CA2184226A1 - Pharmaceutical composition for the treatment of blood coagulation diseases, methods for the production thereof and its use - Google Patents
Pharmaceutical composition for the treatment of blood coagulation diseases, methods for the production thereof and its useInfo
- Publication number
- CA2184226A1 CA2184226A1 CA002184226A CA2184226A CA2184226A1 CA 2184226 A1 CA2184226 A1 CA 2184226A1 CA 002184226 A CA002184226 A CA 002184226A CA 2184226 A CA2184226 A CA 2184226A CA 2184226 A1 CA2184226 A1 CA 2184226A1
- Authority
- CA
- Canada
- Prior art keywords
- factor
- pharmaceutical composition
- coagulation
- factor viia
- feiba
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6437—Coagulation factor VIIa (3.4.21.21)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/647—Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
Abstract
The invention relates to a pharmaceutical composition for the treatment of patients with blood coagulation diseases which are caused by coagulation factor deficiency and/or inhibitors of coagulation factors, whereby the composition has a FEIB-activity and is characterized in that it has Factor VIIa and at least one further active ingredient and the activity of at least 10 Factor VIIa units per unit FEIBA. Additionally, the invention comprises a method for the production of the pharmaceutical preparation and its use.
Description
218~226 , .
ph~ utical composition for the treatment of blood coagulation diseases, methods for the production thereof and its use.
The invention relates to a pharmaceutical composition for the treatment of blood coagulation disorders, especially of :
Factor VIII inhibitor patients. The invention further relates to a method for production of a composition of this type as well as its use.
Blood coagulation is triggered by a series of successive reactions of various proteins and/or enzymes, The formation of fibrin from fibrinogen and, therewith, the closure of wounds is prevented by a lack of blood coagulation factors; ~ -:
the consequence ls hemorrhage. Such a case exists with hemophilia A. This is the most widespread bleeding disorder and is caused by a deficiency in Factor VIII. Factor VIII
containing preparations are used for replacement therapy in hemophilia A. Treatment with these preparations leads to a -rapid cess-ation of bleeding in most cases.
However, there are also patients among which not only a deficiency in Factor VIII occurs, but also who have developed an inhibitor directed against Factor VIII. A further collection of patients posses Factor VIII inhibitors without suffering from hemophilIa A. Depending on the amount of factor A inhibitors present, the effect of administered Factor VIII is inhibited by its neutralization.
Presently, preparations based on a plasma fraction which contains a mixture of coagulation factors are offered for treatment of Factor VIII inhibitor patients. This plasma fraction can contain, for example, factors of the prothrombin , 218g226 complex (Factor II, VII, IX, X). A blood coagulation promoting preparation with Factor VIII inhibitor bypass activity (FEIBA ~) TIM 4, Immuno AG) is obtained, according to AT-0 368 883 for example, by treatment of cryosupernatant.
This preparation also contains coagulation Factors II, VII, IX, X.
The action of a FEIBA preparation is complex due to lts complex composition. Mariani et al (Thromobosis Res. 31, 475-488, (1983) ) mentions Factor VII in its activated form as a principle of action. It was established that a higher content of Factor VIIa in plasma occurs in hemophiliacs after infusion of a FEIBA preparation.
Equally, the role of Factor VIIa in prothrombin complex concentrates with a "Factor VIII-bypassing-activity" is discussed by Teitel (Thrombosis and Haemostasis 66 (5) 559-564, (1991) ) . Simultaneously, the principle of action of Factor Xa in preparations of this type is also dealt with.
The examined prothrombin complex concentrates contained ~:
Factor VIIa, expressed by the ratio of Factor VII activity to Factor VII antigen of 2.1 and 2.5.
The therapeutic composition containing prothrombin produced according to EP-0 044 343-B1 is suitable for treatment of coagulation factor inhibitors and contains an activated prothrombin complex in which the factors are partially activated. The amount of Factor VIIa is between 8-80 units per ml. Factor IX concentration is in the range of 15 to 112 units per ml. Correspondingly, the content of Factor VIIa, with respect to Factor IX, is 0.07 - 5.3 U Factor VIIa/U
Factor IX. Vinazzer (Thromobosis Res. 26:21-29 ~1982) ) demonstrates the difference of the preparations AUTOPLEX, which is produced according to EP-44 343 and FEIBA. As shown there, AUTOPLEX is distinguished by the higher content of thrombin (Factor IIa) measured in NIH units in comparison to FEIBA (see table 1, page 24).
However, highly purified Factor VIIa preparations are also proposed for therapy of coagulation factor inhibitor =
conditions (for example, EP 0 082 182-B1) and Hedner et al.
(Haemostasis 19, 335-343 ~1989) ) .
A method for the production of a concentrate which contains highly purified Factor VIIa is also described in EP 0 547 932-A1. Factor VII is separated from coagulation Factors II, IX and X by anion exchange chromatography of a cryoprecipitate. Factor VII is then subjected to a further purification and activation, whereby the addition of exogenous proteins is avoided. Subsequently, Factor VIIa is treated i-or inactivation of viruses by a treatment with an organic solvent/detergent (TnBP/Tween).
The risk of transmitting infectious agents exists by the use of plasma or a plasma fraction as the starting material for the production of pharmaceutical preparations. Despite selection of donors and tests of the individual plasma donations for possibly present viruses, such as HIV, hepatitis B or hepatitis C virus, the possibility exists that a plasma pool is infectious. This residual infectivity is not to be ruled out because of limited sensitivity of the test systems, but also because of the incubation time until the appearance of measurable infection markers (diagnostic window ) .
' ~ 218g226 Pharmaceutlcal preparations whlch are prepared from plasma or a plasma fraction are therefore su~:lected to diverse treatments for inactivation of viruses potentially present. A
proven method for the inactivation of mernbrane enveloped as well as non-membrane enveloped vlruses is described ln EP 0 159 311-Bl. T}~ereby, a pharmaceutical preparation 18 heated ln a solld state after ad:lustment of a water content to a value of 5-70%. T~lis treatment is also described as a steam (vapor) treatment. Instead of water, methanol or ethanol can also be used.
However, combinatlons of vlrus inactivation methods are equally possible. According to 3P 0 519 901r a treatment wlth highly concentrated tensides is combined wlth a heat t reat ment .
An alm of the lnvent lon 18 to provlde an lltlproved pharmaceutical composltlon for treatment o~ blood coagulation disorders, e~pecially of Factor VIII $nhlbitor patlents as well as a simple method for its production.
The alm 18 achleved according to the invent ion by a pharmaceutlcal composltion with FEIB-activity which contains Factor VIIa and Qt least one furt~-ler active ingredlent and has the activity of 10 Factor VIIa units per unlt FEIBA.
The lnvention also provides corQmercial packages comprlslng such pharmaceutlcal composltlons together wlth instructions for use therefor in rapid termination of hemorrhage in a patlent wlth a coagulation factor deficiency.
The content of Factor VIIa in the compositlon 218~226 .
4a accordlng to the lnventlon is at least 10, preferably 10 to 100, most preferably 10 to 20, especially 10 to 15, units per unlt FEIB-actlYlty. The F~IB-activity i8 determlned by means of a method accordlng to AT-350 726.
218~226 It has turned out that a preparation which contains highly purified Factor VIIa as the sole effective substance does not possess any FEIB-activity. Therefore, it was surprising that addition of Factor VIIa to a preparation with FEIB-activity, such as one of the activated prothrombin complex containing fractions, contributes to a substantial improvement of the effectiveness of a pharmaceutical preparation for the treatment of blood coagulation diseases, especially of Factor VIII inhibitor patients.
The content of Factor VIIa was determined in the following examples with the methods described by van Deij k ~Haemostasis 13: 192-197, 1983) and EP 0 547 932 with bovine thromboplastin. Additionally, Factor VIIa can also be determined by using recombinant, soluble tissue factor according to the methods of Morrissey et al. (Blood 81: 73g-744, (1983) ) and WO 92/18870.
Coagulation Factors II, IX and X and, additionally for example, tissue factor or Xa and phospholipids are to be named as active ingredients in a preparation of FEIB-activity. The above listing of substances in a preparation with FEIB-activity is not complete and, therefore, should not be considered as limiting.
The preparation according to the invention preferably contains coagulation Factors II, IX and X, preferably in a concentration which corresponds to a ratio of 0 . 5 - 2, particularly preferred 0 . 5 - 1. 5 U/U FEIBA for each factor .
The measurement is carried out according to the test description in AT-350 726.
~ 218~226 Therewith, the composition according to the invention differs from known preparations not only in its extraordinary high ratio of Factor VIIa to FEIBA, but also in its ratio of Factor VIIa to Factor IX. For example, the latter is distinctly higher than in EP-O 044 343.
It is of further advantage when the preparation according to the invention additionally contains protein C and/or protein S.
It was surprising that the preparation according to the invention can be produced in a simple and economic manner.
Therefore, the invention also comprises a production method which is characterized by contacting a fraction as illustrated above with an anion exchanger, whereby coagulation Factor VII is at least partially activated.
Conditions are selected thereby which allow for the simultaneous adsorption of Factor VII and/or Factor VIIa with coagulation Factors II, IX and X, for example, by using buffers of low ionic strength. Subsequently, the anion exchanger is separated, for example by sedimentation, and the fraction which contains coagulation Factors II, VIIa, IX and X is isolated. In this connection, the fraction already possesses FEIBA before contact and/or a FEIBA is generated in the fraction during this.
Preferably, a strong anion exchanger, for example a matrix with quaternary ammonium groups such as QAE, TMAE and Q
groups, is used as an anion exchange material. As a suppor~
for the anion exchange groups, cross-linked Dextran, such as Sephadex(~3 or Sepharose~ or synthetic material such as Fraktogel~3) is used.
` ~ 2184226 This method of production is distinguished, among others, by its economic procedure. The method has the advantage that coagulation Factor VII must not be separated at first in order to then be added to the preparation again.
Additionally, the coagulation factor-containing fraction is not only purified, but coagulation Factor VII is also activated in one step. Partial activation of Factor IX and Factor: X, but not of Factor II, also occurs at the same time.
The composition according to the invention is also obtainable by combining a fraction containing a purified prothrombin complex and a fraction containing purified Factor VIIa, and by a treatment for the inactivation of viruses. However, the pharmaceutical composition according to the invention can also be composed of a fraction containing a purified partial prothrombin complex (Factors II, IX and X) and a fraction containing purified Factor VIIa, whereby it is treated for the inactivation of viruses. The prothrombin complex used for the production of the composition according to the invention is preferably present as activated prothrombin complex or as a FEIBA preparation. In an activated prothrombin complex, coagulation Factors II, IX and X are partially activated. However, the activation of Factor II is to be avoided in such a preparation in order to directly exclude prothrombogenic activity.
By using certain conditions such as high ionic strength in the starting solution and/or in the washing buffer and strong anion exchange gels, for example Q-Sepharose(~ Fast Flow (Pharmacia), activated prothrombin complex can be isolated as a FEIBA preparation with higher yields from plasma or plasma supernatant after separation of the cryoprecipitate. Gels of this type then bind Factor VII or Factor VIIa relatively ~ 218~226 poorly. Therefore, Factor VIIa can be isolated from the same starting material in a further process with higher yields.
The fractions obtained are processed to pharmaceutical preparations in the customary manner, for example, by addition of pharmaceutically acceptable carriers and/or adjuvants, sterile filtered and lyophilized to storable pharmaceutical preparations.
The compositlon according to the invention can be produced by a further method with the following steps:
a) isolating coagulation Factors II, IX and X as individual components or as a mixture from one or more starting materials containing one or more of coagulation Factors II, IX and/or X, b) isolating coagulation Factors VII/VIIa from a starting material containing coagulation Factor VIIa and/or coagulation Factor VII, whereby coagulation Factor VII is at least partially activated, c) pretreating or treating the starting materials or the isolated coagulation factors for inactivation of infectious agents, and d) mixing the isolated coagulation factors together with a pharmaceutically acceptable carrier.
In a preferred embodiment, Factor VIIa is added to prothrombin complex, which can also be present as partial prothrombin complex, after purification of the components ard mixed with a pharmaceutically acceptable carrier, and subse~uently subj ected to a treatment for virus inactivation, for example a heat treatment.
The addition and/or content of highly pure Factor VIIa is especially advantageous because less contaminating proteins are introduced into the preparation as impurities therewith.
The specific activity of Factor VIIa in the preparation is preferably more than 10 U/mg to 1, 000 U/mg protein.
The prothrombin complex is preferably present as an activated prothrombin complex or FEIBA preparation.
Preferably, a heat treatment, for example a steam treatment, is carried out for inactivation of infectious agents (viruses). Thereby, it has turned out that a heat treatment of the coagulation factors in a solid state can be conducted without addition of stabilizing proteins, such as albumin.
Factor VIIa is surprisingly stable enough that it can also be treated in a purified state for inactivation of infectious agents .
The heat treatment for inactivation of viruses has the advantage that not only lipid coated viruses are inactivated, but also viruses which are not surrounded with a lipid envelope .
The method according to the invention is characterlzed in that Factor VIIa can be isolated from a fraction which accumulates as a by-product in the production of a fraction containing (partial) prothrombin complex. Therefore, this method is also particularly distinguished by its economic e f f i ciency .
With the method according to the invention, it is possible to obtain all components of the composltion according to the invention from a single starting material, preferably a ` ~lg4226 lo plasma poal or a plasma fraction. Therewith, the safety of the pharmaceutical compositions obtained according to the invention is increased.
In a preferred embodiment, the method according to the invention intends isolation of coagulation Factor VII through chromatographic methods, preferably by means of anion exchangers, such as DEAE-Sephacel~, DEAE-Sephadexg', DEAE-Sepharose~ CL6B, DEAE-Sepharose~ Fast Flow, QAE-Sephadex(~3), QAE-Sepharose~ Fa5t Flow, Q-Sepharose~ High Performance, Q-Sepharose~ Big Beads ~obtainable from Pharmacia); DEAE-Tris Acryl, DEAE-Spherodex~, Q-Hyper-D (obtainable from Sepracor);
Macroprep ~ DEAE, Macroprep Q(8 ~BioRad); DEAE- Toyopearl(~), QAE-Toyopearl~, Poyopearl~ Super-Q (Tosohaas), Protein PAK
DEAE (Waters), Fractogel~ EMD-TMAE, Fractogel~ EMD-DEAE, Fractogel~) EMD-DMAE, Licrospher(~ 1000 TMAE, Licrospher~ 1000 DEAE and Licrospher~ 4000 DMAE (Merck) . A particularly preferred embodiment relates to the isolation of Factor VIIa ~-by hydrophobic chromatography, for example by using the following materials: Butyl-Sepharose(~, Octyl-Sepharose'~, Phenyl-Sepharose(~), Phenyl-Sepharose~ Fast Flow High Sub, Phenyl-Sepharose~ Fast Flow Low Sub, Phenyl-Sepharose~ High Performance (all obtainable from Ph~ r; ~ ); Fractogel~ TSK-Butyl (Merck); Macroprep~-Methyl-HIC-Support, Macroprep(~D-t-Butyl-HIC-Support (BioRad), TSK-Gel Butyl Toyopearl~, TSK-Gel Phenyl Toyopearl(~), TSK-Gel Ether Toyopearl(~) (Tosohaas). A
simultaneous activation - at least partially - during the isolation is advantageous in the purification of coagulation Factors IX and X exactly as with coagulation Factor VII.
However, attention is to be paid that t~le preparation has no `~ 218~226 . .
thrombogenic activityi hence, a content of activated Factor II is to be avoided.
The composition according to the invention advantageously has a FEIB-activity in the range of 1 - 1000 units FEIBA/ml of ready-to-use solution. Preferably, the FEIB-activity is more ~ -than 5 U/ml, most preferably a FEIBA is in the range of 10 to --10 0 U/ml .
The invention also comprises the use of a combination of Factor VIIa and at least one active ingredient for the production of a pharmaceutical composition with FEIB-activity for rapid cessation of hemorrhages in patients with a coagulation factor deficiency. A rapid termination of bleeding which is dependent on inhibitors of coagulation factors is especially effected by this use.
The in vivo effect of the pharmaceutical preparation according to the invention can be demonstrated in an animal model in which rabbits are temporarily placed in a hemophilia A state after treatment with a Factor VIII inhibitor plasma.
In pre-treated animals of this type which have hemorrhagic diathesis corresponding to one of the inhibitor hemophilias, the abnormal hemorrhage behavior can be corrected to that of an untreated rabbit by administering 75 to 100 units FEIBA/kg. In order to demonstrate the improved effectiveness of a FEIBA preparation with a defined higher Factor VIIa content, the pharmaceutical preparation according to the invention containing FEIBA and Factor VIIa in a ratio of 10 units Factor YIIa per unit FEIBA was administered to hemorrhaging rabbits at a dose of 5 U FEIBA/kg (corresponding to 50 U Factor VIIa/kg). The hemorrhage intensity is reduced to the same extent as by the administration of 75 U FEIBA/kg.
As controls, conventional FEIBA ln a dose of 5 U~kg or Factor VIIa in a dose of 50 U/kg 18 admlnlstered which does not lead to any notlceable reductlon of hemorrhage lntensity ln relatlon to the untreated hemop~lllla A lnhlbltor animal ln each case. Therewith, an unexpectedly hlgh effectlveness of tlle pharmaceutlcal compositlon accordlng to the lnvention ls ~OCI l ed. An es6entlal advantage thereby 18 that the tllrombogenlc potentlal of the pharmaceutical cornposltlon accordlng to the lnventlon ls clearly reduced ln comparlson to conventlonal F~IBA.
In a drawlng whlch lllustrates an embodlment of the lnventlon, flgure l shows the reductlon ln bleedlng lntensity in rabblts of composltlons o~ the inventlon The lnventlon ls more closely descrlbed by the f ol lowlng e~amples .
Examples 1 to 8:
Method for the production of a Factor VIIa preparation Example 1: ~3eparation of Cryopreclpltate and F15IBA frolr, Plasma Productlon occurs based on AT-350 726 and AT-368 883.
Fresh, fro~en, human cltrated plasma was thawed at 0 - +4 C and the cryopreclpltate resultlng therefrom was separated by centr~fugatlon at +2 C. At natlve pH-value, 218~226 12a 0 . 5 g of DEAE-Sephadex~ A-50 were added to the cryosupernatant resulting therefrom at +4 C under contlnuous stlrrlng. The suspenslon was stlrred for a further hour and subse~uently allowed to stand such that the proteln-DEAE-Sephadex~ complex sedlmented. Thereby, FEI3A was generated and absorbed onto DEAE-Sephadex~ together wlth the factors of prothrombln 218l226 complex ~II, VII, IX and X) and inert protein. The DEAE-Sephadex~-protein complex was separated from the supernatant after the completed adsorption process by centrifugation or filtration. As described in AT368aa3, FEIBA was isolated by washing and elution of the DEAE-Sephadex~-protein complex.
The supernatant was processed further to Factor VIIa preparation .
xample 2: Separation of Factor VII/VIIa from FEI13A
supernatant After FEIBA separation from example 1, prothrombin complex factors, especially Factors VII and VIIa were then isolated from the supernatant by adsorption to aluminum hydroxide. Per 1 liter FEIBA supernatant, 10 ml of 2% aluminum hydrogel suspension were centrifuged (SORVALL RC3B, Rotor H6~00A, 5000 rpm, 10 minutes, about 4C). The precipitate resulting therefrom was homogeneously mixed with the amount of FEIBA
supernatant to be treated (Ultra Turrax). Subsequently, this was stirred for 30 minutes at 20-22C and centrifuged thereafter (SORVALL RC3B, Rotor H6000A, 5000 rpm, 10 minutes, about 4C).
The centrifuged precipitate was suspended with 3, 5% of the volume of FEIBA supernatant used for the absorption in a solution of 4 g Na3Citrate 2H20/1 and 7 g NaCl/l, p~ 7.5, and stirred for 30 minutes for the removal of protein.
Thereafter, the precipitate was separated by centrifugation (RC3B, Rotor H6000A, 5000 ~rpm, 10 minutes, about 4C) . The supernatant was discarded and the precipitate was used for further processing. Subsequently, a step for the inactivation of possibly present pathogenic substances was .=
`.~ .
conducted according to AT-A1~47/93. Tween~ 80 was presented in an amount of 1. 5 volume % of the FEIE~A supernatant used for the absorption and heated to 55C. The supernatant after the first washing was suspended in the Tween~ 80 solution by means of an Ultra Turrax mixer during 1-2 minutes and stirred for 10 minutes at 55C. Thereafter, this was immediately diluted with 9-fold volumes of water (+4C).
The treated Al (OH) 3-protein complex was separated by centrifugation, the supernatant was discarded, and the precipitate was further processed. Subsequently, two washings each with 3 . 5 voll~lme % of the employed FEIBA
supernatant were conducted by resuspension and renewed centrifugation with citrate buffer (4 g Na3Citrate-2H2O/l and 7g NaCl/l, pH 7.5) .
The fraction containing Factor VII/VIIa was separated from the aluminum hydroxide gel by elution with phosphate buffer.
The protein-aluminum hydroxide complex was stirred for 30 minutes with 1 volume 96 of the FEIBA supernatant used for the absorption of 0.3 M phosph~te buffer, pH 8.6 (53.4 q Na2HPO4 2H2O/l was adjusted to pH 8 . 6 with a solution of 41. 4 g NaH2PO4 H2O/l). Subsequently, the solid phase was separated by centrifugation at 5000 rpm for 10 minutes at 20 - 22C. The supernatant contained Factor VII/VIIa and was further processed for subsequent purification.
Example 3: Activation of Factor VII and ion exchange purification of Factor VIIa The eluate isolated in e~ample 2 was diluted with distilled water i~ a ~atio of l: 1, mixed with 0.25 mM CaCl2 and the pH value was adjusted to 8 . 6. The solution was subsequently mixed with 65 ml/l of Q-Sepharose~ FF, prewashed with buffer, and stirred for 2 hours at 4C. Subsequently, the :
gel to which Factor VIIa was now bound was separated by filtration or centrifugation. The loaded gel was freed from inert protein by resuspension for 15 minutes and subsequently separation from a wash buffer (96.7 g Na2HPO4 2H2O/l and 0.0368 g CaCl2-2H2O/l were adjusted to pH 8.6 with a solution of 20.7 NaH2PO4 H2O/l). Subsequently, Factor VIra was eluded by elution with a solution of 105.7 g (NH4)2SO4/l, 58.5 g NaCl/l and 2.42 g TrisHCl/l, pH 7.4, by suspension for 30 minutes. The gel supernatant ~nnt~ining Factor VIIa was separated by filtration or centrifugation.
xample 4: Purification of Factor VIIa by hydrophobic chromatography The solution containing Factor VIIa from example 3 was chromatographically purified over Phenylsepharose(~ LS. A
column with 50 mm Ir~ and 24 mm bed height was filled with Phenylsepharose~ LS and equilibrated in a buffer containing 2.42 g TrisHCl/l and 105.7 g (NH4)2SO4/l, pH 7.4) . 100 ml of the eluate cPntaining Factor VIIa from example 3 was pumped over the gel with a flow rate of 23 ml/min. Thereby, Factor VIIa was bound to the gel; inert protein was found in the through-flow. The gel was washed with the buffer used for . 218~226 l6 equilibration. Subsequently, Factor VIIa was eluded with a solution of 2 . 42 g TrisHCl/l, pH 7 . 4 . Thereby, fractions were collected. The fractions containing Factor VIIa activity were combined and buf f er exchange was carried out by chromatography over Sephadexg~ G-25 against a buffer containing 2 . 42 g TrisHCl/l, pH 7 . 4 .
xample 5: Purification of Factor VIIa by chromatography on Q-Sepharose(5 Q-Sepharose~ FF, packed in a column with 25 mm ID and 55 mm bed height, was equilibrated with 20 mm TrisHCl/l, pH 7 . 4 .
Subsequently, 43 ml of the solution containing Factor VIIa from example 4 were pumped over the column with a flow rate of 5 . 3 ml/min. Thereby, Factor VIIa was bound to the gel, whereas inert proteins remained in the through-flow.
Subsequently, this was washed with a buffer containing 25 mM
Na3Citrate 2H2O and 80 mM NaCl, pH 6. 0 . Further inert protein was separated thereby. The elution of Factor VIIa was conducted by washing the column with 25 mM
Na3Citrate 2EI20 and 160 mM NaCl, pH 6Ø The protein and the fractions containing Factor VIIa activity of this elution step were pooled. Subsequently, the column was regenerated with a buffer (25mM Na3Citrate-2H20 and 1 M NaCl, pH 6. 0) and used further.
Example 6: Ultraconcentration oi~ Factor VIIa preparation The pool containing Factor VIIa from example 5 was adjusted to pH 7. 0 and mixed with 0.1 % human albumin. Subsequently, this was concentrated to l/10 of the starting volume over 218~226 ultra filtration membranes (Amicon YM 10, cut-off 10, 000 D~
under a pressure of 3 . O bar in an AMICON 80/50 stirring cell .
The concentrated solution containing Factor VIIa was subsequently lyophilized.
Example 7: Heat treatment of lyorhi 1 i 7~1 Factor VIIa For the inactivation of possibly included pathogen, the lyophilized Fact~r VIIa preparation from example 6 was heated accnrding to the method in patent EP 159 311 under elevation of the partial water pressure, and/or following the treatment at 60C, also treated for a further hour at 80C. The yield of Factor VIIa was more than 90 % in each case.
xample 8: Characterization o~ the Factor VIIa preparation Measurement of the degree of activation of Factor VII was carried out according to the method described by van Dei~ k, Haemostasis 13:192-197 (1983). The coagulation test is based on the use of bovine thromboplastin on one hand and human thromboplastin on the other hand in connection with a Factor VII depleted plasma. As a standard, measurement was done with normal human plasma. The specific activity of Factor VIIa for the individual steps of production and purification as well as the activation grade, Factor VII measured with bovine thromboplastin against Factor VII measured with human thromboplastin, are given in Table l. The protein measurements were conducted according to the method of Bradford, Anal. Biochem. 72:248-254 (1976).
l8 Example 9: Combination of FEIBA and Factor VIIa Fresh, frozen, human citrate plasma was thawed at 0 - 4C and the resulting cryoprecipitate was separated by centrifugation at +2C. At native pH-value, 0 . 5 g of DEAE-Sephadex A-50 at +4C was added to the supernatant resulting therefrom under constant stirring. The suspension was stirred for a further hour and subsequently allowed to stand such that the protein-DEAE-Sephadex complex sedimented. Thereby, FEIBA was generated and absorbed to DEAE-Sephadex~D together with the factors of prothrombin complex and inert protein. The DEAE~
Sephadex~-protein complex was separated from the supernatant by filtration after the completed absorption process. As described in AT 368 883, FEIBA was desorbed from DEAE-Sephadex~) by washing with buffer and subsequent elution withsodium chloride solution. The solution containing FEIBA was concentrated to 1/5 of the starting volume by ultrafiltration and subsequently incubated for auto activation of the prothrombin complex factors for 20 hours at room temperature.
Subsequently, the solution containing activated prothrombin complex was lyophilized. Then, the powder was dissolved to a concentration of 30 mg protein/ml in a buffer o~ 2 g Na3Citrate 2H2O and 4 g NaCl, pH 7.2. This solution was filtered through a filter with a pore size of 1 um and re-lyophilized. The powder isolated in this manner was dissolved in a solution containing factor VIIa which was produced as described in example 4. The solution was adjusted such that 10 U FVIIa were contained per 1 U FEIBA, tested according to AT 350 726. This mixture was re-lyophilized according to the method of EP 159 311, and treated for 10 hours at 60C and 1 hour at 80~C. FEIB-` ~ 2184226 activity and the FVIIa activity of the powder were measured before and after the heat treatment. After the heat treatment step, the activity of FVIIa in the preparation was 96 % of the starting material and had decreased by 4 %
therewith; the FEIB-activity was 90 %, corresponding to a loss of 10 %. As a comparison for this, the FVIIa preparation from example 4 was lyophilized and also heat treated. The activity decreased by 48 ~6 thereby.
Example lO: Combination of prothrombin complex and Factor VIIa A preparation containing prothrombin complex was prepared according to the method of H. G. J. Brummelhus, Methods of Plasma Protein Fractionation, J.M. Curling (Hrsg. ), page 117-128, Academic Press 1980. The preparation contained Factor II, IX and X in a similar relationship. Subsequently, the preparation was mixed with Factor VIIa which was produced as described in example 4. The mixture was adjusted in such a manner that 10 U FVIIa were contained per 1 U FX. A freeze- -dried powder of this mixture was treated according to the method of Epn 159 311 for 10~ hours at 60C and 1 hour at 80C. The activity of coagulation Factors II, IX and X and Factor VIIa were measured respectively before and after the heat treatment. The losses of activity were 7 % for Factor II, 12 % for Factor IX, 3 96 for Factor X and 1 % for Factor VI Ia .
As a comparison for this, Factor VIIa preparation from example 4 was lyophilized and also heat treated. The activity decreased by 48 ~ thereby.
~ 218~226 Example 11: Isolation of a Factor VIIa/FEI13A preparation A preparation containing Factor VIIa and FEIB-activity was isolated as follows. Fresh, frozen, human citrate plasma was thawed at 0 - +4C and the resulting cryoprecipitate was separated by centrifugation at +2C. At native pH, the cryosupernatant resulting therefrom was mixed with 0.5 g of QAE-Sephadex~) A50 (Pharmacia~ and stirred at +4C for 15 hours. Subsequently, the gel-protein complex was separated from the supernatant by sedimentation and filtration and the fraction containing Factor VIIa and FEIBA was isolated by elution with 3 96 NaCl solution. Factor VIIa and FEIBA were __ quantitatively measured. The preparation contained 12 . 7 U
FVIIa per unit FEIBA.
Example 12: Isolation of a Factor VIIa/FEIBA preparation FEIBA was isolated as described in example 11. However, Fractogel~ EMD T~E (Merck) was used as an ion exchanger for the absorption in a concentration of 5 ml moist gel per liter cryosupernatant. After elution of the activated prothrombin complex ~actors with 3 % NaCl solution, this contained 54 U
FEIBA/ml as well as 0 . 6 U Factor VIIa/U FEIBA. In order to produce the preparation according to the invention, Factor VIIa according to examples 2 - 4 was isolated from the cryosupernatant and the solution containing FEIBA was added such that at least 10 U Factor VIIa/U FEIBA were finally contained .
Example 13: Isolation of a Factor VIIa/FEIBA preparation 218~22~
.
Analogous to example 12, FEIBA was produced again. As an ion exchanger, Q-Sepharose~ Fast Flow (Pharmacia) was used in a concentration of 5 ml per liter cryosupernatant. The FEIB-activity in the obtained eluate was 1. 280 U/ml and 0 . 3 U
Factor VIIa/U FEIBA. In order to produce a preparation with 20 U Factor VIIa/U FEIBA, Factor VIIa was produced, as in example 11, from the same cryosupernatant from which the FEIBA preparation was isolated and mixed with the solution containing FEIBA.
xample 14: In vivo effectiveness of a Factor VIIa/FEIBA
preparation A combination of FEIBA and Factor VIIa produced according to example 9 was tested in rabbits with Factor VIII inhibitor hemophilia as follows. Approximately 2 kg, white, New Zealand rabbits were anesthetized. After onset of the anesthesia, the right femoral vein of each rabbit was prepared and a permanent venous access was created. Through this, 0 . 5 ml/kg body weight of a human Factor VIII lnhibitor plasma ~1500 Bethesda units~ml) was infused over 10 minutes.
30 minutes after completion of the infusion, the hemorrhage characteristics were measured with a modified method according to Giles et al, Blood 60:727-730 (1982) . For this, the fur around a claw of the hind paw of the rabbit was shaved in order to prevent exiting blood from being absorbed to the fur during later bleeding. The apex of the cutis was injured by means of a claw forceps. Immediately thereafter, filters were arranged underneath the wound in such a manner that the blood could directly drop onto the filter without being drawn up from this by capillary effect. Destruction of a forming blood clot was prevented through this precaution.
The filters were changed every two minutes and the exiting 218~22~
blood was collected in fractions. Blood collection was continued for 30 ~inutes. The qualification of bleeding characterlstics was conducted by extraction of the blood collected in fractions on the filter with 5 ml each of 0. 04 %
ammonium hydroxide s~lution over 5 hours. Thereby, the erythrQcytes which were collected with the blood in the filter were lysed. During a 10 minute ultrasound treatment, hemoglobin was extracted and ~uantitatively measured photometrically at 416 nm against a calibration curve, whereby the calibration curve was established by pipetting rabbit blood volumes between 10 ul and 1 ml onto the filters, extracting as described above, and measuring the hemoglobin photometrically at 416 nm. Accordingly, linear calibration curves could be prepared which made possible a direct conversion of the hemoglobin concentration to the amount of -~
blood per filter. The bleeding characteristics of the nail cut were established as the cumulative loss of blood through additive graphic plotting of the individual blood fractions against time. The increase of cumulative bleeding between 10 and ZO minutes of the experiment was employed as a relevant criterion of bleeding and served as a measurement for the bleeding intensity. The average hemorrhage intensity of animals treated in this method is given in the Figure (Fig.
1) .
For testing the active substances, a solution of a pharmaceutical composition containing the respective active ingredients in a volume of 3D ml was continuously infused after inhibitor-induced bleeding in the rabbit with an infusion rate of 1 ml/min and, simultaneously with the beginning of the infusion, the bleeding intensity was re- -established as described above. Accordingly, a FEIBA
preparation produced according to A 350 726 or A 368 883, was 218~22G
administered in a dose of 75 U/FEIBA/kg to correspondingly pretreated rabbits. This dosage led to a drastic reduction in the bleeding intensity (see Fig. 1).
As a control, a buffer without active ingredient was infused which led to no reduction of bleeding. Equally, the administration of FEIBA in a dose of 5 U/kg led to no significant reduction of bleeding intensity.
In contrast, when a preparation according to the invention corresponding to a dose of 5 U FEIBA/kg and 50 U Factor VIIa/kg was given to the rabbits, a reduction in the bleeding intensity resulted comparable with an effective FEIBA dosage, while 50 U Factor VIla/kg had no effect on the abnormally increase-d hemorrhage intensity.
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ph~ utical composition for the treatment of blood coagulation diseases, methods for the production thereof and its use.
The invention relates to a pharmaceutical composition for the treatment of blood coagulation disorders, especially of :
Factor VIII inhibitor patients. The invention further relates to a method for production of a composition of this type as well as its use.
Blood coagulation is triggered by a series of successive reactions of various proteins and/or enzymes, The formation of fibrin from fibrinogen and, therewith, the closure of wounds is prevented by a lack of blood coagulation factors; ~ -:
the consequence ls hemorrhage. Such a case exists with hemophilia A. This is the most widespread bleeding disorder and is caused by a deficiency in Factor VIII. Factor VIII
containing preparations are used for replacement therapy in hemophilia A. Treatment with these preparations leads to a -rapid cess-ation of bleeding in most cases.
However, there are also patients among which not only a deficiency in Factor VIII occurs, but also who have developed an inhibitor directed against Factor VIII. A further collection of patients posses Factor VIII inhibitors without suffering from hemophilIa A. Depending on the amount of factor A inhibitors present, the effect of administered Factor VIII is inhibited by its neutralization.
Presently, preparations based on a plasma fraction which contains a mixture of coagulation factors are offered for treatment of Factor VIII inhibitor patients. This plasma fraction can contain, for example, factors of the prothrombin , 218g226 complex (Factor II, VII, IX, X). A blood coagulation promoting preparation with Factor VIII inhibitor bypass activity (FEIBA ~) TIM 4, Immuno AG) is obtained, according to AT-0 368 883 for example, by treatment of cryosupernatant.
This preparation also contains coagulation Factors II, VII, IX, X.
The action of a FEIBA preparation is complex due to lts complex composition. Mariani et al (Thromobosis Res. 31, 475-488, (1983) ) mentions Factor VII in its activated form as a principle of action. It was established that a higher content of Factor VIIa in plasma occurs in hemophiliacs after infusion of a FEIBA preparation.
Equally, the role of Factor VIIa in prothrombin complex concentrates with a "Factor VIII-bypassing-activity" is discussed by Teitel (Thrombosis and Haemostasis 66 (5) 559-564, (1991) ) . Simultaneously, the principle of action of Factor Xa in preparations of this type is also dealt with.
The examined prothrombin complex concentrates contained ~:
Factor VIIa, expressed by the ratio of Factor VII activity to Factor VII antigen of 2.1 and 2.5.
The therapeutic composition containing prothrombin produced according to EP-0 044 343-B1 is suitable for treatment of coagulation factor inhibitors and contains an activated prothrombin complex in which the factors are partially activated. The amount of Factor VIIa is between 8-80 units per ml. Factor IX concentration is in the range of 15 to 112 units per ml. Correspondingly, the content of Factor VIIa, with respect to Factor IX, is 0.07 - 5.3 U Factor VIIa/U
Factor IX. Vinazzer (Thromobosis Res. 26:21-29 ~1982) ) demonstrates the difference of the preparations AUTOPLEX, which is produced according to EP-44 343 and FEIBA. As shown there, AUTOPLEX is distinguished by the higher content of thrombin (Factor IIa) measured in NIH units in comparison to FEIBA (see table 1, page 24).
However, highly purified Factor VIIa preparations are also proposed for therapy of coagulation factor inhibitor =
conditions (for example, EP 0 082 182-B1) and Hedner et al.
(Haemostasis 19, 335-343 ~1989) ) .
A method for the production of a concentrate which contains highly purified Factor VIIa is also described in EP 0 547 932-A1. Factor VII is separated from coagulation Factors II, IX and X by anion exchange chromatography of a cryoprecipitate. Factor VII is then subjected to a further purification and activation, whereby the addition of exogenous proteins is avoided. Subsequently, Factor VIIa is treated i-or inactivation of viruses by a treatment with an organic solvent/detergent (TnBP/Tween).
The risk of transmitting infectious agents exists by the use of plasma or a plasma fraction as the starting material for the production of pharmaceutical preparations. Despite selection of donors and tests of the individual plasma donations for possibly present viruses, such as HIV, hepatitis B or hepatitis C virus, the possibility exists that a plasma pool is infectious. This residual infectivity is not to be ruled out because of limited sensitivity of the test systems, but also because of the incubation time until the appearance of measurable infection markers (diagnostic window ) .
' ~ 218g226 Pharmaceutlcal preparations whlch are prepared from plasma or a plasma fraction are therefore su~:lected to diverse treatments for inactivation of viruses potentially present. A
proven method for the inactivation of mernbrane enveloped as well as non-membrane enveloped vlruses is described ln EP 0 159 311-Bl. T}~ereby, a pharmaceutical preparation 18 heated ln a solld state after ad:lustment of a water content to a value of 5-70%. T~lis treatment is also described as a steam (vapor) treatment. Instead of water, methanol or ethanol can also be used.
However, combinatlons of vlrus inactivation methods are equally possible. According to 3P 0 519 901r a treatment wlth highly concentrated tensides is combined wlth a heat t reat ment .
An alm of the lnvent lon 18 to provlde an lltlproved pharmaceutical composltlon for treatment o~ blood coagulation disorders, e~pecially of Factor VIII $nhlbitor patlents as well as a simple method for its production.
The alm 18 achleved according to the invent ion by a pharmaceutlcal composltion with FEIB-activity which contains Factor VIIa and Qt least one furt~-ler active ingredlent and has the activity of 10 Factor VIIa units per unlt FEIBA.
The lnvention also provides corQmercial packages comprlslng such pharmaceutlcal composltlons together wlth instructions for use therefor in rapid termination of hemorrhage in a patlent wlth a coagulation factor deficiency.
The content of Factor VIIa in the compositlon 218~226 .
4a accordlng to the lnventlon is at least 10, preferably 10 to 100, most preferably 10 to 20, especially 10 to 15, units per unlt FEIB-actlYlty. The F~IB-activity i8 determlned by means of a method accordlng to AT-350 726.
218~226 It has turned out that a preparation which contains highly purified Factor VIIa as the sole effective substance does not possess any FEIB-activity. Therefore, it was surprising that addition of Factor VIIa to a preparation with FEIB-activity, such as one of the activated prothrombin complex containing fractions, contributes to a substantial improvement of the effectiveness of a pharmaceutical preparation for the treatment of blood coagulation diseases, especially of Factor VIII inhibitor patients.
The content of Factor VIIa was determined in the following examples with the methods described by van Deij k ~Haemostasis 13: 192-197, 1983) and EP 0 547 932 with bovine thromboplastin. Additionally, Factor VIIa can also be determined by using recombinant, soluble tissue factor according to the methods of Morrissey et al. (Blood 81: 73g-744, (1983) ) and WO 92/18870.
Coagulation Factors II, IX and X and, additionally for example, tissue factor or Xa and phospholipids are to be named as active ingredients in a preparation of FEIB-activity. The above listing of substances in a preparation with FEIB-activity is not complete and, therefore, should not be considered as limiting.
The preparation according to the invention preferably contains coagulation Factors II, IX and X, preferably in a concentration which corresponds to a ratio of 0 . 5 - 2, particularly preferred 0 . 5 - 1. 5 U/U FEIBA for each factor .
The measurement is carried out according to the test description in AT-350 726.
~ 218~226 Therewith, the composition according to the invention differs from known preparations not only in its extraordinary high ratio of Factor VIIa to FEIBA, but also in its ratio of Factor VIIa to Factor IX. For example, the latter is distinctly higher than in EP-O 044 343.
It is of further advantage when the preparation according to the invention additionally contains protein C and/or protein S.
It was surprising that the preparation according to the invention can be produced in a simple and economic manner.
Therefore, the invention also comprises a production method which is characterized by contacting a fraction as illustrated above with an anion exchanger, whereby coagulation Factor VII is at least partially activated.
Conditions are selected thereby which allow for the simultaneous adsorption of Factor VII and/or Factor VIIa with coagulation Factors II, IX and X, for example, by using buffers of low ionic strength. Subsequently, the anion exchanger is separated, for example by sedimentation, and the fraction which contains coagulation Factors II, VIIa, IX and X is isolated. In this connection, the fraction already possesses FEIBA before contact and/or a FEIBA is generated in the fraction during this.
Preferably, a strong anion exchanger, for example a matrix with quaternary ammonium groups such as QAE, TMAE and Q
groups, is used as an anion exchange material. As a suppor~
for the anion exchange groups, cross-linked Dextran, such as Sephadex(~3 or Sepharose~ or synthetic material such as Fraktogel~3) is used.
` ~ 2184226 This method of production is distinguished, among others, by its economic procedure. The method has the advantage that coagulation Factor VII must not be separated at first in order to then be added to the preparation again.
Additionally, the coagulation factor-containing fraction is not only purified, but coagulation Factor VII is also activated in one step. Partial activation of Factor IX and Factor: X, but not of Factor II, also occurs at the same time.
The composition according to the invention is also obtainable by combining a fraction containing a purified prothrombin complex and a fraction containing purified Factor VIIa, and by a treatment for the inactivation of viruses. However, the pharmaceutical composition according to the invention can also be composed of a fraction containing a purified partial prothrombin complex (Factors II, IX and X) and a fraction containing purified Factor VIIa, whereby it is treated for the inactivation of viruses. The prothrombin complex used for the production of the composition according to the invention is preferably present as activated prothrombin complex or as a FEIBA preparation. In an activated prothrombin complex, coagulation Factors II, IX and X are partially activated. However, the activation of Factor II is to be avoided in such a preparation in order to directly exclude prothrombogenic activity.
By using certain conditions such as high ionic strength in the starting solution and/or in the washing buffer and strong anion exchange gels, for example Q-Sepharose(~ Fast Flow (Pharmacia), activated prothrombin complex can be isolated as a FEIBA preparation with higher yields from plasma or plasma supernatant after separation of the cryoprecipitate. Gels of this type then bind Factor VII or Factor VIIa relatively ~ 218~226 poorly. Therefore, Factor VIIa can be isolated from the same starting material in a further process with higher yields.
The fractions obtained are processed to pharmaceutical preparations in the customary manner, for example, by addition of pharmaceutically acceptable carriers and/or adjuvants, sterile filtered and lyophilized to storable pharmaceutical preparations.
The compositlon according to the invention can be produced by a further method with the following steps:
a) isolating coagulation Factors II, IX and X as individual components or as a mixture from one or more starting materials containing one or more of coagulation Factors II, IX and/or X, b) isolating coagulation Factors VII/VIIa from a starting material containing coagulation Factor VIIa and/or coagulation Factor VII, whereby coagulation Factor VII is at least partially activated, c) pretreating or treating the starting materials or the isolated coagulation factors for inactivation of infectious agents, and d) mixing the isolated coagulation factors together with a pharmaceutically acceptable carrier.
In a preferred embodiment, Factor VIIa is added to prothrombin complex, which can also be present as partial prothrombin complex, after purification of the components ard mixed with a pharmaceutically acceptable carrier, and subse~uently subj ected to a treatment for virus inactivation, for example a heat treatment.
The addition and/or content of highly pure Factor VIIa is especially advantageous because less contaminating proteins are introduced into the preparation as impurities therewith.
The specific activity of Factor VIIa in the preparation is preferably more than 10 U/mg to 1, 000 U/mg protein.
The prothrombin complex is preferably present as an activated prothrombin complex or FEIBA preparation.
Preferably, a heat treatment, for example a steam treatment, is carried out for inactivation of infectious agents (viruses). Thereby, it has turned out that a heat treatment of the coagulation factors in a solid state can be conducted without addition of stabilizing proteins, such as albumin.
Factor VIIa is surprisingly stable enough that it can also be treated in a purified state for inactivation of infectious agents .
The heat treatment for inactivation of viruses has the advantage that not only lipid coated viruses are inactivated, but also viruses which are not surrounded with a lipid envelope .
The method according to the invention is characterlzed in that Factor VIIa can be isolated from a fraction which accumulates as a by-product in the production of a fraction containing (partial) prothrombin complex. Therefore, this method is also particularly distinguished by its economic e f f i ciency .
With the method according to the invention, it is possible to obtain all components of the composltion according to the invention from a single starting material, preferably a ` ~lg4226 lo plasma poal or a plasma fraction. Therewith, the safety of the pharmaceutical compositions obtained according to the invention is increased.
In a preferred embodiment, the method according to the invention intends isolation of coagulation Factor VII through chromatographic methods, preferably by means of anion exchangers, such as DEAE-Sephacel~, DEAE-Sephadexg', DEAE-Sepharose~ CL6B, DEAE-Sepharose~ Fast Flow, QAE-Sephadex(~3), QAE-Sepharose~ Fa5t Flow, Q-Sepharose~ High Performance, Q-Sepharose~ Big Beads ~obtainable from Pharmacia); DEAE-Tris Acryl, DEAE-Spherodex~, Q-Hyper-D (obtainable from Sepracor);
Macroprep ~ DEAE, Macroprep Q(8 ~BioRad); DEAE- Toyopearl(~), QAE-Toyopearl~, Poyopearl~ Super-Q (Tosohaas), Protein PAK
DEAE (Waters), Fractogel~ EMD-TMAE, Fractogel~ EMD-DEAE, Fractogel~) EMD-DMAE, Licrospher(~ 1000 TMAE, Licrospher~ 1000 DEAE and Licrospher~ 4000 DMAE (Merck) . A particularly preferred embodiment relates to the isolation of Factor VIIa ~-by hydrophobic chromatography, for example by using the following materials: Butyl-Sepharose(~, Octyl-Sepharose'~, Phenyl-Sepharose(~), Phenyl-Sepharose~ Fast Flow High Sub, Phenyl-Sepharose~ Fast Flow Low Sub, Phenyl-Sepharose~ High Performance (all obtainable from Ph~ r; ~ ); Fractogel~ TSK-Butyl (Merck); Macroprep~-Methyl-HIC-Support, Macroprep(~D-t-Butyl-HIC-Support (BioRad), TSK-Gel Butyl Toyopearl~, TSK-Gel Phenyl Toyopearl(~), TSK-Gel Ether Toyopearl(~) (Tosohaas). A
simultaneous activation - at least partially - during the isolation is advantageous in the purification of coagulation Factors IX and X exactly as with coagulation Factor VII.
However, attention is to be paid that t~le preparation has no `~ 218~226 . .
thrombogenic activityi hence, a content of activated Factor II is to be avoided.
The composition according to the invention advantageously has a FEIB-activity in the range of 1 - 1000 units FEIBA/ml of ready-to-use solution. Preferably, the FEIB-activity is more ~ -than 5 U/ml, most preferably a FEIBA is in the range of 10 to --10 0 U/ml .
The invention also comprises the use of a combination of Factor VIIa and at least one active ingredient for the production of a pharmaceutical composition with FEIB-activity for rapid cessation of hemorrhages in patients with a coagulation factor deficiency. A rapid termination of bleeding which is dependent on inhibitors of coagulation factors is especially effected by this use.
The in vivo effect of the pharmaceutical preparation according to the invention can be demonstrated in an animal model in which rabbits are temporarily placed in a hemophilia A state after treatment with a Factor VIII inhibitor plasma.
In pre-treated animals of this type which have hemorrhagic diathesis corresponding to one of the inhibitor hemophilias, the abnormal hemorrhage behavior can be corrected to that of an untreated rabbit by administering 75 to 100 units FEIBA/kg. In order to demonstrate the improved effectiveness of a FEIBA preparation with a defined higher Factor VIIa content, the pharmaceutical preparation according to the invention containing FEIBA and Factor VIIa in a ratio of 10 units Factor YIIa per unit FEIBA was administered to hemorrhaging rabbits at a dose of 5 U FEIBA/kg (corresponding to 50 U Factor VIIa/kg). The hemorrhage intensity is reduced to the same extent as by the administration of 75 U FEIBA/kg.
As controls, conventional FEIBA ln a dose of 5 U~kg or Factor VIIa in a dose of 50 U/kg 18 admlnlstered which does not lead to any notlceable reductlon of hemorrhage lntensity ln relatlon to the untreated hemop~lllla A lnhlbltor animal ln each case. Therewith, an unexpectedly hlgh effectlveness of tlle pharmaceutlcal compositlon accordlng to the lnvention ls ~OCI l ed. An es6entlal advantage thereby 18 that the tllrombogenlc potentlal of the pharmaceutical cornposltlon accordlng to the lnventlon ls clearly reduced ln comparlson to conventlonal F~IBA.
In a drawlng whlch lllustrates an embodlment of the lnventlon, flgure l shows the reductlon ln bleedlng lntensity in rabblts of composltlons o~ the inventlon The lnventlon ls more closely descrlbed by the f ol lowlng e~amples .
Examples 1 to 8:
Method for the production of a Factor VIIa preparation Example 1: ~3eparation of Cryopreclpltate and F15IBA frolr, Plasma Productlon occurs based on AT-350 726 and AT-368 883.
Fresh, fro~en, human cltrated plasma was thawed at 0 - +4 C and the cryopreclpltate resultlng therefrom was separated by centr~fugatlon at +2 C. At natlve pH-value, 218~226 12a 0 . 5 g of DEAE-Sephadex~ A-50 were added to the cryosupernatant resulting therefrom at +4 C under contlnuous stlrrlng. The suspenslon was stlrred for a further hour and subse~uently allowed to stand such that the proteln-DEAE-Sephadex~ complex sedlmented. Thereby, FEI3A was generated and absorbed onto DEAE-Sephadex~ together wlth the factors of prothrombln 218l226 complex ~II, VII, IX and X) and inert protein. The DEAE-Sephadex~-protein complex was separated from the supernatant after the completed adsorption process by centrifugation or filtration. As described in AT368aa3, FEIBA was isolated by washing and elution of the DEAE-Sephadex~-protein complex.
The supernatant was processed further to Factor VIIa preparation .
xample 2: Separation of Factor VII/VIIa from FEI13A
supernatant After FEIBA separation from example 1, prothrombin complex factors, especially Factors VII and VIIa were then isolated from the supernatant by adsorption to aluminum hydroxide. Per 1 liter FEIBA supernatant, 10 ml of 2% aluminum hydrogel suspension were centrifuged (SORVALL RC3B, Rotor H6~00A, 5000 rpm, 10 minutes, about 4C). The precipitate resulting therefrom was homogeneously mixed with the amount of FEIBA
supernatant to be treated (Ultra Turrax). Subsequently, this was stirred for 30 minutes at 20-22C and centrifuged thereafter (SORVALL RC3B, Rotor H6000A, 5000 rpm, 10 minutes, about 4C).
The centrifuged precipitate was suspended with 3, 5% of the volume of FEIBA supernatant used for the absorption in a solution of 4 g Na3Citrate 2H20/1 and 7 g NaCl/l, p~ 7.5, and stirred for 30 minutes for the removal of protein.
Thereafter, the precipitate was separated by centrifugation (RC3B, Rotor H6000A, 5000 ~rpm, 10 minutes, about 4C) . The supernatant was discarded and the precipitate was used for further processing. Subsequently, a step for the inactivation of possibly present pathogenic substances was .=
`.~ .
conducted according to AT-A1~47/93. Tween~ 80 was presented in an amount of 1. 5 volume % of the FEIE~A supernatant used for the absorption and heated to 55C. The supernatant after the first washing was suspended in the Tween~ 80 solution by means of an Ultra Turrax mixer during 1-2 minutes and stirred for 10 minutes at 55C. Thereafter, this was immediately diluted with 9-fold volumes of water (+4C).
The treated Al (OH) 3-protein complex was separated by centrifugation, the supernatant was discarded, and the precipitate was further processed. Subsequently, two washings each with 3 . 5 voll~lme % of the employed FEIBA
supernatant were conducted by resuspension and renewed centrifugation with citrate buffer (4 g Na3Citrate-2H2O/l and 7g NaCl/l, pH 7.5) .
The fraction containing Factor VII/VIIa was separated from the aluminum hydroxide gel by elution with phosphate buffer.
The protein-aluminum hydroxide complex was stirred for 30 minutes with 1 volume 96 of the FEIBA supernatant used for the absorption of 0.3 M phosph~te buffer, pH 8.6 (53.4 q Na2HPO4 2H2O/l was adjusted to pH 8 . 6 with a solution of 41. 4 g NaH2PO4 H2O/l). Subsequently, the solid phase was separated by centrifugation at 5000 rpm for 10 minutes at 20 - 22C. The supernatant contained Factor VII/VIIa and was further processed for subsequent purification.
Example 3: Activation of Factor VII and ion exchange purification of Factor VIIa The eluate isolated in e~ample 2 was diluted with distilled water i~ a ~atio of l: 1, mixed with 0.25 mM CaCl2 and the pH value was adjusted to 8 . 6. The solution was subsequently mixed with 65 ml/l of Q-Sepharose~ FF, prewashed with buffer, and stirred for 2 hours at 4C. Subsequently, the :
gel to which Factor VIIa was now bound was separated by filtration or centrifugation. The loaded gel was freed from inert protein by resuspension for 15 minutes and subsequently separation from a wash buffer (96.7 g Na2HPO4 2H2O/l and 0.0368 g CaCl2-2H2O/l were adjusted to pH 8.6 with a solution of 20.7 NaH2PO4 H2O/l). Subsequently, Factor VIra was eluded by elution with a solution of 105.7 g (NH4)2SO4/l, 58.5 g NaCl/l and 2.42 g TrisHCl/l, pH 7.4, by suspension for 30 minutes. The gel supernatant ~nnt~ining Factor VIIa was separated by filtration or centrifugation.
xample 4: Purification of Factor VIIa by hydrophobic chromatography The solution containing Factor VIIa from example 3 was chromatographically purified over Phenylsepharose(~ LS. A
column with 50 mm Ir~ and 24 mm bed height was filled with Phenylsepharose~ LS and equilibrated in a buffer containing 2.42 g TrisHCl/l and 105.7 g (NH4)2SO4/l, pH 7.4) . 100 ml of the eluate cPntaining Factor VIIa from example 3 was pumped over the gel with a flow rate of 23 ml/min. Thereby, Factor VIIa was bound to the gel; inert protein was found in the through-flow. The gel was washed with the buffer used for . 218~226 l6 equilibration. Subsequently, Factor VIIa was eluded with a solution of 2 . 42 g TrisHCl/l, pH 7 . 4 . Thereby, fractions were collected. The fractions containing Factor VIIa activity were combined and buf f er exchange was carried out by chromatography over Sephadexg~ G-25 against a buffer containing 2 . 42 g TrisHCl/l, pH 7 . 4 .
xample 5: Purification of Factor VIIa by chromatography on Q-Sepharose(5 Q-Sepharose~ FF, packed in a column with 25 mm ID and 55 mm bed height, was equilibrated with 20 mm TrisHCl/l, pH 7 . 4 .
Subsequently, 43 ml of the solution containing Factor VIIa from example 4 were pumped over the column with a flow rate of 5 . 3 ml/min. Thereby, Factor VIIa was bound to the gel, whereas inert proteins remained in the through-flow.
Subsequently, this was washed with a buffer containing 25 mM
Na3Citrate 2H2O and 80 mM NaCl, pH 6. 0 . Further inert protein was separated thereby. The elution of Factor VIIa was conducted by washing the column with 25 mM
Na3Citrate 2EI20 and 160 mM NaCl, pH 6Ø The protein and the fractions containing Factor VIIa activity of this elution step were pooled. Subsequently, the column was regenerated with a buffer (25mM Na3Citrate-2H20 and 1 M NaCl, pH 6. 0) and used further.
Example 6: Ultraconcentration oi~ Factor VIIa preparation The pool containing Factor VIIa from example 5 was adjusted to pH 7. 0 and mixed with 0.1 % human albumin. Subsequently, this was concentrated to l/10 of the starting volume over 218~226 ultra filtration membranes (Amicon YM 10, cut-off 10, 000 D~
under a pressure of 3 . O bar in an AMICON 80/50 stirring cell .
The concentrated solution containing Factor VIIa was subsequently lyophilized.
Example 7: Heat treatment of lyorhi 1 i 7~1 Factor VIIa For the inactivation of possibly included pathogen, the lyophilized Fact~r VIIa preparation from example 6 was heated accnrding to the method in patent EP 159 311 under elevation of the partial water pressure, and/or following the treatment at 60C, also treated for a further hour at 80C. The yield of Factor VIIa was more than 90 % in each case.
xample 8: Characterization o~ the Factor VIIa preparation Measurement of the degree of activation of Factor VII was carried out according to the method described by van Dei~ k, Haemostasis 13:192-197 (1983). The coagulation test is based on the use of bovine thromboplastin on one hand and human thromboplastin on the other hand in connection with a Factor VII depleted plasma. As a standard, measurement was done with normal human plasma. The specific activity of Factor VIIa for the individual steps of production and purification as well as the activation grade, Factor VII measured with bovine thromboplastin against Factor VII measured with human thromboplastin, are given in Table l. The protein measurements were conducted according to the method of Bradford, Anal. Biochem. 72:248-254 (1976).
l8 Example 9: Combination of FEIBA and Factor VIIa Fresh, frozen, human citrate plasma was thawed at 0 - 4C and the resulting cryoprecipitate was separated by centrifugation at +2C. At native pH-value, 0 . 5 g of DEAE-Sephadex A-50 at +4C was added to the supernatant resulting therefrom under constant stirring. The suspension was stirred for a further hour and subsequently allowed to stand such that the protein-DEAE-Sephadex complex sedimented. Thereby, FEIBA was generated and absorbed to DEAE-Sephadex~D together with the factors of prothrombin complex and inert protein. The DEAE~
Sephadex~-protein complex was separated from the supernatant by filtration after the completed absorption process. As described in AT 368 883, FEIBA was desorbed from DEAE-Sephadex~) by washing with buffer and subsequent elution withsodium chloride solution. The solution containing FEIBA was concentrated to 1/5 of the starting volume by ultrafiltration and subsequently incubated for auto activation of the prothrombin complex factors for 20 hours at room temperature.
Subsequently, the solution containing activated prothrombin complex was lyophilized. Then, the powder was dissolved to a concentration of 30 mg protein/ml in a buffer o~ 2 g Na3Citrate 2H2O and 4 g NaCl, pH 7.2. This solution was filtered through a filter with a pore size of 1 um and re-lyophilized. The powder isolated in this manner was dissolved in a solution containing factor VIIa which was produced as described in example 4. The solution was adjusted such that 10 U FVIIa were contained per 1 U FEIBA, tested according to AT 350 726. This mixture was re-lyophilized according to the method of EP 159 311, and treated for 10 hours at 60C and 1 hour at 80~C. FEIB-` ~ 2184226 activity and the FVIIa activity of the powder were measured before and after the heat treatment. After the heat treatment step, the activity of FVIIa in the preparation was 96 % of the starting material and had decreased by 4 %
therewith; the FEIB-activity was 90 %, corresponding to a loss of 10 %. As a comparison for this, the FVIIa preparation from example 4 was lyophilized and also heat treated. The activity decreased by 48 ~6 thereby.
Example lO: Combination of prothrombin complex and Factor VIIa A preparation containing prothrombin complex was prepared according to the method of H. G. J. Brummelhus, Methods of Plasma Protein Fractionation, J.M. Curling (Hrsg. ), page 117-128, Academic Press 1980. The preparation contained Factor II, IX and X in a similar relationship. Subsequently, the preparation was mixed with Factor VIIa which was produced as described in example 4. The mixture was adjusted in such a manner that 10 U FVIIa were contained per 1 U FX. A freeze- -dried powder of this mixture was treated according to the method of Epn 159 311 for 10~ hours at 60C and 1 hour at 80C. The activity of coagulation Factors II, IX and X and Factor VIIa were measured respectively before and after the heat treatment. The losses of activity were 7 % for Factor II, 12 % for Factor IX, 3 96 for Factor X and 1 % for Factor VI Ia .
As a comparison for this, Factor VIIa preparation from example 4 was lyophilized and also heat treated. The activity decreased by 48 ~ thereby.
~ 218~226 Example 11: Isolation of a Factor VIIa/FEI13A preparation A preparation containing Factor VIIa and FEIB-activity was isolated as follows. Fresh, frozen, human citrate plasma was thawed at 0 - +4C and the resulting cryoprecipitate was separated by centrifugation at +2C. At native pH, the cryosupernatant resulting therefrom was mixed with 0.5 g of QAE-Sephadex~) A50 (Pharmacia~ and stirred at +4C for 15 hours. Subsequently, the gel-protein complex was separated from the supernatant by sedimentation and filtration and the fraction containing Factor VIIa and FEIBA was isolated by elution with 3 96 NaCl solution. Factor VIIa and FEIBA were __ quantitatively measured. The preparation contained 12 . 7 U
FVIIa per unit FEIBA.
Example 12: Isolation of a Factor VIIa/FEIBA preparation FEIBA was isolated as described in example 11. However, Fractogel~ EMD T~E (Merck) was used as an ion exchanger for the absorption in a concentration of 5 ml moist gel per liter cryosupernatant. After elution of the activated prothrombin complex ~actors with 3 % NaCl solution, this contained 54 U
FEIBA/ml as well as 0 . 6 U Factor VIIa/U FEIBA. In order to produce the preparation according to the invention, Factor VIIa according to examples 2 - 4 was isolated from the cryosupernatant and the solution containing FEIBA was added such that at least 10 U Factor VIIa/U FEIBA were finally contained .
Example 13: Isolation of a Factor VIIa/FEIBA preparation 218~22~
.
Analogous to example 12, FEIBA was produced again. As an ion exchanger, Q-Sepharose~ Fast Flow (Pharmacia) was used in a concentration of 5 ml per liter cryosupernatant. The FEIB-activity in the obtained eluate was 1. 280 U/ml and 0 . 3 U
Factor VIIa/U FEIBA. In order to produce a preparation with 20 U Factor VIIa/U FEIBA, Factor VIIa was produced, as in example 11, from the same cryosupernatant from which the FEIBA preparation was isolated and mixed with the solution containing FEIBA.
xample 14: In vivo effectiveness of a Factor VIIa/FEIBA
preparation A combination of FEIBA and Factor VIIa produced according to example 9 was tested in rabbits with Factor VIII inhibitor hemophilia as follows. Approximately 2 kg, white, New Zealand rabbits were anesthetized. After onset of the anesthesia, the right femoral vein of each rabbit was prepared and a permanent venous access was created. Through this, 0 . 5 ml/kg body weight of a human Factor VIII lnhibitor plasma ~1500 Bethesda units~ml) was infused over 10 minutes.
30 minutes after completion of the infusion, the hemorrhage characteristics were measured with a modified method according to Giles et al, Blood 60:727-730 (1982) . For this, the fur around a claw of the hind paw of the rabbit was shaved in order to prevent exiting blood from being absorbed to the fur during later bleeding. The apex of the cutis was injured by means of a claw forceps. Immediately thereafter, filters were arranged underneath the wound in such a manner that the blood could directly drop onto the filter without being drawn up from this by capillary effect. Destruction of a forming blood clot was prevented through this precaution.
The filters were changed every two minutes and the exiting 218~22~
blood was collected in fractions. Blood collection was continued for 30 ~inutes. The qualification of bleeding characterlstics was conducted by extraction of the blood collected in fractions on the filter with 5 ml each of 0. 04 %
ammonium hydroxide s~lution over 5 hours. Thereby, the erythrQcytes which were collected with the blood in the filter were lysed. During a 10 minute ultrasound treatment, hemoglobin was extracted and ~uantitatively measured photometrically at 416 nm against a calibration curve, whereby the calibration curve was established by pipetting rabbit blood volumes between 10 ul and 1 ml onto the filters, extracting as described above, and measuring the hemoglobin photometrically at 416 nm. Accordingly, linear calibration curves could be prepared which made possible a direct conversion of the hemoglobin concentration to the amount of -~
blood per filter. The bleeding characteristics of the nail cut were established as the cumulative loss of blood through additive graphic plotting of the individual blood fractions against time. The increase of cumulative bleeding between 10 and ZO minutes of the experiment was employed as a relevant criterion of bleeding and served as a measurement for the bleeding intensity. The average hemorrhage intensity of animals treated in this method is given in the Figure (Fig.
1) .
For testing the active substances, a solution of a pharmaceutical composition containing the respective active ingredients in a volume of 3D ml was continuously infused after inhibitor-induced bleeding in the rabbit with an infusion rate of 1 ml/min and, simultaneously with the beginning of the infusion, the bleeding intensity was re- -established as described above. Accordingly, a FEIBA
preparation produced according to A 350 726 or A 368 883, was 218~22G
administered in a dose of 75 U/FEIBA/kg to correspondingly pretreated rabbits. This dosage led to a drastic reduction in the bleeding intensity (see Fig. 1).
As a control, a buffer without active ingredient was infused which led to no reduction of bleeding. Equally, the administration of FEIBA in a dose of 5 U/kg led to no significant reduction of bleeding intensity.
In contrast, when a preparation according to the invention corresponding to a dose of 5 U FEIBA/kg and 50 U Factor VIIa/kg was given to the rabbits, a reduction in the bleeding intensity resulted comparable with an effective FEIBA dosage, while 50 U Factor VIla/kg had no effect on the abnormally increase-d hemorrhage intensity.
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Claims (29)
1. Pharmaceutical composition with FEIB-activity for treatment of a patient with a blood coagulation disorder caused by a coagulation factor deficiency or inhibitor of a coagulation factor, which composition comprises Factor VIIa and at least one further active ingredient and has an activity of at least 10 Factor VIIa units per unit FEIBA.
2. Pharmaceutical composition according to claim 1, having an activity in the range of 10 to 100 Factor VIIa units per unit FEIBA.
3. Pharmaceutical composition according to claim 2, having an activity in the range of 10 to 20 Factor VIIa units per unit FEIBA.
4. Pharmaceutical composition according to claim 2, having an activity in the range of 10 to 15 Factor VIIa units per unit FEIBA.
5. Pharmaceutical composition according to anyone of claims 1 to 4, additionally comprising at least one of coagulation Factors II, IX and X as a further active ingredient.
6. Pharmaceutical composition according to claim 5, wherein each of said coagulation Factors II, IX and X is present in a ratio of 0.5 to 2 units per unit FEIBA.
7. Pharmaceutical composition according to claims 1 to 4, additionally comprising, besides Factor VIIa, at least one of coagulation Factors II, IX and X as a further active ingredient.
8. Pharmaceutical composition according to claim 7, wherein each of said coagulation Factors II, IX and X is present in a ratio of 0.5 to 1.5 units per unit FEIBA.
9. Pharmaceutical composition according to anyone of claims 1 to 8, further comprising protein C or protein S.
10. Pharmaceutical composition according to claim 1, comprising a fraction which contains purified prothrombin complex and a fraction which contains purified Factor VIIa, wherein the specific activity of Factor VIIa amounts to > 10 U/mg protein, and is treated for the inactivation of viruses.
11. Pharmaceutical composition according to claim 1, comprising a fraction which contains purified partial prothrombin complex and a fraction which contains purified Factor VIIa, whereby the specific activity of Factor VIIa is from < 10 U/mg protein to 1,000 U/mg protein and is treated for the inactivation of viruses.
12. Pharmaceutical composition according to claim 10 or 11, wherein the prothrombin complex is present as activated prothrombin complex or FEIBA preparation.
13. Pharmaceutical composition according to claim 10 or 11, wherein the factors containing prothrombin complex as well as Factor VIIa originate from the same plasma pool.
14. Method for producing a pharmaceutical composition according to any one of claims 1 to 9, which method comprises:
- contacting a fraction which contains coagulation Factor VII with an anion exchanger, whereby coagulation Factor VII is at least partially activated, - separating the anion exchanger and isolating the fraction containing coagulation Factor VIIa, whereby the fraction possesses a FEIBA before contact or a FEIBA is generated in the fraction.
- contacting a fraction which contains coagulation Factor VII with an anion exchanger, whereby coagulation Factor VII is at least partially activated, - separating the anion exchanger and isolating the fraction containing coagulation Factor VIIa, whereby the fraction possesses a FEIBA before contact or a FEIBA is generated in the fraction.
15. Method according to claim 14, wherein the anion exchanger has quaternary ammonium groups.
16. Method according to claim 15, wherein the anion exchanger is a QAE, TAE or Q type anion exchanger.
17. Method for producing a pharmaceutical composition with FEIB-activity according to one of claims 10 to 12, which method comprises the following steps:
a) isolating coagulation Factors II, IX and X as individual components or as a mixture from one or more starting materials which contain(s) these coagulation factors, and b) isolating coagulation Factors VII/VIIa from a starting material which contains coagulation Factor VII, whereby coagulation Factor VII is at least partially activated to VIIa, and c) either pretreating or treating the starting materials or the respective coagulation factors for inactivation of infectious agents, and d) mixing the isolated coagulation factors with a pharmaceutically acceptable carrier.
a) isolating coagulation Factors II, IX and X as individual components or as a mixture from one or more starting materials which contain(s) these coagulation factors, and b) isolating coagulation Factors VII/VIIa from a starting material which contains coagulation Factor VII, whereby coagulation Factor VII is at least partially activated to VIIa, and c) either pretreating or treating the starting materials or the respective coagulation factors for inactivation of infectious agents, and d) mixing the isolated coagulation factors with a pharmaceutically acceptable carrier.
18. Method according to claim 17, wherein the coagulation factors are isolated from a single starting material.
19. Method according to claim 18, wherein said single starting material is a plasma pool or plasma fraction.
20. Method according to claim 17, wherein a heat treatment is carried out for the inactivation of infectious agents.
21. Method according to claim 20, wherein a heat treatment of the coagulation factors is carried out in a solid state.
22. Method according to claim 20, wherein the heat treatment occurs in the absence of albumin.
23. Method according to any one of claims 17 to 22, wherein treatment for inactivation of infectious agents is carried out after the mixing of the coagulation factors with the pharmaceutically acceptable carrier.
24. Method according to one or more of claims 17 to 23, wherein coagulation Factor VIIa is isolated by hydrophobic chromatography.
25. Method according to claim 17 to 23, wherein coagulation Factors IX, X as well as VII are at least partially activated during isolation.
26. Use of a combination of Factor VIIa and at least one further active ingredient for production of a pharmaceutical composition with FEIB-activity according to any one of claims 1 to 13 for rapid termination of hemorrhage in a patient with coagulation factor deficiency.
27. Use according to claim 26, wherein the composition is suitable for rapid cessation of hemorrhaging caused by an inhibitor of a coagulation factor.
28. A commercial package comprising a pharmaceutical composition according to any of one of claims 1 to 13 together with restrictions for use thereof is rapid termination of hemorrhage in a patient with coagulation factor deficiency.
29. A commercial package according to claim 28, wherein said composition is suitable for rapid cessation of hemorrhaging caused by an inhibitor of a coagulation factor.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19531637A DE19531637A1 (en) | 1995-08-28 | 1995-08-28 | Pharmaceutical composition for the treatment of blood coagulation disorders, method for the production thereof and their use |
| DE19531637.1 | 1995-08-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2184226A1 true CA2184226A1 (en) | 1997-03-01 |
Family
ID=7770599
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002184226A Abandoned CA2184226A1 (en) | 1995-08-28 | 1996-08-27 | Pharmaceutical composition for the treatment of blood coagulation diseases, methods for the production thereof and its use |
Country Status (6)
| Country | Link |
|---|---|
| US (2) | US5891843A (en) |
| EP (1) | EP0765669A1 (en) |
| JP (1) | JPH09110715A (en) |
| CA (1) | CA2184226A1 (en) |
| DE (1) | DE19531637A1 (en) |
| NO (1) | NO963568L (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU758152B2 (en) * | 1998-06-17 | 2003-03-13 | Baxalta GmbH | Pharmaceutical factor VII preparation |
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| CZ81997A3 (en) * | 1996-03-20 | 1997-10-15 | Immuno Ag | Pharmaceutical preparation for treating disorders connected with blood clotting, process of its preparation and use |
| AT405608B (en) * | 1997-04-08 | 1999-10-25 | Immuno Ag | METHOD FOR INACTIVATING PATHOGENS, ESPECIALLY VIRUSES, IN A BIOLOGICAL MATERIAL |
| AU737566B2 (en) | 1997-04-08 | 2001-08-23 | Baxalta GmbH | An immunotolerant prothrombin complex preparation |
| US20050032690A1 (en) * | 1997-09-10 | 2005-02-10 | Rojkjaer Lisa Payne | Factor VII polypeptides for preventing formation of inhibitors in subjects with haemophilia |
| ATA159597A (en) * | 1997-09-19 | 2000-09-15 | Immuno Ag | PREPARATION FOR TREATING BLOOD COagulation disorders |
| US6747003B1 (en) | 1997-10-23 | 2004-06-08 | Regents Of The University Of Minnesota | Modified vitamin K-dependent polypeptides |
| US7247708B2 (en) | 1997-10-23 | 2007-07-24 | Regents Of The University Of Minnesota | Modified vitamin K-dependent polypeptides |
| JP4676585B2 (en) | 1999-12-24 | 2011-04-27 | 一般財団法人化学及血清療法研究所 | Pharmaceutical composition for treatment / prevention of diseases based on abnormal blood coagulation |
| DE60138364D1 (en) | 2000-02-11 | 2009-05-28 | Bayer Healthcare Llc | CLEANING FACTOR VII OR VIIA CONJUGATE |
| DE10012732A1 (en) * | 2000-03-18 | 2001-09-20 | Aventis Behring Gmbh | Thrombin composition, for use as hemostatic or as a component of fibrin glues, comprises non-covalently bonded inhibitor for stabilization |
| US7220837B1 (en) | 2000-04-28 | 2007-05-22 | Regents Of The University Of Minnesota | Modified vitamin K-dependent polypeptides |
| US7812132B2 (en) | 2000-04-28 | 2010-10-12 | Regents Of The University Of Minnesota | Modified vitamin K-dependent polypeptides |
| US20030211094A1 (en) | 2001-06-26 | 2003-11-13 | Nelsestuen Gary L. | High molecular weight derivatives of vitamin k-dependent polypeptides |
| US20030203845A1 (en) * | 2001-02-05 | 2003-10-30 | Knudsen Jens Bjerre | Combined use of factor VII polypeptides and factor IX polypeptides |
| EP1359936B1 (en) * | 2001-02-05 | 2008-02-20 | Novo Nordisk Health Care AG | Combined use of factor vii polypeptides and factor viii polypeptides |
| EP1407780B1 (en) * | 2001-07-10 | 2013-05-01 | The Chemo-Sero-Therapeutic Research Institute | Pharmaceutically stable hemostatic compositions |
| BR0215216A (en) | 2001-12-21 | 2004-11-16 | Novo Nordisk Healthcare Ag | Liquid aqueous composition, method for preparing a liquid aqueous composition of a factor vii polypeptide, use of a liquid aqueous composition, and method for treating a factor vii response syndrome |
| NZ536340A (en) * | 2002-04-30 | 2006-09-29 | Maxygen Holdings Ltd | Factor VII or VIIa polypeptide variants with N-glycosylation |
| ES2523655T5 (en) | 2002-06-21 | 2018-04-23 | Novo Nordisk Health Care Ag | Solid stabilized compositions of Factor VIIa polypeptides |
| AU2003277034A1 (en) * | 2002-09-30 | 2004-04-23 | Socratech L.L.C. | Protein s protects the nervous system from injury |
| US20040198660A1 (en) * | 2002-11-06 | 2004-10-07 | Petersen Lars Christian | Tissue factor antagonist and protein C polypeptide compositions |
| US20070142272A1 (en) * | 2003-01-24 | 2007-06-21 | Zlokovic Berislav V | Neuroprotective activity of activated protein c independent of its anticoagulant activity |
| CN101818138A (en) * | 2003-03-18 | 2010-09-01 | 诺和诺德医疗保健公司 | Method for the production of GLA-residue containing serine proteases |
| AU2004221761B2 (en) * | 2003-03-20 | 2010-01-07 | Bayer Healthcare Llc | FVII or FVIIa variants |
| US7897734B2 (en) | 2003-03-26 | 2011-03-01 | Novo Nordisk Healthcare Ag | Method for the production of proteins |
| CA2465004A1 (en) * | 2003-05-19 | 2004-11-19 | National Institute For Biological Standards And Control | Composition |
| EP1628677B1 (en) | 2003-05-23 | 2010-01-13 | Novo Nordisk Health Care AG | Protein stabilization in solution |
| PT1644504E (en) * | 2003-06-19 | 2010-03-22 | Bayer Healthcare Llc | Factor vii or viia gla domain variants |
| ATE547114T1 (en) | 2003-06-25 | 2012-03-15 | Novo Nordisk Healthcare Ag | LIQUID COMPOSITIONS OF FACTOR VII POLYPEPTIDES |
| EP1656158B1 (en) | 2003-08-14 | 2016-03-09 | Novo Nordisk Health Care AG | Liquid, aqueous pharmaceutical composition of factor vii polypeptides |
| EP2311437A1 (en) | 2003-12-19 | 2011-04-20 | Novo Nordisk Health Care AG | Stabilised compositions of factor VII polypeptides |
| ATE455861T1 (en) | 2004-05-04 | 2010-02-15 | Novo Nordisk Healthcare Ag | O-LINKED GLYCOFORMS OF FACTOR VII AND METHOD FOR THE PRODUCTION THEREOF |
| US20070037966A1 (en) * | 2004-05-04 | 2007-02-15 | Novo Nordisk A/S | Hydrophobic interaction chromatography purification of factor VII polypeptides |
| JP2008507561A (en) * | 2004-07-23 | 2008-03-13 | ザ ユニバーシティ オブ ロチェスター | Inhibition of adverse effects of plasminogen activator in the brain by activated protein C |
| WO2006035060A2 (en) * | 2004-09-29 | 2006-04-06 | Novo Nordisk Health Care Ag | Purification of a bulk of a factor vii polypeptide by fractionated elution from an anion-exchange material |
| US20070129298A1 (en) * | 2005-07-22 | 2007-06-07 | Maxygen Holdings, Ltd. | In-solution activation of factor vii |
| US7807435B2 (en) * | 2005-08-11 | 2010-10-05 | Baxter International Inc. | Method for the purification of alpha-1 proteinase inhibitor (a1PI) |
| ES2257225B1 (en) * | 2006-02-17 | 2007-03-16 | Grifols, S.A | THERAPEUTIC PREPARATION OF FVIIA OF VERY HIGH PURITY AND METHOD FOR OBTAINING IT. |
| AR102331A1 (en) * | 2014-08-12 | 2017-02-22 | Baxalta Inc | ACTIVATION OF FACTOR X |
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| AT154793B (en) | 1936-09-03 | 1938-10-25 | Telefunken Gmbh | Multigrid tube. |
| AT350726B (en) * | 1976-08-30 | 1979-06-11 | Immuno Ag | PROCESS FOR THE PRODUCTION OF A BLOOD CLOTIFYING PREPARATION FROM HUMAN BLOOD PLASMA |
| WO1981002105A1 (en) * | 1980-01-28 | 1981-08-06 | Baxter Travenol Lab | Therapeutic compositions & methods for manufacture and use |
| US4287180A (en) * | 1980-01-28 | 1981-09-01 | Baxter Travenol Laboratories, Inc. | Method for treating blood clotting factor inhibitors |
| US4364861A (en) * | 1980-05-27 | 1982-12-21 | Cutter Laboratories, Inc. | Blood-coagulation-promoting products and methods of preparing them |
| AT368883B (en) * | 1980-07-22 | 1982-11-25 | Immuno Ag | METHOD FOR PRODUCING A NEW HUMAN PROTEIN-BASED PREPARATION PROCESSING BLOOD |
| US4382083A (en) * | 1981-06-25 | 1983-05-03 | Baxter Travenol Laboratories, Inc. | Therapeutic method for treating blood-clotting defects with factor VIIa |
| ATE36457T1 (en) * | 1983-05-02 | 1988-09-15 | Immuno Ag | METHOD OF INACTIVATING REPRODUCTIVE pathogens. |
| AT389815B (en) * | 1984-03-09 | 1990-02-12 | Immuno Ag | METHOD FOR INACTIVATING VARIABLE FILTERABLE DISEASERS IN BLOOD PRODUCTS |
| DE3740520A1 (en) * | 1987-11-30 | 1989-06-08 | Hans Dr Rer Nat Scheefers | Rapid process for obtaining a factor VII preparation and its use |
| FR2632524B1 (en) * | 1988-06-09 | 1992-03-13 | Fondation Nale Transfusion San | PROCESS FOR THE PREPARATION OF A CONCENTRATED FRACTION IN FACTOR VIIA AND ITS APPLICATION AS A MEDICAMENT |
| US5472850A (en) * | 1991-04-10 | 1995-12-05 | Oklahoma Medical Research Foundation | Quantitative clotting assay for activated factor VII |
| DE69023829T2 (en) * | 1989-08-04 | 1996-07-04 | Komori Printing Mach | Printing plate attachment for a sheet printing machine. |
| AT402891B (en) * | 1991-06-20 | 1997-09-25 | Immuno Ag | METHOD FOR PRODUCING AN INACTIVATED BLOOD PRODUCT |
| FR2684999A1 (en) * | 1991-12-16 | 1993-06-18 | Aquitaine Dev Transf Sanguine | PROCESS FOR MANUFACTURING HIGH-PURITY ACTIVE FACTOR VII CONCENTRATE ESSENTIALLY HAVING DEPENDENT VITAMIN K FACTORS AND VIIICAG FACTORS |
| AT402151B (en) * | 1993-08-03 | 1997-02-25 | Immuno Ag | Process for the production of a virus-safe biological product |
-
1995
- 1995-08-28 DE DE19531637A patent/DE19531637A1/en not_active Withdrawn
-
1996
- 1996-07-09 EP EP96111052A patent/EP0765669A1/en not_active Withdrawn
- 1996-08-22 US US08/701,755 patent/US5891843A/en not_active Expired - Lifetime
- 1996-08-27 CA CA002184226A patent/CA2184226A1/en not_active Abandoned
- 1996-08-27 NO NO963568A patent/NO963568L/en not_active Application Discontinuation
- 1996-08-28 JP JP8226561A patent/JPH09110715A/en active Pending
-
1997
- 1997-09-11 US US08/926,845 patent/US6013620A/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU758152B2 (en) * | 1998-06-17 | 2003-03-13 | Baxalta GmbH | Pharmaceutical factor VII preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH09110715A (en) | 1997-04-28 |
| US5891843A (en) | 1999-04-06 |
| NO963568L (en) | 1997-03-03 |
| US6013620A (en) | 2000-01-11 |
| EP0765669A1 (en) | 1997-04-02 |
| NO963568D0 (en) | 1996-08-27 |
| DE19531637A1 (en) | 1997-03-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |
Effective date: 20030827 |