa for the preparation of a composition for the treatment of patients suffering from bleeding disorders not caused by coagulation factor defects or coagulation factor inhibitors, which composition contains an effective hemostatic amount of factor VIIa.

The composition may also comprise a physiologically acceptable carrier or thinner or an adjuvant. Suitable excipients include albumin, calcium, non-reducing sugars, polyalcohols, polysaccharides and antioxidants.

The composition described herein is particularly effective in treating patients suffering from thrombocytopenia, von Willebrand's disease, as well as other platelet defects. In addition, the composition can be used to treat patients suffering from gastrointestinal or nasal-oral bleeding.

In a preferred embodiment, the composition is administered intravenously and in an amount of from ca. 100 units to 1000 units, more preferably 100 to 500 units of factor Vlla per kg body weight. Preferably, the composition is administered over a period of approx. 24 hours.

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Preferably, the composition described herein will contain purified factor VIIa at a concentration of at least 25 µρ / µπ.

It will be appreciated by those skilled in the art that in order to facilitate administration it will be preferable to use a concentration of factor V1a of approx. 25 μg / ml - 500 μρ / πιΐ, and more preferably a concentration of 25 μρ / πιΐ - 200 μg / ml, although considerably higher concentrations can be used. Use of concentrations as described above allows for 10 appropriate doses of 1 to 5 ml per dose.

Other aspects of the present invention will be readily apparent with reference to the following detailed description.

The composition may contain non-activated factor 15 VII and other non-activated blood coagulation factors such as factor IX, which may enhance the activity of factor VIIa. The Factor IX concentration should preferably be within a range corresponding to a given dose of approx. 10 units per kg body weight. It is preferred that factor VIIa is not accompanied by 20 blood coagulation factors other than factor IX.

Human purified factor VIIa is mainly prepared according to the methods described by Broze and Majerus, J. Biol.Chem. 255 (4): 1242 - 1247, 1980 and Hedner and Kisiel, J. Clin. Invest. TJLi 1836 - 1841, 1983. These methods give factor VII without detectable amounts of other blood coagulation factors. An even further purified Factor VII preparation can be obtained by introducing an additional gel filtration as the final purification step. Factor VII is then converted to activated factor V1a in a known manner, e.g. using various plasma proteins, such as factor XIIa, IXa or Xa. As described by Bjoern et al. (Research Disclos- ure Hours, 269, September 1986, p.

564 - 565), factor VII can also be activated by passing it through an ion exchange chromatography column, such as Mono Q® (Pharmacia Fine Chemicals) or the like. It will be obvious to one skilled in the art that a suitable factor VIIa for use in the present invention may also be prepared by DNA recombination.

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nant technology, i.e. by inserting cDNA or the gene encoding Factor VII (Hagen et al., Proc.Natl.Acad.Sci USA 83: 2412-2416, 1986) into a suitable vector, transforming suitable cell lines with the vector and culturing the transformed cells in a suitable medium, after which the expressed product is isolated and activated to factor VIIa. Factor VIIa produced by DNA recombinant technique may be authentic factor VIIa or a more or less modified factor VIIa, provided that such a modified factor VIIa has essentially the same biological activity for blood coagulation as authentic factor VIIa. Such a modified factor VIIa can be produced by modifying the DNA sequence encoding factor VII either by altering the amino acid codons or by removing some of the amino acid codons in the natural gene in a known manner, e.g. by site-specific mutagenesis.

It is obvious that the practice of the method described herein is independent of how the purified factor VIIa is prepared, and the present invention thus encompasses the use of any factor VIIa preparation for the preparation of a composition for treating the Claim 1, specified coagulation disorders.

Factor VIIa has been shown to be able to stop bleeding in patients who have virtually no circulating platelets. Purified Factor IIa was injected into rabbits made thrombocytopenic by anti-platelet serum, and experiments showed that small amounts of purified human Factor VIIa effectively stopped bleeding in the thrombocytopenic animals. Therefore, factor VIIIa is able to bypass the primary hemostasis and can cause local hemostasis without platelet involvement and the initial coagulation phase. Factor Vlla has also been shown to cause local hemostasis in people suffering from thrombocytopenia.

Patients suffering from widespread tissue damage with a massive cell destruction may develop complex hemostatic disorders as a result of the release of various

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enzymes from the destroyed cells. Such enzymes can influence both the coagulant and fibrinolytic systems and cause the breakdown of several factors involved in one or the other system. It may also be advantageous to use factor VIIa for these patients because the ability of factor V1a to cause the formation of a haemostatic plug by activating the last phases of the coagulation system. In this regard, treatment involving the use of factor VIIa may be by intravenous injection or topical application and may be combined with anti-fibrinolytic therapy. Furthermore, in many cases of bleeding, e.g. gastrointestinal or nasal-oral bleeding or during surgery may be appropriate to use factor VIIa at substantially the same concentrations as described herein and thereby cause local hemostasis. Factor VIIa can also be used locally or intravenously in these situations.

Factor VIIa is preferably administered by intravenous injections and in an amount of approx. 100 to 1000 units / kg of body weight, and preferably in an amount of approx. 100 - 500 units per kg body weight corresponding to approx. 2-5 µg / kg / dose, which may need to be given again 2-4 times over 24 hours.

The term "unit" as used in this specification is defined as the amount of factor VII present in 1 ml of normal plasma corresponding to ca. 0.5 µg protein.

After activation, 50 units correspond to approx. 1 µg protein.

The term "a haemostatic effect" or "amount" as used in this specification is defined as a significant opher of bleeding within 15 minutes of administration of about 30 minutes. 100 - 1000 units per kg body weight of pure factor VIIa.

The present invention further relates to the use of factor VIIa for the preparation of a pharmaceutical composition for treatment disorders, in which factor VIIa, preferably in purified form, is admixed with suitable auxiliaries or a suitable carrier or thinner. Suitable physiologically acceptable carriers or thinners include sterile

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water and salt water. Acceptable excipients in this context include calcium, albumins or other inactive proteins to stabilize the purified factor VIIa. Other physiologically compatible excipients are non-reducing sugars, 5 polyalcohols (such as sorbitol or glycerol), polysaccharides such as low molecular weight dextrins, amino acids and antioxidants (such as bisulfite and ascorbate). The adjuvants are generally present in a concentration of from 0.1 to 3% w / v.

The pharmaceutical composition may also contain protease inhibitors, e.g. aprotinin. In a particularly preferred embodiment, calcium is used in combination with other selected adjuvants in the pharmaceutical composition. The amount of calcium is preferably 5 - 50 mM and more preferably 10-20 mM.

The following examples illustrate the effect of compositions containing factor VIIa.

Example 1

Rabbits were rendered thrombocytopenic by administration of sheep antibodies to platelets from rabbits prepared according to the methods described by Busch et al., Acta Chir.Scand., 140: 255, 1974. Hemostatic plug formation in the rabbits' mesenteric microcarts was investigated according to the procedure described. by Bergqvist and Arfors, Thromb.Diathes.Haemorrh., 30: 586, 1973. For each observation, three arterioles and three small veins (diameter 20 - 40 μπι) were cut and the time until hemostatic plug formation was measured and the frequency of post-bleeding. noted. The time needed for primary haemostatic plug formation was defined as "the period between cut-off and the first stop in bleeding". The sum of 30 and all post-bleeding times is called "total haemostatic plug formation time" (THT). The platelet count of the rabbits decreased to a minimum of 15 - 60 minutes after administration of the anti-platelet serum and remained low throughout the observation period. In the control group, a decrease of 263 x 10/1 9 occurred

DK 156699B

(mean) to 10 x 10/1 (mean). Before platelet antibodies were administered, THT in the arterioles (THT-A) showed a mean of 54 s. A more than threefold extension of THT-A was observed for 179 as well as 15 minutes as 60 minutes after administration of antibody. THT in the small veins (THT-V) ranged between 202 and 394 s (mean 274 s) before antibody administration. In parallel with the extension of THT-A, an extension of THT-V (mean 768 s) was observed 15 minutes after antibody administration, and it remained relatively constant throughout the 10 observation time.

Three rabbits were made thrombocytopenic in the same manner as the control group, and human factor VIIa (50 units per kg body weight) was administered (30 minutes after antibody). Ten minutes after the injection of factor VIIa, the 15 THT-A values showed a significant decrease in each rabbit (mean 114 seconds, average after antibody administration 260 seconds before factor VIIa). However, the decrease was transient (average 30 minutes after factor VIIa was 219 s). THT-V showed a similar sample with a decrease 10 minutes after factor VIIa (average of antibody administration 698 seconds and 10 minutes after factor VIIa 499 seconds). The minor decrease was transient, and 30 minutes after the factor IIa injection, THT-V showed an average of 672 s.

A further 5 thrombocytopenic rabbits were then given 25 twice as much factor Vla (100 units per kg body weight) and 10 minutes after administration of factor Vla, THT-A showed a marked decrease from an average of 256 s after antibody administration to an average of 89 s in 10 minutes. by factor V11a. This decrease lasted throughout the observation period. THT-V 30 clearly decreased after injection of factor VIIa (from a mean of 591 s after antibody administration to a mean of 390 s). After 30 minutes, normalization of THT-V (average 255 s) and the same sauce occurred after 60 minutes (mean 299 s).

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No effect was observed on the THT values of 5 non-thrombocytopenic rabbits. When factor VII was administered instead of factor VIIa, no effect on THT values was seen.

5 The results are shown in the following table.

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DK 156699 B

The present experiments demonstrate the important role of factor VIIa in initiating the coagulation process in vivo. By contrast, factor VII had little effect in this process, if any at all. Furthermore, factor VIIa was able to initiate the coagulation process even in the absence of platelets. As a result, the phospholipid, which is usually delivered at the damaged site by the platelets, is made available by the damaged endothelial cells which also provide the tissue factor.

Example 2

Treatment of two patients with thrombocytopenia using pure factor VIIa

Two patients with haematological disorders (respectively Waldenstrom macroglobulinemia and chronic lymphocytic leukemia) complicated with severe thrombocytopenia (platelet count 9 less than 10 x 10/1) received factor IIa, purified from human plasma on essentially that of Hedner and Silicon (supra) described.

The first patient was treated for severe nasal bleeding. The bleeding time (BT) according to Duke was greater than 15 minutes for the injection of factor VIIa as a result of severe thrombocytopenia. A dose of 100 units / kg body weight (2 μg / kg body weight) was given intravenously, and 15 minutes after the end of the injection, 25 Duke BT was normalized (4 minutes, normal range: less than 5 9

minutes). The plate number remained the same (10 x 10/1) throughout the observation time. The nasal bleeding stopped immediately and the solidified blood could be removed without bleeding. A minor bleeding started again later but stopped spontaneously. No effect on pus, temperature or blood pressure was observed. The factor VII level in plasma increased from 0.66 units / ml to 2.07 units / ml and was again 0.60 8 hours after injection. There was no effect on the plasma level of factor X

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(1.12 units / ml before and 1.12 units / ml after injection) soin remained the same through 8 hours of observation time. No fibrin / fibrinogen degradation products appeared in the blood circulation and the ethanol gelation test remained negative.

Furthermore, no changes were seen in ATIII or OAP.

Patient # 2 had a plate number less than 9 10 x 10/1 and a Duke BT greater than 15 minutes for the injection of factor V11a. The patient was bleeding profusely from Duke blood sampling in the ear, a bleeding that had to be stopped 10 manually by compression and topical application of thrombin. Pure factor VIIa was administered intravenously at a dose of ca. 100 units per kg body weight (2 μg / kg body weight), and Duke BT was ground 15 minutes after the end of the injection. Duke BT was then 10 minutes and the formation a visible coagulation saw 15 at the incision site. There was no change in plate number and no effect on pus, blood pressure or body temperature. The plasma level of factor VII increased from 0.57 units / ml to 2.17 units / ml. There was no change in the factor X level (0.73 units / ml before and 0.81 units / ml after injection) and no change in ATIII or α 1 AP.

All in all, intravenously injected purified factor Vlla shortened the prolonged BT in patients with severe thrombocytopenia. In parallel, there was an increase in the plasma level of factor VII. There was no evidence of a general effect on the coagulation mechanism.

Claims (11)

DK 156699B Use of Factor VIIa for the preparation of a composition for treating patients with coagulation disorders not due to coagulation factor defects or coagulation factor inhibitors, the composition comprising Factor IIa in an effective hemostatic amount. Use according to claim 1 for the treatment of patients with thrombocytopenia or platelet disorders, von Willebrand's disease or tissue damage. Use according to claim 1, characterized in that the composition contains purified factor Vlla at a concentration of at least 25 μg / ml. Use according to claim 1, characterized in that the hemostatic amount comprises from approx. 100 units to 1000 units factor VIIa per kg body weight, preferably from approx. 100 to 500 units factor Vlla per kg body weight. Use according to claim 1, characterized in that the composition further comprises factor IX. 16 DK 156699 B Use according to claim 1, characterized in that the composition further comprises a physiologically acceptable carrier or diluent and an adjuvant. Use according to claim 6, characterized in that the excipient is calcium. Use according to claim 6, characterized in that the excipient is selected from the group consisting of albumin, non-reducing sugars, polyalcohols, amino acids, polysaccharides and antioxidants. Use according to claim 1, characterized in that the composition contains calcium together with one or more excipients selected from the group consisting of albumin, non-reducing sugars, polyalcohols, amino acids, polysaccharides and antioxidants. »S r,