CN1596122A - Pharmaceutical composition comprising factor vii polypeptides and pai-1 polypeptides - Google Patents
Pharmaceutical composition comprising factor vii polypeptides and pai-1 polypeptides Download PDFInfo
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- CN1596122A CN1596122A CNA028236777A CN02823677A CN1596122A CN 1596122 A CN1596122 A CN 1596122A CN A028236777 A CNA028236777 A CN A028236777A CN 02823677 A CN02823677 A CN 02823677A CN 1596122 A CN1596122 A CN 1596122A
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Abstract
The present invention relates to a composition comprising factor VII or a factor VII-related polypeptide, and PAI-1 or a PAI-1-related polypeptide, and the use thereof for treating bleeding episodes.
Description
Invention field
The present invention relates to comprise the compositions of factor VII or factor VII related polypeptide and PAI-1 or PAI-1 related polypeptide.The preparation that is combined in that the invention still further relates to factor VII or factor VII related polypeptide and PAI-1 or PAI-1 related polypeptide is used for the treatment of the experimenter who suffers from bleeding episodes or prevents application in their medicine of bleeding episodes.The invention still further relates to the Therapeutic Method of experimenter's bleeding episodes, and relate to the method that strengthens grumeleuse formation in the experimenter.The invention still further relates to the test kit that contains these chemical compounds.
Background of invention
Form complex between the FVIIa in the circulation and start hemostasis because of vascular damaged contacts the tissue factor (TF) of blood circulation and is present in the amount that is equivalent to about 1% total FVII albumen quality.This species complex is anchored on the cell that contains TF and makes FX on the cell surface activate into Fxa and activates into FIXa with FIX.FXa activates into thrombin with thrombinogen, and thrombin makes FVIII, FV, FXI and FXIII activation.In addition, the limited amount thrombin that forms in the hemostatic initial step also makes platelet activation.After thrombin was to platelet function, these platelet changed shape and expose its lip-deep charged phospholipid.This activatory platelet surface has formed the template of further FX activation and whole thrombin generations.The lip-deep further FX activation of activated blood platelet is to take place by the FIXa-FVIIIa complex that forms on the activated blood platelet surface, and FXa changes into thrombin with thrombinogen then, still keeps from the teeth outwards simultaneously.Thrombin changes into fibrinogen insoluble subsequently and makes initial platelet plug keep stable fibrin.This process is a compartmentation, and promptly this process is positioned TF expression or exposure portion, thus the activatory harm of the general of blood coagulation system is reduced to bottom line.The insoluble fibrin that forms described platelet plug is crosslinked and further stabilisation by the catalytic fibrin fiber of FXIII-.
FVIIa is present in the blood plasma mainly as the strand proenzyme, and it is cracked into its double-stranded activated form FVIIa by FXa.The activation factor VIIa (rFVIIa) that will recombinate is developed as short hemorrhage (pro-haemostatic agent).Give rFVIIa and can in suffering from the hemorrhage hemophiliac that to treat with the thrombin product because of antibody formation, provide fast short hemostasis reaction (pro-haemostatic response) efficiently.It is normal but hemorrhage over-drastic experimenter is taken place successfully to treat the hemorrhage experimenter of factor VII deficiency or blood coagulation system with FVIIa.In these researchs, do not find any adverse side effect (particularly taking place under the situation of thromboembolism) of rFVIIa as yet.
The outside gives the formation that FVIIa can increase thrombin on the activated blood platelet surface extraly.This situation can take place in the hemophiliac of the effective way that lacks FIX or FVIII, lost that complete thrombin forms thus.In addition, few or exist under the hematoblastic situation of functional defect in the platelet amount, extra FVIIa has increased thrombin formation.
(Novo NordiskA/S Denmark) sells the commodity preparation of recombined human FVIIa as NovoSeven .The indication of Novoseven has treatment A type and haemophilia B patient's bleeding episodes.Novoseven is unique reorganization FVIIa that can obtain on market, be used for effectively and reliably treating bleeding episodes.
Confirmed that plasminogen activator inhibitor (PAI-1) plays an important role in the Proteolytic enzyme of regulating the fibrinolysin mediation.The PAI-1 of integumentary pattern plasminogen activator inhibitor (e-PAI) can suppress urokinase type plasminogen activator (u-PA) and tissue plasminogen activator (t-PA) in being also referred to as, generally available from human endothelial cell, human blood platelets and rat hepatocytes oncocyte (HTC).The PAI-1 inhibitor can be as U.S. Pat 6, described in 271,352 in the human fibrosarcoma cell of HT-1080 strain purification, or as van Mourik, J.A. etc. (1984) at " journal of biological chemistry " (J.Biol.Chem.) 259, described in the 14914-14921 from cattle endotheliocyte purification.PAI-1 is the strand glycoprotein (Van Mourik J A etc. " journal of biological chemistry " are (1984) 259:14914-14921 (J.Biol.Chem.)) with 50kDa molecular weight, and it is at present known tPA and the effective inhibitors of uPA " european journal of biological chemistry " (Eur J Biochem) (1989) 186:523-533 such as () Lawrence D to strand and double chain form.PAI-1 also suppresses (Biochemistry) (1988) 27:2911-2918 such as fibrinolysin and trypsin Hekman C M " biochemistrys "), but also Trombin inhibiting and activated protein C, but effect is extremely low.
As everyone knows, the easier generation complication of any hemorrhage experimenter does not take place than those in the generation excessive hemorrhage and experimenter that need transfuse blood relevant with operation or severe trauma.Yet, need give human blood or blood products (be used for the treatment of coagulation defect etc. platelet, leukocyte, derive from the concentrate of blood plasma) the hemorrhage relevant complication of harm that shifts with Human virus's (hepatitis, HIV, parvovirus and other unknown at present virus) of also may causing of moderate.The extensive hemorrhage generation that can cause comprising the multiple organ failure, MOF of lung and renal function injury that needs big blood transfusion.In case the experimenter has been developed these severe complications, then begin to occur a succession of incident that relates to many cytokines and inflammatory reaction, make that extremely difficulty is carried out any treatment, and unsuccessful lamentedly usually.Therefore, perform the operation and treat main purpose in the big histologic lesion and be to avoid hemorrhage or it is reduced to bottom line.Hemorrhage or it is reduced to bottom line for fear of this class, importantly guarantee to form to belong to and be difficult for by the dissolved stable and firm tampon of fibrinoclase (haemostatic plugs).In addition, importantly guarantee to form fast and effectively this class plug or grumeleuse.
At present, because the half-life of FVIIa short (2.5 hours), it is above so that keep certain hemostatic capability level to need to be administered once, so treat the experimenter that bleeding episodes takes place several times by injection or infusion FVIIa usually, comprise wound victim and the hemorrhage experimenter relevant with operation.Make hemorrhage stopping more rapidly having important beneficial effect for this class experimenter.So need to reduce administration number of times to stop hemorrhage and to keep hemostasis.
European patent EP 225,160 (Novo Nordisk) relates to and being used for the treatment of not is the FVIIa compositions and the method for the hemorrhage that caused by deficiency of coagulation factors or blood coagulation factor inhibitors.
European patent EP 82,182 (Baxter Travenol Lab.) relates to and is used to resist experimenter's deficiency of coagulation factors or the inhibitor factor VIIa compositions to the influence of thrombin.
International Patent Publication No. WO 93/06855 (Novo Nordisk) relates to the topical application of FVIIa.
Still the experimenter who needs in this area the experience bleeding episodes improves the demand of therapy, comprises the experimenter with following situation: the bleeding episodes that causes because of the histologic lesion of operation, wound or other form; The coagulopathy that brings out comprises the experimenter's that repeatedly transfuses blood coagulopathy; Congenital or acquired blood coagulation or hemorrhage comprise liver function decline (" hepatopathy "); Platelet function defective or platelet counts reduce; Essential blood coagulation " chemical compound " (for example platelet or Feng's von willebrand's factor albumen) lacks or is unusual; Fibrinolysis increases; Anticoagulant therapy or thrombolytic therapy; Or stem cell transplantation.
Still need in this area can strengthen solidify, strengthen or guarantee to stablize tampon (haemostatic plugs) but form or strengthen subject experimenter's convenience or the experimenter, particularly have among the unusual experimenter of thrombin generation a method that realizes the improved reliable and extensive use of complete hemostatic.Also need the method that has shortened bleeding stopping period.
Summary of the invention
An object of the present invention is to provide the compositions that can be effective to treat or prevent bleeding episodes and disorders of blood coagulation.
Second purpose of the present invention provides the compositions that can be effective to treat or prevent bleeding episodes or be used as the single unit dosage forms of Procoagulants.Another object of the present invention provides and shows synergistic compositions, Therapeutic Method or test kit.
Another object of the present invention provides and do not show significant side effects, such as the activatory compositions of high-level general coagulation system, Therapeutic Method or test kit.
When reading this description, obviously can draw other purpose of the present invention.
The present invention provides the pharmaceutical composition that comprises factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide in aspect first.
The present invention provides the test kit that contains the ingredient that is used for the treatment of bleeding episodes in aspect second, comprising:
A) can accept the preparation of carrier on the factor VII of the effective dose of first unit dosage forms or relevant polypeptide of factor VII and the medicine;
B) can accept the preparation of carrier on the PAI-1 of the effective dose of second unit dosage forms or PAI-1 related polypeptide and the medicine; With
C) be used to comprise the container apparatus of described first and second unit dosage forms.
The present invention provides in aspect the 3rd the preparation that is combined in of factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide to be used for the treatment of application in the medicine of the bleeding episodes among the experimenter.The present invention provides in one aspect of the method as any described compositions among the claim 1-18 and has been used for the treatment of application in the medicine of bleeding episodes among the experimenter in preparation.
In the different embodiment of the present invention, described medicine can be used for reducing acquisition stop blooding fully the required time, reduce and keep the required time of hemostasis, reduce setting time, prolong EGCT and increase clot strength.
In different embodiments, described medicine is used for the treatment of the experimenter who experiences bleeding episodes because of following situation: the histologic lesion of operation, wound or other form; Coagulopathy comprises the experimenter's that repeatedly transfuses blood coagulopathy; Congenital or acquired blood coagulation or hemorrhage comprise liver function decline (" hepatopathy "); Platelet function defective or platelet counts reduce; Essential blood coagulation " chemical compound " (for example platelet or Feng's von willebrand's factor albumen) lacks or is unusual; Fibrinolysis increases; Anticoagulant therapy or thrombolytic therapy; Or stem cell transplantation.In a series of embodiments, hemorrhage occurring in such as brain, interior ear field, eye, liver, lung, tumor tissues, the such organ of gastrointestinal tract; In another serial embodiment, hemorrhage is that dispersivity is hemorrhage, such as hemorrhagic gastritis and flooding.In another serial embodiment, bleeding episodes be with have acute hemarthrosis (intraarticular is hemorrhage), chronic hemophilic arthosis, hematoma (for example behind muscle, the peritoneum, after Sublingual and the pharynx), other tissue in the experimenter of hemorrhage, hematuria (hemorrhage from the kidney road), cerebral hemorrhage, operation (for example hepatectomy), exodontia and gastrointestinal hemorrhage (for example UGI is hemorrhage) in operation or relevant hemorrhage of wound.In one embodiment, described medicine is used for the treatment of because of experimenter's wound or operation or platelet count or the active bleeding episodes that is caused that reduces.
The present invention provides the Therapeutic Method of experimenter's internal hemorrhage outbreak in one aspect of the method, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can effectively be treated hemorrhage together.
The present invention provides the method that reduces setting time among the experimenter in one aspect of the method, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can effectively reduce setting time together.
The present invention provides hemostatic method in the enhancing experimenter in one aspect of the method, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can strengthen hemostasis together effectively.
The present invention provides the method that prolongs EGCT in the experimenter in one aspect of the method, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can effectively prolong EGCT together.
The present invention provides the method that increases clot strength in the experimenter in one aspect of the method, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can increase clot strength together effectively.
In the embodiment of a series of described methods, give described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide with single dosage form.
In the embodiment of another serial described method, give described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide with first dosage form of the preparation that contains factor VII or the relevant polypeptide of factor VII and the form of second dosage form of the preparation that contains PAI-1 or PAI-1 related polypeptide.In a series of its embodiments, give described first dosage form and the interval that gives between described second dosage form is no more than 15 minutes.
The present invention provides the test kit that contains the treatment articles that is useful on bleeding episodes in one aspect of the method, comprising:
D) can accept carrier on the PAI-1 of the factor VII of the effective dose of single unit dosage forms or relevant polypeptide of factor VII and effective dose or PAI-1 related polypeptide and the medicine; With
E) be used to comprise the container apparatus of described single unit dosage forms.
In a series of embodiments of the present invention, described factor VII or the relevant polypeptide of factor VII are the relevant polypeptide of factor VII.In a series of embodiments of the present invention, the relevant polypeptide of described factor VII is a factor VII aminoacid sequence variant.In one embodiment, when measuring with as described in this manual " extracorporeal hydrolysis test ", the ratio of the activity of the activity of the relevant polypeptide of described factor VII and natural human factor VIIa (wild type FVIIa) is at least about 1.25.
In a series of embodiments of the present invention, described factor VII or the relevant polypeptide of factor VII are factor VII.In one embodiment, described factor VII is the factor VII of cattle, pig, dog, horse, Mus or salmon.In another embodiment, factor VII prepares with recombination form.In another embodiment, described factor VII derives from blood plasma.In preferred embodiments, described factor VII is the reorganization human factor VII.In a series of embodiments of the present invention, described factor VII or the relevant polypeptide of factor VII are its activated form.In a preferred embodiment of the invention, described factor VII is the recombined human factor VIIa.
In a series of embodiments, described PAI-1 or PAI-1 related polypeptide are the PAI-1 related polypeptides.In one embodiment, described PAI-1 related polypeptide is a PAI-1 aminoacid sequence variant.In one embodiment, when measuring with as described in this manual " PAI-1 test ", the ratio of the activity of the activity of described PAI-1 related polypeptide and natural human plasma PAI-1 (wild type PAI-1) is at least about 1.25.In one embodiment, described PAI-1 or PAI-1 related polypeptide are the PAI-1 polypeptide.In one embodiment, described PAI-1 is people PAI-1.In one embodiment, described PAI-1 is the PAI-1 of cattle, pig, dog, horse, Mus or rat.In preferred embodiments, described PAI-1 prepares with recombination form.In one embodiment, described PAI-1 derives from platelet; In another embodiment, it derives from endotheliocyte.In preferred embodiments, described PAI-1 is recombined human PAI-1.In one embodiment, described PAI-1 related polypeptide is the PAI-1 fragment.In one embodiment, described PAI-1 related polypeptide is a heterozygosis PAI-1 polypeptide, for example pig/people's heterozygote.
In one embodiment, the weight ratio of described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide existence is about 100: about 1: 100 (w/w factor VII: PAI-1) of 1-.
In one embodiment, described VII related polypeptide is (promptly to have U.S. Pat 4 with wild type factor VII, the polypeptide of disclosed aminoacid sequence in 784,950) compares and have the aminoacid sequence variant that is no more than 20 aminoacid replacement, disappearance or insertions.In another embodiment, described factor VII variant is compared with wild type factor VII to have and is no more than 15 aminoacid replacement, disappearance or insertions; In other embodiments, described factor VII variant compare with wild type factor VII have be no more than 10 aminoacid, such as 8,6,5 or 3 aminoacid replacement, disappearance or insertions.In one embodiment, described factor VII variant is selected from L305V-FVIIa, L305V/M306D/D309S-FVIIa, L305I-FVIIa, L305T-FVIIa, F374P-FVIIa, V158T/M298Q-FVIIa, V158D/E296V/M298Q-FVIIa, K337A-FVIIa, M298Q-FVIIa, V158D/M298Q-FVIIa, L305V/K337A-FVIIa, V158D/E296V/M298Q/L305V-FVIIa, V158D/E296V/M298Q/K337A-FVIIa, V158D/E296V/M298Q/L305V/K337A-FVIIa, K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII, the group of V158D/M298K-FVII and S336G-FVII.
In another embodiment, the VII related polypeptide is compared the tissue factor dependent/non-dependent activity with increase with natural human proconvertin a.In another embodiment, this increase active and without the change of substrate specificity.In another embodiment of the invention, the combination of VII related polypeptide and tissue factor does not suffer damage, and the VII related polypeptide has the activity of wild type factor VIIa at least when combining with tissue factor.
In preferred embodiments, described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide are recombined human factor VIIa and recombined human PAI-1.
In one embodiment, the setting time of mammalian obtains reducing.In another embodiment, the hemostasis in the mammalian is enhanced.In another embodiment, the EGCT in the mammalian obtains prolonging.In another embodiment, the clot strength in the mammalian is improved.In one embodiment, described mammalian is a human blood.In another embodiment, described mammalian is a normal human blood.In one embodiment, described blood is from the unusual experimenter of thrombin generation.In one embodiment, described blood is from the experimenter's of one or more deficiencies of coagulation factors blood wherein; In another embodiment, described blood is from the blood that has at the experimenter of the inhibitor of one or more thrombins; In one embodiment, described blood is from having the experimenter that fibrinogen concentration reduces; In one embodiment, described blood is PAI-1-defect type human blood.In a series of embodiments, described blood is blood plasma.
In one embodiment of the invention, described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide are included in and in the described compositions hemorrhage are only arranged.In another embodiment, described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide are included in and in the described compositions active hemorrhage are only arranged.In another embodiment, described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide be give the experimenter thrombin only arranged.In one embodiment of the invention, described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide be give the patient activating agent only arranged.In one embodiment, described compositions is substantially free of thrombin or thrombinogen; In another embodiment, described compositions is substantially free of FX; In another embodiment, described compositions is substantially free of FXa.
In another embodiment, pharmaceutical composition is formulated into and is used for intravenous administration, preferably injection or infusion, particularly injection.In one embodiment, described compositions contains at least a pharmaceutically acceptable excipient or carrier.
In one embodiment of the invention, described compositions is single unit dosage forms, and wherein this single unit dosage forms contains this two kinds of thrombins.In one embodiment of the invention, described compositions is the preparation that comprises VII or the relevant polypeptide of factor VII as the preparation of first unit dosage forms and PAI-1 or PAI-1 related polypeptide as second unit dosage forms and comprise the test kit of the container apparatus that is used to comprise described first and second unit dosage forms.In one embodiment, described compositions or test kit can suitably further comprise the description that is used for giving respectively described compositions or independent element.
In one embodiment of the invention, give described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide with single dosage form.In one embodiment of the invention, give described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide with first unit dosage forms of the preparation that comprises VII or the relevant polypeptide of factor VII and the form of second unit dosage forms of the preparation that comprises PAI-1 or PAI-1 related polypeptide.
In one embodiment of the invention, give described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide simultaneously.In another embodiment, give described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide successively.In one embodiment, be no more than 15 minutes, preferred 10 minutes, more preferably 5 minutes, more preferably 2 minutes interval gives described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide.In one embodiment, with 2 hours at the most, preferred 1-2 hour, more preferably at the most 1 hour, more preferably 30 minutes-1 hour, more preferably at the most 30 minutes, more preferably 15-30 minute interval gives described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide.
In one embodiment, the effective dose of described factor VII or the relevant polypeptide of factor VII is about 0.05mg/ days-Yue 500mg/ days (70-kg experimenter).In one embodiment, the effective dose of the preparation of PAI-1 or PAI-1 related polypeptide is about 0.01mg/ days-Yue 500mg/ days (70-kg experimenter).
In one embodiment, described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide are about 100 with weight ratio: (w/w factor VII: ratio PAI-1) existed 1-in about 1: 100.
In one embodiment of the invention, described pharmaceutical composition is single unit dosage forms, mainly is made up of with the branch that becomes that one or more are selected from the group that can accept carrier, stabilizing agent, detergent, neutral salt, antioxidant, antiseptic and protease inhibitor on the medicine the preparation of preparation, PAI-1 or the PAI-1 related polypeptide of factor VII or the relevant polypeptide of factor VII.
In another embodiment of the invention, the kit form that described pharmaceutical composition is made up of ingredient, wherein comprise first unit dosage forms and second unit dosage forms, described first unit dosage forms mainly is selected to become to be grouped in the group that can accept carrier, stabilizing agent, detergent, neutral salt, antioxidant, antiseptic and protease inhibitor on the medicine by the preparation of factor VII or the relevant polypeptide of factor VII and one or more; Described second unit dosage forms mainly is selected to become to be grouped in the group that can accept carrier, stabilizing agent, detergent, neutral salt, antioxidant, antiseptic and protease inhibitor on the medicine by the preparation of PAI-1 or PAI-1 related polypeptide and one or more.
In another embodiment, described experimenter is the people; In another embodiment, described experimenter to have thrombin generation unusual; In one embodiment, described experimenter's fibrinogen plasma concentration reduces (for example experimenter who repeatedly transfuses blood); In one embodiment, Factor IX or FIX plasma concentration reduce among the described experimenter.
The present invention relates in one aspect of the method and treats the experimenter to comparing enhancing hemostatic method in suffering from the reactive syndromic experimenter of factor VII with factor VII as unique coagulated protein, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can strengthen hemostasis together effectively.
The present invention relates to the method that strengthens the formation of experimenter's intravascular coagulation enzyme in one aspect of the method, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can strengthen thrombin together effectively and form.
The present invention relates in one aspect of the method comparing when treating the experimenter with factor VII as unique coagulated protein in suffering from the reactive syndromic experimenter of factor VII and strengthens the method that thrombin forms, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can strengthen thrombin together effectively and form.
The present invention relates in one aspect of the method comparing when treating the experimenter with factor VII as unique coagulated protein in suffering from the reactive syndromic experimenter of factor VII and reduces the method for finishing the required thrombin administration number of times of hemostasis, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can reduce the coagulation factor protein administration number of times together effectively.
The present invention relates in one aspect of the method to suffering from the hemorrhage method for the treatment of among the reactive syndromic experimenter of factor VII, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can be treated hemorrhage together effectively.
In one embodiment, described factor VII is people's recombinant factor VIIa (rFVIIa).In another embodiment, rFVIIa be NovoSeven (NovoNordisk A/S, Bagsvaerd, Denmark).
The present invention relates to being combined in of factor VII or relevant polypeptide of factor VII and PAI-1 in one aspect of the method and prepares the application that can be used for strengthening in the medicine that the fibrin piece forms in the mammalian plasma.
The present invention relates to the method that strengthens fibrin piece formation among the experimenter in one aspect of the method, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can be treated hemorrhage together effectively.
The accompanying drawing catalogue
Accompanying drawing 1: add FVIIa and cause EGCT to prolong in the dose dependent mode.This 10nM FVIIa that acts on is best down.
Accompanying drawing 2: under the situation that has 10nM FVIIa to exist, add PAI-1 and cause EGCT further to prolong.This effect is a dose dependent, and is best under 10nM PAI-1.
Accompanying drawing 3: utilize combination that thrombelastography (roTEG) measured value analyzes FVIIa and PAI-1 to maximum grumeleuse hardness (Maximal Clot Firmness, MCF) and the influence of grumeleuse dissolved toleration that t-PA is mediated.In the human normal plasma, add PAI-1 (30nM) and can not change MCF, but the half EGCT can be extended to 16.1 minutes.The time scale Minute.
Accompanying drawing 4: utilize the influence of the dissolved toleration that combination that thromboelastogram tracing (roTEG) measured value analyzes FVIIa and PAI-1 mediates t-PA maximum grumeleuse hardness (MCF) and grumeleuse.In the blood plasma of hemophiliac, add PAI-1 separately MCF is increased to 5mm, and the half EGCT is extended to 32.4 minutes.The time scale Minute.
Detailed Description Of The Invention
Because of the operation or large-scale wound is excessively hemorrhage, need thus the experimenter who transfuses blood than those easier generation complication of any hemorrhage experimenter not to take place. Yet, if need to give human blood or blood product (be used for the treatment of blood coagulation defective etc. blood platelet, leucocyte, derive from the concentrate of blood plasma), then moderate is hemorrhage also can cause complication, and this is because it is relevant with transfer Human virus (hepatitis, HIV, parvovirus and other present unknown virus) and the harm of non-viral pathogen. The extensive hemorrhage generation that can cause comprising the MOF of lung and kidney function damage that needs big blood transfusion. In case these serious complication take place in the experimenter, then begin to occur a succession of event that relates to many cell factors and inflammatory reaction, so that extremely difficulty is carried out any treatment, and usually lamentedly unsuccessful. It is unstable that the patient of taking place to lose blood greatly becomes clinically. This class patient has the risk that atrial fibrillation takes place, and this can cause, and fatefulue cardiac function stops, extravasation of fluid (so-called " wet lung " or ARDS) in renal dysfunction or the lung. Therefore, the main purpose of operation and the infringement for the treatment of Main Tissues is to avoid hemorrhage or it is reduced to bottom line. Be reduced to bottom line for fear of do not expect hemorrhage of this class or with it, importantly guarantee to form and be difficult for stable, the firm tampon (haemostatic plugs) that dissolved by fibrinoclase. In addition, importantly guarantee to form fast and effectively this class tampon or grumeleuse.
The experimenter who suffers from decrease of platelet (platelet count or active descend) also exists the unusual and fibrin plug of thrombin generation to be difficult to stable problem, thereby makes tampon tend to too early dissolving. In addition, large-scale wound or organ injury take place thereby the experimenter that frequently transfused blood usually has the low platelet counting and hangs down fibrinogen, Factor IX and other coagulated protein level. Thrombin generation unusual (or low) among these experimenters. Therefore, there is defective in these experimenters or renders a service lowlyer at hemostatic capability, and the fibrin plug that causes forming can be easy to and be dissolved by proteolytic enzyme prematurely, and this fermentoid is also extensively discharging in the situation of feature take extensive wound and organ injury.
The hemorrhage hemotoncus that can also cause in the tissue forms. The size of hemotoncus (particularly between cranium and vertebra hemotoncus) and nervous function forfeiture, recover difficulty and/or recover seriousness and the degree of rear nervous function permanent damage closely related. When hemotoncus is arranged in brain, can be observed severest consequences, even can cause death.
Therefore, treat hemorrhage main purpose and be to reach hemostasis in the shortest time, making thus loses blood remains on the floor level.
The present invention provides useful composition, application and the methods for the treatment of of the experimenter's internal haemorrhage outbreak that can be used for treating these needs thus. Described composition, application and method can be brought beneficial effect, such as: before obtaining hemostasis, just reduce and lose blood; The blood flow volume that needs in surgical procedure reduces; Blood pressure is remained on the acceptable level until obtain hemostasis; Make blood pressure keep more quickly stable; The patient who treats is shortened recovery time; Reduce hemotoncus and form or form littler hemotoncus (comprising intracerebral hematoma); Prevent more quickly hemorrhage; Reduce and make stopped bleeding and keep the required administration number of times of hemostasis.
With give factor VIIa separately or PAI-1 compares, setting time shortens when uniting the preparation of the preparation that gives factor VII or the relevant polypeptide of factor VII, for example factor VIIa and PAI-1 or PAI-1 related polypeptide, and grumeleuse is hard and Fibrinolytic tolerance increased.
When uniting the preparation of the preparation that gives factor VII or the relevant polypeptide of factor VII (for example factor VIIa) and PAI-1 or PAI-1 related polypeptide, compare with the situation that gives separately one of factor VIIa or PAI-1, the former reaches the time decreased of hemostasis, and keeps the required administration number of times of hemostasis and reduce. The invention provides simultaneously or give successively the beneficial effect of the preparation of the preparation of factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide. Pharmaceutical composition of the present invention can be single composition forms, or it can be the form of multicomponent kit (kit that multicomponent forms). Composition of the present invention can be used as and comprises such as the treatment in the mammal of the such primate of people and preventative Procoagulants. The present invention further provides the method (comprising prophylactic treatment or prevention) that comprises the bleeding episodes in the human experimenter for the treatment of.
When no matter mentioning the first or second or the 3rd UD etc. in the context of this specification in which kind of situation, this dosage does not represent the preferred sequence of administration, and only is for convenience of explanation.
The combination of the preparation of the preparation of factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide is the favourable product of guaranteeing that setting time shortens, tampon forms fast and forming stable tampon (haemostatic plugs). The present inventor has been found that the combination of the preparation of the preparation of factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide is the favourable product of guaranteeing that tampon firm, stable and that form fast forms.
The present inventor is verified, can more effectively increase the hardness of grumeleuse with the combination that independent factor VIIa or PAI-1 compare factor VIIa and PAI-1. Also confirm to compare with independent factor VIIa or PAI-1, the combination of factor VII or the relevant polypeptide of factor VII and PAI-1 can more effectively prolong human normal plasma's external EGCT. Confirm in addition to compare with independent factor VIIa or PAI-1, the combination of factor VII or the relevant polypeptide of factor VII and PAI-1 can more effectively prolong the half EGCT among the human normal plasma. Also confirm to compare with one of independent factor VIIa or PAI-1, the combination of factor VII or the relevant polypeptide of factor VII and PAI-1 can more effectively prevent grumeleuse generation fibrinolysis among the human normal plasma, particularly the fibrinolysis of tPA-mediation. Therefore, by strengthening freezing action, can in the experimenter, more effectively treat hemorrhage.
Do not wish to be subjected to theory, it is believed that complete thrombin generation be form solid, stable tampon, to keep hemostasis institute thus requisite. The fibrin structure of this class plug depend on formation fibrin ferment amount and the beginning thrombin generation speed. In the unusual situation of thrombin generation, form the porous fibre albumen plug of high osmosis. Usually the fibrinoclase that exists on the fibrin surface is easy to dissolve this class fibrin plug. The formation of stable fibrin plug also depends on by the existence of the factor XIIIa of activated by thrombin, also depends on thus complete thrombin generation. In addition, but the fibrin ferment activated fiber protein dissolution inhibitor TAFI that recently describes requires quite high fibrin ferment amount to be used for its activation. Therefore, under the formation of fibrin ferment was not fully suitable situation, TAFI may not activate, and caused forming usually being easier to the tampon that dissolved by normal fiber protein dissolution activity. Descend, be in the situation of thrombopenia at platelet counts, can start the generation of rapid blood coagulation enzyme by giving extra exogenous factor VIIa. Yet, even total factor VIIa also can not make thrombin generation normalization under high concentration.
In the low experimenter of the PC of fibrinogen (because of many wounds or extensively perform the operation cause the experimenter that repeatedly transfuses blood), complete activated by thrombin can not take place. More effective hemostasis gives factor VII or the relevant polypeptide of factor VII and PAI-1 and reaches by uniting.
It is unusual to suffer among the thrombocytopenic experimenter thrombin generation, and the stabilisation defectiveness of fibrin plug causes tampon (haemostatic plugs) to tend to too early dissolving. In addition, the experimenter who big wound or organ injury take place, frequently transfuses blood thus usually has the low platelet counting and hangs down fibrinogen, Factor IX and other coagulated protein level. These experimenters' thrombin generation unusual (or low). In addition, the fibrinogen levels of its reduction has produced negative interference effect to the activation of FXIII. Therefore, there is defective in these experimenters' hemostatic capability or renders a service lowlyer, and causing forming can be easily and prematurely by the fibrin plug that proteolytic enzyme dissolved, and this fermentoid is also extensively discharging in the situation of feature take extensive wound and organ injury.
In order to be conducive to form the plug with complete stability of keeping the complete ability of stopping blooding in the experimenter, give composition of the present invention. Said composition is especially favourable to the low experimenter of the blood plasma level of the low experimenter of platelet counts and fibrinogen and/or other coagulated protein.
Factor VII polypeptides:
In implementing process of the present invention, can use effective prevention or treat hemorrhage any factor VII polypeptides. Comprise the factor VII polypeptide that derives from blood or blood plasma or produce by recombination form.
The present invention includes factor VII polypeptides, such as: for example those have U.S. Pat 4,784, the factor VII (wild type human factor VII) of disclosed amino acid sequence in 950. In certain embodiments, described factor VII polypeptides is such as U.S. Pat 4,784, disclosed human factor VII a (wild type factor VII) in 950. In a series of embodiments, factor VII polypeptides comprise the specific biological activity that shows human factor VII a at least about 10%, preferably at least about 30%, more preferably at least about 50% and most preferably at least about 70% polypeptide. In a series of embodiments, factor VII polypeptides comprise the specific biological activity that shows human factor VII a at least about 90%, preferably at least about 100%, preferably at least about 120%, more preferably at least about 140% and most preferably at least about 160% polypeptide. In a series of embodiments, factor VII polypeptides comprises and showing and U.S. Pat 4, in 784,950 disclosed wild type factor VII sequence have at least about 70%, preferably at least about 80%, more preferably at least about 90% and most preferably at least about the polypeptide of 95% homogeneity.
" factor VII polypeptides " used herein is including, but not limited to the relevant polypeptide with factor VII of factor VII. Term " factor VII " is in order to including, but not limited to having the wild type human factor VII (such as U.S. Pat 4,784,950 is disclosed) polypeptide of 1-406 amino acids sequence and the wild type factor VII that derives from other kind, such as the factor VII from ox, pig, dog, mouse or salmon, described factor VII derives from blood or blood plasma, perhaps produces by recombination form. This term further comprises the natural equipotential version of the factor VII that can exist and occur between individuality. In addition, the degree of glycosylation or other posttranslational modification can change with the different of character of selected host cell and host cell environment with the position. Term " factor VII " also has been processed into the polypeptide of its corresponding biologically active form in order to the factor VII polypeptides that comprises not cracking (proenzyme) form with those through proteolysis, can be with they called after factor VIIas. In general, the 152nd of factor lytic VII the-the 153rd residue and obtain factor VIIa.
" the relevant polypeptide of factor VII " is including, but not limited to respect to having carried out chemical modification for the human factor VII and/or with respect to the factor VII polypeptides that contains one or more amino acid sequences for the human factor VII and change (being factor VII variant) and/or contain the amino acid sequence (being factor VII fragment) of brachymemma for human factor VII. The relevant polypeptide of this class factor VII can show the characteristic different from human factor VII, comprises the specific activity of stability, phospholipids incorporate, change etc. Term " the relevant polypeptide of factor VII " has been processed into the polypeptide of its corresponding biologically active form in order to the polypeptide that comprises not cracking form of this class (proenzyme) with those through proteolysis, can be with their called afters " factor VIIa related polypeptide " or " factor VII related polypeptide of activation ".
" the relevant polypeptide of factor VII " used herein is including, but not limited to showing bioactive polypeptide substantially the same or that improve with the wild type human factor VII, and the polypeptide that the factor VIIa biologically active has changed or reduced in fact for wild type human factor VII a. These polypeptide are including, but not limited to the factor VII that carries out chemical modification or factor VIIa and imported and change or destroy the factor VII variant that the bioactive specific amino acid sequence of described polypeptide changes.
The polypeptide that wherein further comprises the amino acid sequence with slight modification, for example, with the N-end of modifying, comprise-terminal amino acid disappearance or the polypeptide that adds and/or the polypeptide that for human factor VII a, carries out chemical modification.
The relevant polypeptide of factor VII that comprises factor VII variant, no matter be to show substantially the same or better biologically active with wild type factor VII, or compare with wild type factor VII and to show the biologically active that changes or reduce in fact, all including, but not limited to because inserting, lack or replacing the polypeptide that one or more amino acid have the amino acid sequence that is different from wild type factor VII sequence.
When using aforesaid thrombotest, proteolysis test or TF be during in conjunction with one or more tests in the test, the relevant polypeptide of factor VII that comprises variant comprise the specific activity that shows the wild type factor VIIa that in the same cell type, produces at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120% or at least about 130% those polypeptides.
When with the test of aforesaid thrombotest, proteolysis or TF during in conjunction with one or more tests in the test, with wild type factor VIIa have the relevant polypeptide of bioactive factor VII (comprising variant) substantially the same or that make moderate progress comprise the specific activity that shows the wild type factor VIIa that in the same cell type, produces at least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120% and most preferably at least about 130% those polypeptides.
When with the test of aforesaid thrombotest, proteolysis or TF during in conjunction with one or more tests in the test, compare with wild type factor VIIa have the relevant polypeptide of bioactive factor VII (comprising variant) that reduces in fact be show the wild type factor VIIa that in the same cell type, produces specific activity be lower than about 10%, more preferably less than about 5% and most preferably be lower than about 1% those polypeptides. Compare with wild type factor VII and to have the bioactive factor VII variant that changes in fact including, but not limited to the factor VII variant that shows TF dependent/non-dependent factor X protein hydrolysing activity and those factor VII variant of factor lytic X in conjunction with TF but not.
In certain embodiments, factor VII polypeptides is the relevant polypeptide of factor VII, and particularly (" its activity is at least about 1.25 variant with the ratio of the activity of natural human factor VIIa (wild type FVIIa) during test ") test referring to following using " extracorporeal hydrolysis test "; In other embodiments, this ratio is at least about 2.0; In other embodiments, this ratio is at least about 4.0. In certain embodiments of the invention, factor VII polypeptides is the relevant polypeptide of factor VII, and particularly (" its activity is at least about 1.25 variant with the ratio of the activity of natural human factor VIIa (wild type FVIIa) during test ") test referring to following using " external proteolysis test "; In further embodiment, this ratio is at least about 2.0; In further embodiment, this ratio is at least about 4.0; In further embodiment, this ratio is at least about 8.0.
In certain embodiments, described factor VII polypeptides is such as U.S. Pat 4,784, disclosed human factor VII (wild type factor VII) in 950. In certain embodiments, described factor VII polypeptides is human factor VII a. In a series of embodiments, factor VII polypeptides be show human factor VII a the specific biological activity at least about 10%, preferably at least about 30%, more preferably at least about 50% and most preferably at least about the relevant polypeptide of 70% factor VII. In certain embodiments, described factor VII polypeptides has because inserting, lack or replacing the amino acid sequence that one or more amino acid are different from the sequence of wild type factor VII.
Have substantially the same with wild type factor VIIa or the limiting examples of bioactive factor VII variant that is better than it including, but not limited to the factor VII variant described in Danish Patent Application PA 2,000 00734 and PA 2,000 01360 (being equivalent to WO 01/83725) and the PA 2,000 01361 (being equivalent to WO 02/22776). Limiting examples with bioactive factor VII variant of or improvement substantially the same with wild type factor VII comprises: S52A-FVII, S60A-FVII (lino etc. " biochemistry and biophysics archives " (Arch. Biochem.Biophys.) 352:182-192,1998); L305V-FVII, L305V/M306D/D309S-FVII, L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII, V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII, L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII, V1 58D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII, K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII, V158D/M298K-FVII and S336G-FVII; Such as U.S. Pat 5,580, the disclosed FVIIa variant that shows the proteolysis stability of increase in 560; Carried out the factor VIIa (Mollerup etc. " Biotechnology and Bioengineering " (Biotechnol. Bioeng.), 48:501-505,1995) that proteolysis cuts at 291 residues of 290-or 315-316 position residue; Oxidised form (Kornfelt etc. " biochemistry and biophysics archives " are 363:43-54 (Arch.Biochem.Biophys.), 1999) with factor VIIa. The limiting examples of comparing the bioactive factor VII variant that has in fact reduction or change with wild type factor VII comprises R152E-FVIIa, and (Wildgoose etc. " biochemistry " are 29:3413-3420 (Biochem), 1990), S344A-FVIIa (Kazama etc. " journal of biological chemistry " (J.Biol. Chem.) 270:66-72,1995), (Hoist etc. " operation magazine in European blood vessel and the blood vessel " are 15:515-520 (Eur.J.Vasc.Endovasc.Surg.) for FFR-FVIIa, 1998) and lack the factor VIIa (Nicolaisen etc. " FEBS communication " (FEBS Letts.) 317:245-249,1993) of Gla domain. The factor VII polypeptides of chemical modification and the limiting examples of sequence variants for example are described in the U.S. Pat 5,997,864.
The biologically active of factor VIIa in blood clotting derive from its (i) bind tissue factor (TF) and (ii) catalytic factor IX or factor X the proteolysis cutting and produce the ability of activation factor IX or X (being respectively factors IX a or Xa).
For the purposes of the present invention, can be such as U.S. Pat 5, come the biologically active (" factor VII biologically active ") to factor VII polypeptides quantitative by the ability of measuring preparation enhancing blood clotting with the blood plasma that lacks factor VII and tissue thromoboplastin described in 997,864. In this test, biologically active is expressed as the minimizing of comparing setting time with control sample, by changing into " factor VII unit " with the merging human serum standard items comparison that contains 1 unit/ml factor VII activity. Perhaps, can be through the following steps quantitative to the factor VIIa biologically active:
(i) measure factor VIIa or factor VIIa related polypeptide produce activation factor X (factor Xa) in the system that comprises the TF that is embedded in the adipose membrane and factor X ability. (Persson etc. " journal of biological chemistry " are 272:19919-19924 (J.Biol.Chem.), 1997);
(ii) in aqueous system, measure factor X hydrolysis (" external proteolysis test " vide infra);
(iii) use based on the Instrument measuring factor VIIa of surperficial cytogene resonance or the physical bond (Persson, " FEBS communication " (FEBS Letts.) 413:359-363,1997) of factor VIIa related polypeptide and TF; With
(iv) measure factor VIIa and/or factor VIIa related polypeptide to the hydrolysis (" extracorporeal hydrolysis test " vide infra) of synthetic substrate; With
(v) generation of fibrin ferment in the non-TF dependence vitro system of mensuration.
Term " factor VII biologically active " or " factor VII activity " are in order to comprise the ability that produces fibrin ferment; This term also is included in the ability that produces fibrin ferment in the situation that does not have the tissue factor existence on the surface of activated blood platelet.
The factor VIIa preparation is but is not limited to NovoSeven (Novo Nordisk A/S, Bagsvaerd, Denmark) used according to the present invention.
The PAI-1 polypeptide：
The present invention includes the application of PAI-1 polypeptide, such as: Gils etc. had in " biochemistry and biophysics journal " (Biochim Biophys Acta.) on September 8th, 1998 such as those; 1387 (1-2): 291-7, Sui etc. are in " biologically active magazine " (Biochem J.) 1998 April 15; 331 (Pt2): 409-15, Ginsburg etc. are at " Journal of Clinical Investigation " (J.Clin. Invest.), and the 78th rolls up the PAI-1 polypeptide of disclosed amino acid sequence among the pp.1673-1680 (1986); Those are described in U.S. Pat 6,303,338, United States Patent (USP) 6,103,498 or such as Pannekoek etc. at " European molecular biology association magazine " (JOURNAL EMBO J.) 5 (10), disclosed PAI-1 polypeptide (wild type PAI-1) among the 2539-2544 (1986).
In implementing process of the present invention, can use effective prevention or treat hemorrhage any PAI-1 polypeptide. This comprises the PAI-1 polypeptide that derives from blood or blood plasma or produce by recombination form. Term " PAI-1 " is in order to the PAI-1 polypeptide of the natural appearance that comprises activity form (comprising the constitutive activity form, for example PAI-1 (14-1b, Molecular Innovations)) and potential conformation.
" PAI-1 polypeptide " used herein is including, but not limited to PAI-1 and PAI-1-related polypeptide.
Term " PAI-1 " in order to including, but not limited to: have Pannekoek etc. at " European molecular biology association magazine " (JOURNAL EMBO J.) 5 (10), 2539-2544 (1986), Gils etc. are in " biochemistry and biophysics journal " (Biochim Biophys Acta.) 1998 September 8; 1387 (1-2): 291-7, Sui etc. are in " biologically active magazine " (Biochem J.) 1998 April 15; 331 (Pt2): 409-15, Ginsburg etc. at " Journal of Clinical Investigation " (J.Clin.Invest.), the 78th the volume, the polypeptide of the amino acid sequence described in the pp.1673-1680 (1986); Or those are described in U.S. Pat 6,303,338, the polypeptide in the United States Patent (USP) 6,103,498; With the wild type PAI-1 that derives from other species, such as ox, pig, dog, mouse and P of Rats AI-1.
The natural equipotential version that wherein further comprises the PAI-1 that may between individuality, exist and occur. In addition, the degree of glycosylation or other posttranslational modification can change with the different of host cell environmental properties with selected host cell with the position. Term " PAI-1 " also has been processed into the polypeptide of its corresponding biologically active form in order to the PAI-1 polypeptide that comprises zymogen forms with those.
" PAI-1 related polypeptide " is including, but not limited to respect to having carried out chemical modification for the people PAI-1 and/or with respect to the PAI-1 polypeptide that contains one or more amino acid sequences for the people PAI-1 and change (being the PAI-1 variant) and/or contain the amino acid sequence (being the PAI-1 fragment) of brachymemma for people PAI-1. This class PAI-1 related polypeptide can show the characteristic different from people PAI-1, comprises the specific activity of stability, phospholipids incorporate, change etc.
Term " PAI-1 related polypeptide " has been processed into the polypeptide of its corresponding biologically active form in order to this class polypeptide that comprises zymogen forms with those.
" PAI-1 related polypeptide " used herein including, but not limited to showing bioactive polypeptide substantially the same with wild type people PAI-1 or that make moderate progress, and compare the polypeptide that its PAI-1 biologically active has changed or reduced in fact with the activity of wild type people PAI-1. These polypeptide are including, but not limited to the PAI-1 of chemical modification and imported and change or destroy the PAI-1 variant that the bioactive specific amino acid sequence of described polypeptide changes.
The polypeptide that wherein further comprises the amino acid sequence with slight modification, for example, with the N-end of modifying, comprise-terminal amino acid disappearance or the polypeptide that adds and/or the polypeptide that for people's factor PAI-1, has carried out chemical modification.
The factor PAI-1 related polypeptide that comprises factor PAI-1 variant, no matter be to show substantially the same with wild type PAI-1 or be better than its biologically active, or compare with wild type PAI-1 and to show the biologically active that changes or reduce in fact, include but be not limited to have because inserting, lack or replacing the polypeptide of amino acid sequence that one or more amino acid are different from the sequence of wild type PAI-1.
When testing with the PAI-1 activity test described in this description, the PAI-1 related polypeptide that comprises variant comprise the specific activity that shows the wild type PAI-1 that in the same cell type, produces at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120% with at least about 130% those polypeptides.
Having bioactive PAI-1 related polypeptide (comprising variant) substantially the same or that improve with wild type PAI-1 comprises, with one or more whens test in the aforesaid specificity PAI-1 activity test, show the specific biological of the wild type people PAI-1 that in the same cell type, produces active at least about 25%, preferably at least about 50%, more preferably at least about 75%, more preferably at least about 100%, more preferably at least about 110%, more preferably at least about 120% and most preferably at least about 130% those polypeptides.For the purposes of the present invention, the ability of the clot dissolution that can mediate by the clot dissolution or the fibrinolysin of for example mensuration preparation inhibition tPA-as described herein mediation comes the biological activity of PAI-1 is carried out quantitatively.In two kinds of tests, biological activity is expressed as the minimizing of comparing EGCT with control sample.Perhaps, can be by Chandler for example etc. at " clinical chemistry " (Clinical Chemistry), described in 35 (5) 787-793 (1989) or be used for the enzyme-linked immunosorbent assay (HPAIKT, Molecular Innovations) of functional human PAI-1 or other test determination PAI-1 as known in the art and/or PAI-1 equivalent and come the biological activity of PAI-1 quantitative to the inhibitory action of tPA amidohydrolase activity (amidolytic activity).
When with one or more whens test in the aforesaid specificity PAI-1 activity test, have substance be lower than the bioactive PAI-1 related polypeptide of wild type PAI-1 (comprising variant) be show the wild type PAI-1 that in the same cell type, produces specific activity be lower than about 25%, preferably be lower than about 10%, more preferably less than about 5% and most preferably be lower than about 1% those polypeptides.
The limiting examples of PAI-1 equivalent comprises for example PAI-1 equivalent described in the U.S. Pat 6103498.
In certain embodiments, described PAI-1 polypeptide is when using " PAI-1 test " (Chandler etc. " clinical chemistry " (Clinical Chemistry), 35 (5) 787-793 (1989) are referring to above) ratio of the activity of its active and natural human PAI-1 (wild type PAI-1) is at least about 1.25 PAI-1 equivalent during test; In other embodiments, this ratio is at least about 2.0; In further embodiment, this ratio is at least about 4.0.
The PAI-1 related polypeptide also comprises the PAI-1 of its characteristic hemostasis related activity of maintenance or the fragment of PAI-1 related polypeptide.For example, can use the PAI-1 activity test described in this description to measure the hemostasis related activity of PAI-1 polypeptide.
Definition
In the context of the present specification, use trigram or single-letter aminoacid representation according to the conventional sense shown in the table 1.Unless describe in detail, aminoacid mentioned herein is L-aminoacid.Should understand for example natural aminoacid that appears on the wild type factor VII assigned address of the letter of first in K337 representative, and for example [K337A]-FVIIa refers to wherein to appear at aminoacid on the assigned address by the FVII-variant by the aminoacid replacement of single-letter code A representative by single-letter code K representative natural.
Table 1: amino acid abbreviations:
| Aminoacid | The trigram code | The single-letter code |
| Glycine Gly G proline Pro P alanine Ala A valine Val V leucine Leu L isoleucine Ile I methionine Met M cysteine Cys C phenylalanine Phe F tyrosine Tyr Y tryptophan Trp W histidine H lysine Lys K arginine Arg R glutamine Gln Q asparagine Asn N glutamic acid Glu E aspartic acid Asp D | ||
Term " factor VIIa " or " FVIIa " can exchange use.
In the context of the present specification, " have the unusual experimenter of thrombin generation " and refer to the experimenter that can not on the activated blood platelet surface, thrombin be broken out fully, the generation that comprises thrombin is less than the experimenter that subject intravascular coagulation enzyme with normal haemostatic system that function is arranged fully produces, and the described normal haemostatic system that function arranged fully comprises the normal amount and the function (for example in the human normal plasma who compiles) of thrombin, platelet and fibrinogen; Including, but not limited to: the experimenter who lacks Factor IX; Have low platelet quantity or have the hematoblastic experimenter (for example thrombocytopenia or glanzmann's thrombasthenia or excessively hemorrhage experimenter) of functional defect; Experimenter with low-level thrombinogen, FX or FVII; The level of several thrombins reduces the experimenter of (for example because of wound or extensive perform the operation and cause excessively hemorrhage); The experimenter low (for example experimenter who repeatedly transfuses blood) with the fibrinogen plasma concentration.
So-called " thrombin generation level " or " normal thrombin generation " refers to the generation level of comparing patient's intravascular coagulation enzyme with the intravital level of health volunteer.This water-glass is shown as the percentage ratio of normal level.If suitable, these terms can exchange use.
Term " strengthens the hemostasis system " and refers to the ability that produces thrombin that strengthens.Term " strengthen hemostasis " is when being included in identical thrombin generation experimental test, respectively with the control sample that only contains factor VII or the relevant polypeptide of factor VII or PAI-1 or PAI-1 related polypeptide in separately thrombin generation compare, contain the situation that the thrombin generation measured in the specimen of preparation of the preparation of factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide obtains prolonging.Can described in the test of the thrombin generation in this description (referring to " test portion "), detect thrombin generation.
" the only having " activating agent used herein or the factor refer to factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide each other jointly for being included in unique hemorrhage or active hemorrhage or the thrombin in pharmaceutical composition or the test kit, or in the particular treatment process, such as the situation of the unique hemorrhage that in specific bleeding episodes process, the patient is given or active hemorrhage or thrombin.Should understand these situations and comprise that other hemorrhage or thrombin be not in use with amount or active be enough to appreciable impact one or more solidify those situations that parameter exists.
EGCT, clot strength, fibrin piece form and setting time is in order to detect the stop blooding clinical parameter of system mode of patient.With proper spacing blood sampling in patient's body, and as following document described in detect in these parameters one or more by for example thrombelastography: " blood coagulation and fibrinolysis " (Blood Coagulation﹠amp such as Meh for example; Fibrinolysis) 2001; 12:627-637; Vig etc. " hematology " (Hematology), the 6th the volume (3) pp.205-213 (2001); Vig etc. " blood coagulation and fibrinolysis " (Blood Coagulation ﹠amp; Fibrinolysis), the 12nd roll up (7) pp.555-561 (2001) October; Glidden etc. " clinical and application thrombosis/hemostasis " (Clinical andapplied thrombosis/hemostasis), the 6th rolls up (4) pp.226-233 (2000) October; McKenzie etc. " cardiology " (Cardiology), the 92nd volume (4) pp.240-247 (1999) April; Or " U.S. nephrology association magazine " (Journal of theAmerican Society of Nephrology) such as Davis, the 6th volume (4) pp.1250-1255 (1995).
When term " prolong EGCT " is tested in the identical clot dissolution test in order to be included in, contain the situation that the EGCT measured value of the preparation of factor VII or the relevant polypeptide of factor VII and the specimen of the preparation of PAI-1 or PAI-1 related polypeptide prolongs to some extent with respect to each EGCT than the control sample that only contains factor VII or the relevant polypeptide of factor VII or PAI-1 or PAI-1 related polypeptide respectively.Can detect EGCT as mentioned above.
When term " increase clot strength " is tested in the identical clot strength test in order to be included in, the clot strength that the specimen of the preparation that contains factor VII or the relevant polypeptide of factor VII and the preparation of PAI-1 or PAI-1 related polypeptide is measured, the situation that for example mechanical strength increased to some extent than each grumeleuse cracking time of only containing the control sample of factor VII or the relevant polypeptide of factor VII or PAI-1 or PAI-1 related polypeptide.Can be as (Carr ME, Zekert SL. " mensuration that platelet-mediated power takes place in the plasma clot forming process " (Measurement of platelet-mediated force development duringplasma clot formation.)-" united states drug science magazine " (AM J MED SCI) 1991 as described in the Carr etc. 1991; 302:13-8) or as mentioned above detect clot strength by thrombelastography.
When term " strengthen fibrin grumeleuse form " was tested in the identical thrombotest in order to be included in, the fibrin grumeleuse that the specimen of the preparation that contains factor VII or the relevant polypeptide of factor VII and the preparation of PAI-1 or PAI-1 related polypeptide is measured formed fibrin grumeleuse that control sample that speed or degree comparison only contain factor VII or the relevant polypeptide of factor VII or PAI-1 or PAI-1 related polypeptide measures and forms the situation that speed or degree increase to some extent.Can detect the fibrin grumeleuse as mentioned above forms.
When term " shorten setting time " is tested in the identical thrombotest in order to be included in, contain the situation that grumeleuse formation time (setting time) measured value of the preparation of factor VII or the relevant polypeptide of factor VII and the specimen of PAI-1 or PAI-1 related polypeptide preparation increases to some extent than the individual setting time that only contains the control sample of factor VII or the relevant polypeptide of factor VII or PAI-1 or PAI-1 related polypeptide respectively.Can pass through the known standard P T og aPTT test determination setting time of those of ordinary skills.
Term " platelet count or the activity that reduce " refers to the platelet counts (platelet) and the hematoblastic blood coagulation associated biomolecule of this class activity that are present in experimenter's blood plasma.The counting that reduces may because for example platelet destruction increases, platelet produce reduce and spleen in compiled and be higher than normal platelet level branch and cause.For example, thrombocytopenia is defined as platelet count and is lower than 150,000 platelet/microlitres; Be limited to 350,000-450,000 platelet/microlitre on the general orthoplastocyte counting.Can measure platelet count by automatization's platelet counter; This is the well-known methods of those skilled in the art.Because of platelet count reduces the syndrome caused including, but not limited to thrombocytopenia, coagulopathy." activity is " including, but not limited to hematoblastic gathering, adhesion and coagulation activity.Active reduction may be because for example glycoprotein unusually, film-cytoskeleton interaction is unusual, the granule of platelet is unusual, the platelet coagulation activity is unusual, signal conducts and diacrisis causes.Can measure biologically active pdgf by method known in those skilled in the art, comprise gathering, adhesion and coagulation activity, for example, referring to " hemocyte that-practical means " (Platelets.A Practical Approach), Ed.S.P.Watson ﹠amp; K.S.Authi: " clinicing aspect of blood platelet disorder " (Clinical Aspects ofPlatelet Disorders) be 15:299-318 (K.J.Clemetson), and 1996, OxfordUniversity Press; WilliamsHematology, the 6th edition, Eds.Butler, Lichtman, Coller, Kipps ﹠amp; Selig-sohn, 2001, McGraw-Hill.Because of biologically active pdgf reduces the syndrome cause including, but not limited to glanzmann's thrombasthenia, Bai-Suo syndrome, anticoagulant therapy and thrombolytic therapy." reduce " referring to when in identical test, measuring, compare, counting in the specimen or active the reduction with counting or the activity compiled normally in the plasma sample.
Term used herein " hemorrhagic disease " has reflected any defective in the cell that shows bleeding episodes or molecule source, can be geneogenous, acquired or bringing out property.The example of hemorrhagic disease is including, but not limited to: deficiency of coagulation factors (for example blood coagulation factor VIII, IX, XI or VII lack); Thrombin suppresses; Platelet function defective (for example glanzmann's thrombasthenia and Bai-Suo syndrome); Thrombocytopenia; Feng's von Willebrand disease; And coagulopathy, such as increase and reduce the coagulopathy that (for example carried out operation or take place among the patient of repeatedly blood transfusion of wound) caused because of coagulated protein dilution, fibrinolysis because of hemorrhage and/or blood transfusion cause platelet counts.
The hemorrhage blood that refers to exosmoses from any composition of blood circulation.Term " bleeding episodes " is in order to comprising relevant unwanted, not controlled and normally over-drastic hemorrhage of histologic lesion with operation, wound or other form, and suffers among the experimenter of hemorrhagic disease unwanted hemorrhage.Bleeding episodes can have basically normal coagulation system but the experimenter of (temporary) coagulopathy is taken place and suffer from congenital or acquired solidify or the experimenter of hemorrhagic disease in take place.In the experimenter who suffers from the platelet function defective, hemorrhage may to hemophilia cause hemorrhage similar, this is that the blood coagulation " chemical compound " (for example platelet or Feng's von willebrand's factor protein) that wherein hemostasis system shortage is essential or such chemical compound are unusual because the same with hemophilia.In the experimenter that for example relevant with operation or a large amount of wound extensive histologic lesion takes place, normal hemostatic mechanism may not satisfy hemostatic needs at once, therefore, although these mechanism basically (wound before or operation before) normal, they still may take place excessively hemorrhage.Usually this class experimenter who further transfuses blood can take place to cause (temporary) coagulopathy (promptly increasing and the platelet count reduction because of coagulated protein dilution, fibrinolysis hemorrhage and/or that blood transfusion causes) because of hemorrhage and/or blood transfusion.Hemorrhage can also in the organ such, the generation such as brain, interior ear field and eye; They are that the surgical hemostasis probability is very little, thereby are difficult to obtain the zone of gratifying haemostatic effect.Similar problem may produce in the process of getting biopsy from different organs (liver, lung, tumor tissues, gastrointestinal tract), also may be behind laparoscopic surgery and pubis prostate eradicate in the process of art (radical retropubic prostatectomy) and produce.With regard to all these situations, commonly be difficult to by surgical technic (stitching thread, pincers etc.) hemostasis, when hemorrhage also be like this when being dispersivity (for example hemorrhagic gastritis and uterus massive hemorrhage).Hemorrhage can also the generation in the experimenter who carries out anticoagulant therapy wherein induced the hemostasis defective because of the therapy that gives; These are hemorrhage normally acute with a large amount of.Anticoagulant therapy is normally for prevention of thromboembolic disorders.This class therapy can comprise the vitamin K-antagonist of Dan Baijutang, warfarin or other form of using heparin, other form and aspirin and other anticoagulant, such as the active antibody of GP IIb/IIIa or other inhibitor.Hemorrhage can also be because of so-called thrombolytic therapy takes place, this thrombolytic therapy comprises with antiplatelet drug (for example aspirin), anticoagulant (for example heparin) and fibrinolytic agent (for example tPA of tissue plasminogen activator) and carries out conjoint therapy.Bleeding episodes also in order to including, but not limited to the experimenter who suffers from following disease in operation or relevant not controlled and over-drastic hemorrhage of wound: hemorrhage, the hematuria (hemorrhage from the kidney road) in acute hemarthrosis (intraarticular is hemorrhage), chronic hemophilic arthosis, hematoma (for example behind muscle, the peritoneum, after Sublingual and the pharynx), other tissue, brain hemorrhage, operation (for example hepatectomy), exodontia and gastrointestinal hemorrhage (for example UGI is hemorrhage).Bleeding episodes may be relevant with following situation: the inhibitor of anti-Factor IX; Haemophilia A; Inhibitor is in conjunction with haemophilia A; Haemophilia B; Factor VII deficiency; PAI-1 lacks; Thrombocytopenia; Feng's von willebrand's factor lacks (Feng's von Willebrand's disease); Serious histologic lesion; Severe trauma; The hands art; Laparoscopic surgery; Hemorrhagic gastritis; Get biopsy; Anticoagulant therapy; Upper gastro-intestinal tract hemorrhage (UGI); Or stem cell transplantation.Bleeding episodes can be uterus massive hemorrhage, occur in the less organ of machinery hemostasis probability hemorrhage, occur in brain hemorrhage, occur in lug areas hemorrhage or occur in the hemorrhage of ophthalmic.If suitable, term " bleeding episodes " and " hemorrhage " can exchange use.
In the context of the present specification, term " treatment " in order to comprise that prevention estimates hemorrhage, such as hemorrhage in the operation with regulate taken place hemorrhage, hemorrhage such as in the wound, purpose is to suppress hemorrhage or it is minimized." estimate hemorrhage " mentioned above can be estimate to take place in particular organization or organ hemorrhage, maybe can be unspecified hemorrhage.Therefore, preventatively give the preparation of factor VII or the relevant polypeptide of factor VII and the preparation of PAI-1 or PAI-1 related polypeptide is included in the scope of term " treatment ".
Term used herein " experimenter " is in order to refer to any animal, particularly mammal, such as the people, if suitable then can exchange with term " patient " and use.The present invention also comprises factor VII or FVII related polypeptide and the tPA inhibitor application in veterinary's operation.
Can simultaneously or give successively as defined factor VII in this description or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide.The described factor can be provided as single dosage form, wherein said single dosage form contains this two kinds of thrombins; Maybe the described factor can be provided as ingredient test kit (kit-of-parts) form, the preparation that comprises factor VII or the relevant polypeptide of factor VII in the described ingredient test kit is as first unit dosage forms, and the preparation that comprises PAI-1 or PAI-1 related polypeptide is as second unit dosage forms.No matter under which kind of situation, mention in the context of this description first second or the third etc. during unit dose, this dosage is not represented the preferred sequence of administration, and only is for convenience of explanation.
The preparation that so-called " simultaneously " gives the preparation of factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide refers to single dosage form and gives these coagulation factor proteins, or give first thrombin earlier, subsequently to be no more than 15 minutes, preferably to be no more than 10 minutes, to give second coagulation factor protein more preferably no more than 2 minutes intervals.Can at first give in two kinds of factors any.
So-called " successively " administration refers to and gives first coagulation factor protein, gives second coagulation factor protein subsequently, and its interval is 2 hours at the most, preferred 1-2 hour, more preferably at the most 1 hour, more preferably 30 minutes-1 hour, more preferably at the most 30 minutes, more preferably 15-30 minute.Can at first give wherein any unit dosage forms or coagulation factor protein.Preferably inject this two kinds of products by identical intravenous route.
So-called " level of PAI-1 " or " PAI-1 level " refers to the PAI-1 activity level of comparing the patient with health volunteer's level.This water-glass is shown the percentage ratio of normal level.If suitable, these terms can exchange use.
So-called " reduction of PAI-1 level " or " the PAI-1 level that reduces " refers to the mean P AI-1 level in the population of subjects that does not have PAI-1 shortage or PAI-1 inhibitor and compares, and exist level or the activity of PAI-1 descends in the blood flow.Can measure the level of cycle P AI-1 by coagulant or immunity test.Proofread and correct the ability of the setting time of PAI-1 shortage type blood plasma by patient's blood plasma and determine that (for example APTT tests the PAI-1 activity, vide infra; In addition referring to " test portion " of this description).
The PAI-1 of 1 unit has been defined as the amount (being equivalent to 100% PAI-1 level) of PAI-1 contained in 1 milliliter of normal (compiling) human plasma.
The factor VII of 1 unit is defined as the amount of factor VII contained in 1 milliliter of normal plasma, has been equivalent to about 0.5 μ g protein.After the activation, 50 units are equivalent to about 1 μ g protein.
So-called " shortages " refers to compare with the normal health individuality in the blood plasma and has level or its active decline such as PAI-1.If suitable, this term can exchange with " the PAI-1 level that reduces " and use.
So-called " APTT " or " aPTT " refers to portion of tissue thrombokinase soak time, and (for example by Proctor RR, Rapaport SI is described: " using kaolinic portion of tissue thrombokinase time-be used for the simple screening test that the phase I plasma coagulation factors lacks " (The partial thromboplastin time with kaolin; A simple screeningtest for first-stage plasma clotting factor deficiencies.)-and " U.S.'s clinical pathology magazine " (Am J Clin Pathol) 36:212,1961).
So-called " the reactive syndrome of PAI-1 " refers to having this experimenter who needs to give can to prevent, cure or improve behind the exogenous PAI-1 any expectation that is caused by this syndrome or the syndrome of already present any symptom, situation or disease.Including, but not limited to reduce the syndrome caused because of the PAI-1 level, the hemorrhagic disease that causes by the inhibitor of PAI-1 for example.Can also be with the reactive syndrome of combination treatment PAI-1-of the present invention.
So-called " the reactive syndrome of factor VII-" refers to having this experimenter who needs to give can to prevent, cure or improve behind exogenous factor VII, the preferred factor VIIa any expectation that is caused by this syndrome or the syndrome of already present any symptom, situation or disease.Comprise, but be not limited to because of blood coagulation factor VIII, IX, XI or VII level reduce, blood coagulation factor inhibitors, platelet function defective (for example glanzmann's thrombasthenia and Bai-Suo syndrome), thrombocytopenia, Feng's von Willebrand's disease and such as because of coagulated protein dilution, fibrinolysis increases and reduces because of hemorrhage and/or blood transfusion cause platelet counts the syndrome that coagulopathy caused that (for example in the patient of the repeatedly blood transfusion of having carried out operation or generation wound) causes.
The plasma concentration that " half-life " refers to factor VII or the relevant polypeptide of factor VII or PAI-1 or PAI-1 related polypeptide is reduced to this from particular value and is worth half required time.
So-called " preliminary hemostasis " refers to beginning by FXa and TF: factor VIIa produces thrombin, subsequently platelet activation and form first portion loose and activation, adhesion but as yet not by the platelet plug of fibrin stabilisation, and stable by crosslinked fibrin at last.If by the fibrin stabilisation (keeping hemostasis) that forms in the hemostasis process in second step, it is not easy to be dissolved by fibrinolytic system so.
So-called " secondary hemostasis " or " keeping hemostasis " refers on activatory platelet surface generation and by Factor IX a and secondary, complete and a large amount of outburst or the generations of the catalytic thrombin of Factor IX a, forms fibrin subsequently, stablizes initial stage platelet plug.Fibrin can cause stopping blooding fully to the stabilisation of this plug.
So-called " hemostasis fully " refers to and form stable and firm fibrin grumeleuse or plug on damage location, and this grumeleuse or plug can make and hemorrhagely effectively stop and being not easy to being dissolved by fibrinolytic system.In the context of the present specification, the term hemostasis is used to represent aforesaid hemostasis fully.
Can be by general known method, for example measure proteinic total amount in the preparation by measuring optical density.Can measure the amount of PAI-1-or factor VII protein (" antigen ") by general known method, such as standard Elisa immunity test.In general, carry out this class test through the following steps: the anti--thrombomodulin antibody on for example containing the proteic formulation soln of PAI-1 and being fixed on the elisa flat board is contacted, the anti-PAI-1 antibody of fixed antibody-PAI-1 complex and the secondary that carries labelling is contacted, in the 3rd step, measure their amount.Can be in a similar way, use the amount of suitable each thrombin of TPPA.Measure coagulation factor protein total amount contained in the preparation by adding a certain amount of each coagulation factor protein.In one embodiment, described preparation comprises isolating thrombin.In another embodiment, described preparation is substantially free of prothrombin and prothrombin a (thrombinogen and thrombin) and/or factor X or Xa.
Term used herein " isolating " refers to that (thrombin of for example having separated in the cell of PAI-1 or PAI-1 related polypeptide or in their culture medium of natural existence (for example blood plasma or blood) is as PAI-1 or PAI-1 related polypeptide from synthetic thrombin.Can be by method isolated polypeptide from derived cell of any known in this area, including, but not limited to from the adhesive cell culture, taking out the cell culture medium that contains required product, centrifugal or remove by filter the cell etc. of not adhesion.Can from their culture medium of natural existence, separate described polypeptide by the method for any known in this area, including, but not limited to: affinity chromatography, such as the affinity chromatography that on anti-factor VII or anti-PAI-1 antibody column, carries out respectively; Hydrophobic interaction chromatography; Ion exchange chromatography; The size exclusion chromatography; Electrophoresis method (for example preparation type isoelectrofocusing (IEF)); Differential dissolubility (for example ammonium sulfate precipitation); Or extraction method etc.
In the present invention, " effective dose " of factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide is defined as and is enough to together to prevent or reduces hemorrhage or lose blood to cure, to alleviate disease and complication thereof or to make factor VII that its part suppresses or the consumption of the relevant polypeptide of factor VII (for example FVIIa) and PAI-1 or PAI-1 related polypeptide.
Term " activity of factor VIIa " or " factor VIIa activity " comprise the ability that produces thrombin; This term also is included in the ability that produces thrombin under the situation that does not have tissue factor to exist on activatory platelet surface.
Abbreviation
The TF tissue factor
FVII strand, the factor VII of activated form not
The factor VII of FVIIa activated form
The recombinant factor VII of rFVIIa activated form
TAFI proenzyme, the TAFI of activated form not
The PAI-1 plasminogen activator inhibitor
The preparation of chemical compound:
Preferably by Hagen for example etc. at " NAS's journal " (Proc.Natl.Acad.Sci.USA) 83:2412-2416,1986 or as European patent EP 200.421 (ZymoGenetics, the DNA recombinant technique preparation described in Inc.) is applicable to purification human factor VII a of the present invention.
Can also be by Broze and Majerus at " journal of biological chemistry " (J.Biol.Chem.) 255 (4): 1242-1247,1980 and Hedner and Kisiel at " Journal of Clinical Investigation " (J.Clin.lnvest.), 71:1836-1841, the method production factor VII described in 1983.These methods have obtained factor VII, but and do not have other thrombin of detection limit.Filter as the final purification step even the factor VII goods that can obtain being further purified by comprising additional gel.By known way, for example use several different plasma proteins, factor VII changed into activation factor VIIa then such as PAI-1Ia, IXa or Xa.On the other hand, as Bjoern etc. (research open (Research Disclosure), in JIUYUE, 269,1986, pp.564-565) described, by making factor VII by ion-exchange chromatography, such as MonoQ
(Pharmacia fine Chemicals) etc. makes its activation.
Can be by modifying wild type factor VII or passing through the relevant polypeptide of recombinant technique production factor VII.Can produce the relevant polypeptide of factor VII of comparing aminoacid sequence with wild type factor VII by the nucleotide sequence of modifying encoding wild type factor VII with change, wherein said modification step both can be undertaken by changing amino acid code, also can be undertaken by some amino acid code of through known way, for example removing by direct mutagenesis on the nucleic acid of the natural factor VII of coding.
Those skilled in the art obviously can relatively replace and still can obtain active polypeptide in the outside in crucial zone factor VIIa or PAI-1-molecular function.Can according to method as known in the art, such as direct mutagenesis or alanine scanning mutagenesis identify essential to the active institute of factor VII or the relevant polypeptide of factor VII or PAI-1 or PAI-1 related polypeptide, therefore preferably not substituted amino acid residue is (for example, referring to Cunningham and Wells, 1989, " science " be 244:1081-1085 (Science)).In a kind of technology in back, each positively charged residue place introduces sudden change in molecule, the gained mutant molecule is tested the crosslinking active of corresponding coagulant, to identify the amino acid residue crucial for the activity of this molecule.Can also by analyze such as determine by the three dimensional structure of this class technical measurement of nuclear magnetic resonance spectroscopy, crystallography or photoaffinity labeling substrate-enzyme interacting site (for example, referring to de Vos etc., 1992, " science " be 255:306-312 (Science); Smith etc., 1992, " molecular biology magazine " (Journal of Molecular Biology) 224:899-904; Wlodaver etc., 1992, " FEBS communication " (FEBS Letters) 309:59-64).
Can be by direct mutagenesis, use any means as known in the art importing nucleotide sequence that will suddenly change to change nucleotide.Useful especially superhelix double-stranded DNA carrier and insert interested and two methods that contain the synthetic primer of required sudden change of being to use.In the temperature cycles process, extend by the Pfu archaeal dna polymerase with the complementary oligonucleotide primers of the opposite strand of described carrier separately.When mixing primer, generate the mutant plasmid that contains staggered cut.After temperature cycles, use to have specific Dpnl processing product so that digestion parental DNA template also selects to contain the synthetic DNA of sudden change with hemimethylation DNA to methylating.Can also use other method that becomes known for producing, identify and separating variant in this area, such as: for example gene is reorganized method or display technique of bacteriophage.
Can pass through method isolated polypeptide from derived cell of any known in this area, including, but not limited to: the cell culture medium that contains required product from the adhesive cell culture, taken out; Centrifugal or filter with the cell of removing not adhesion etc.
Randomly, can be further purified factor VII or the relevant polypeptide of factor VII.Can use the method for any known in this area to carry out purification, including, but not limited to: affinity chromatography, such as the affinity chromatography that on anti--factor VII antibody column, carries out (for example, referring to (J.Biol.Chem.) 261:11097 such as Wakabayashi " journal of biological chemistry ", 1986; With (Biochem.) 27:7785 such as Thim " biochemistry ", 1988); Hydrophobic interaction chromatography; Ion exchange chromatography; The size exclusion chromatography; Electrophoresis method (for example preparation type isoelectrofocusing (IEF)); Differential dissolubility (for example ammonium sulfate precipitation); Or extraction method etc.Generally referring to Scopes, " protein purification " (Protein Puri-fication), Springer-Verlag, NewYork, 1982; " protein purification " (Protein Purification), J.C.Janson and Lars Ryden edit, VCH Publishers, New York, 1989.Behind the purification, preferably contain in these goods be lower than about 10% weight, more preferably less than about 5% weight, most preferably be lower than the non-factor VII that derives from host cell or the relevant polypeptide of factor VII of about 1% weight.
Can cut by Proteolytic enzyme, use PAI-1Ia or other to have the specific protease of trypsin-like (such as factors IX a, kallikrein, factor Xa and thrombin) and make factor VII or the relevant polypeptide activation of factor VII.For example, referring to (Biochem.) 11:2853 (1972) such as Osterud " biochemistry "; Thomas, U.S. Pat 4,456,591; With (J.Clin.Invest.) 71:1836 (1983) such as Hedner " Journal of Clinical Investigation ".Perhaps, can be by making factor VII or the relevant polypeptide of factor VII by ion-exchange chromatography, make its activation such as MonoQ (Pharmacia) etc.Preparation as described below then also gives the activation factor VII or the relevant polypeptide of factor VII of gained.
For example, can from platelet or endotheliocyte, separate according to known method and be used for PAI-1 of the present invention; But, the preferred reorganization PAI-1 that uses is to avoid using the blood or the tissue derived product of pathophoresis danger.Separating PAI-1 is known with the method for preparing the PAI-1 that recombinates in the art; For example, referring to Pannekoek etc. " European molecular biology association magazine " (JOURNAL EMBO J.) 5 (10), 2539-2544 (1986); Gils etc. " biochemistry and biophysics's journal " (Biochim Bio-phys Acta.) 1998Sep 8; 1387 (1-2): 291-7; Sui etc. " journal of biological chemistry " (Biochem J.) 1998 Apr 15; 331 (Pt2): 409-15; Gins-burg etc. " Journal of Clinical Investigation " (J.Clin.Invest.), the 78th the volume, pp.1673-1680 (1986); Or U.S. Pat 6,303,338 and US6,103,498.Can produce the PAI-1 variant by aforesaid direct mutagenesis.
Can be by modifying wild type PAI-1 or producing the PAI-1 related polypeptide by recombinant technique.Can produce the PAI-1 related polypeptide of comparing aminoacid sequence with wild type PAI-1 by the nucleotide sequence of modifying encoding wild type PAI-1 with change, the step of wherein said modification both can be undertaken by changing amino acid code, also can be undertaken by some amino acid code that known way, the direct mutagenesis that for example above describes in detail are removed on the nucleic acid of the natural PAI-1 of coding.Can pass through method isolated polypeptide from derived cell of any known in this area, including, but not limited to: the cell culture medium that contains required product from the adhesive cell culture, taken out; Centrifugal or filter with the cell of removing not adhesion etc.Randomly, can be further purified PAI-1 or PAI-1 related polypeptide.Can use the method for any known in this area to carry out purification, including, but not limited to the affinity chromatography that above describes in detail, such as the affinity chromatography that on anti--PAI-1 antibody column, carries out; Hydrophobic interaction chromatography; Ion exchange chromatography; The size exclusion chromatography; Electrophoresis method (for example preparation type isoelectric focusing (IEF)); Differential dissolubility (for example ammonium sulfate precipitation); Or extraction method etc.Behind the purification, preferably contain in these goods be lower than about 10% weight, more preferably less than about 5% weight, most preferably be lower than the non--PAI-1 that derives from host cell or the PAI-1 related polypeptide of about 1% weight.Then can preparation as described below and give the activation PAI-1 or the PAI-1 related polypeptide of gained.
Just as will be understood by the skilled person in the art, the preferred use and homologous PAI-1 polypeptide of experimenter and factor VII polypeptides is so that reduce the danger of induction of immunity reaction.The present invention also comprises this class PAI-1 polypeptide and the application of factor VII polypeptides in the veterinary.
Pharmaceutical composition and using method
Preparation of the present invention can be used for the treatment of the reactive syndrome of any factor VII, such as, hemorrhagic disease for example, including, but not limited to: the syndrome that causes because of blood coagulation factor VIII, IX, XI or the reduction of VII level, blood coagulation factor inhibitors, platelet function defective (for example glanzmann's thrombasthenia and Bai-Suo syndrome); Thrombocytopenia; Feng's von Willebrand's disease; With such as increase and reduce the coagulopathy that (for example carried out operation or take place among the patient of repeatedly blood transfusion of wound) caused because of coagulated protein dilution, fibrinolysis because of hemorrhage and/or blood transfusion cause platelet counts.The pharmaceutical composition that comprises the preparation of the preparation of factor VII of the present invention or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide is mainly used in to prevent and/or treat uses parenterai administration.Preferably give this pharmaceutical composition, promptly by intravenous, subcutaneous or intramuscular administration, most preferably intravenous administration through non-intestinal.Can also be by continuous or the infusion administration of beating.
Pharmaceutical composition of the present invention or preparation comprise factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide, they can be mixed with single unit dosage forms or ingredient kit form, preferably be dissolved in pharmaceutically acceptable carrier, preferred aqueous carrier or the diluent.Briefly, by being prepared, the combination of factor VII or the relevant polypeptide of factor VII or PAI-1 or factor VII or relevant polypeptide of factor VII and PAI-1 (preferred purified form) and suitable adjuvant and suitable carriers or mixing diluents be applicable to pharmaceutical composition of the present invention.Can use various aqueous carriers, such as water, buffered water, 0.4% saline, 0.3% glycine etc.Can also use nonaqueous carrier to prepare preparation of the present invention, for example be mixed with that gel or liposome form are transported or targeting to damage location.The Liposomal formulation general description is at for example U.S. Pat 4,837,028, US4, in 501,728 and US4,975,282.Can be by these these compositions sterilizations of conventional well-known sterilization technology.Can be with the obtained aqueous solution packaging applications, or under aseptic condition, filter and lyophilizing, before administration, lyophilized formulations is mixed with aseptic aqueous solution.
These compositionss can contain pharmaceutically acceptable auxiliary substance or adjuvant, including, but not limited to pH regulator agent and buffer agent and/or tension regulator, such as: for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride etc.
Preparation may further include one or more diluent, emulsifying agent, antiseptic, buffer agent, excipient etc., and they can be made such as dosage forms such as liquid, powder, Emulsion, controlled releases.Those skilled in the art can suitable way and are prepared compositions of the present invention according to the practice of accepting, edit such as being disclosed in " RemingtonShi pharmaceutical science " (Remington ' sPharmaceutical Sciences) Gennaro, Mack Publishing Co., Easton, PA, those modes in 1990.Therefore, the typical pharmaceutical compositions that intravenous infusion can be used is made the form that contains aseptic Ringer's solution of 250ml and 10mg preparation.
The compositions that can contain preparation of the present invention is used to prevent and/or treat Sex therapy.In treatment was used, the experimenter of disease gave compositions to suffering from as mentioned above, and its consumption is enough to cure, alleviate or partly suppress the clinical manifestation of disease and complication thereof.The consumption that will be enough to reach this purpose is defined as " treatment effective dose ".The effective dose that is used for each purpose depends on the order of severity of i or I and experimenter's body weight and general situation.Should understand and to use normal experiment, determine optimal dose by the difference that makes up numerical matrix and test in this matrix.
For example, can be by spraying, perfusion, double balloon catheter, stent, mix blood vessel graft or stent, be used to apply the hydrogel of balloon catheter or method that other is fully set up is come the local preparation of the present invention of delivering, such as local application.Under any circumstance, described pharmaceutical composition all should provide the preparation consumption that is enough to effectively treat described disease.
The concentration that is combined in these preparations of factor VII or the relevant polypeptide of factor VII, PAI-1 or PAI-1 related polypeptide or factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide can extensively change, promptly, mainly select according to fluid volume, viscosity etc. and according to selected specific administration mode from being lower than about 0.5% weight, being generally or being at least about 1% weight to reaching 15 or 20% weight.Preferably, particularly inject by injection or the administration of infusion mode.Therefore, factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide are made the form that is suitable for intravenous administration, such as a kind of like this preparation, promptly in a kind of dosage form, not only contained factor VII or the relevant polypeptide of factor VII but also contained PAI-1 or the dissolved freeze-dried powder preparation or the liquid preparation of PAI-1 related polypeptide, and perhaps in a kind of dosage form, contained the dissolved lyophilized powder of factor VII or the relevant polypeptide of factor VII or liquid preparation and in another kind of dosage form, contain the dissolved lyophilized powder or the liquid preparation of PAI-1 or PAI-1 related polypeptide.
Should understand the amount of factor VII or the relevant polypeptide of factor VII and the amount of PAI-1 or PAI-1 related polypeptide and constitute the total effective dose for the treatment of bleeding episodes jointly.
Must be noted that material of the present invention generally can be used for serious i or I state, i.e. life-threatening or life is had the situation of potential threat.In this class situation, owing to foreign body has been reduced to bottom line and the overall immunogenicity that lacks factor VIIa and PAI-1 in human body, can give these excessive in fact compositionss by the clinicist who participates in treatment, and might compare ideal like this.
In prophylactic applications, to susceptible or be in the disease situation or the experimenter of damage in the danger contained the compositions of preparation of the preparation of factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide to strengthen experimenter's self blood coagulation ability.This class consumption is defined as " prevention effective dose ".Should understand the amount of factor VII or the relevant polypeptide of factor VII and the amount of PAI-1 or PAI-1 related polypeptide and constitute the total effective dose that prevents bleeding episodes jointly.
Can use dosage level and the scheme selected by the clinicist who participates in treatment that described compositions is carried out single or multiple dosing.Can every day or give one or many with described compositions weekly.The effective dose of this class pharmaceutical composition is for providing the consumption of clinical remarkable effect to bleeding episodes.This class consumption depends in part on the specified disease of being treated, experimenter's age, body weight and general health situation and the other factors that it will be apparent to those skilled in the art.
Generally estimate hemorrhage before or begin to give when hemorrhage the present composition of single dose.Yet, also repeatedly (dosage several times), preferably give compositions of the present invention with 2-4-6-12 hour interval, this depends on the dosage that gives and experimenter's situation.
In order to be used for intervening relevant treatment, generally give factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide in about 24 hours before intervening, and after this continue more than 7 days or 7 days with plan.Can be as described herein by all means with it as the coagulant administration.
Compositions can be the two the unitary agent form (single dosage form) of preparation that comprises the preparation of the factor VII of suitable concentration or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide.Said composition can also be the ingredient kit form, comprising first unit dosage forms of the preparation of factor VII or the relevant polypeptide product of factor VII with comprise second unit dosage forms of the preparation of PAI-1 or PAI-1 related polypeptide goods.In this case, should be successively, preferred each interval is less than in about 15 minutes, for example in 10 minutes or preferably in 5 minutes or more preferably gave factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide each other in 2 minutes.Can at first give in two kinds of unit dosage forms any.
Described test kit comprises two kinds of independently pharmaceutical compositions at least.This test kit comprises the container apparatus that is used to comprise each independent group compound, such as bottle that separates or the little paper tinsel bag that separates.This test kit generally comprises the description that is used to give independent element.When preferred give each independent element with different dosage form, when giving independent element at interval with various dose maybe when opening each composition in need determining to make up of clinicist according to prescription, the test kit dosage form is particularly advantageous.
The amount of the factor VII that gives according to the present invention or the amount of the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide can be about 1: change between the ratio of 100-about 100: 1 ( w/w ) .VIIPAI-1：1∶1001∶901∶801∶701∶601∶501∶401∶301∶201∶101∶51∶21∶12∶15∶110∶120∶130.140∶150∶160∶170∶180∶190∶1100∶11∶90-1∶11∶80-1∶21∶70-1∶51∶60-1∶101∶50-1∶251∶40-1∶3090∶1-1∶180∶1-2∶170∶1-5∶160∶1-10∶150∶1-25∶140∶1-30∶1。
With regard to 70-kg experimenter, the scope that is equivalent to the about 500mg/ of about 0.05mg-days wild type factor VII as the load and the dosage of maintenance dose factor VII or the relevant polypeptide of factor VII, for example about 1mg-about 200mg/ days or for example about 5mg-about 175mg/ days, this depends on the order of severity of experimenter's body weight, disease and disease.
With regard to 70-kg experimenter, the scope that is equivalent to the about 500mg/ of about 0.05mg-days wild type PAI-1 as the dosage of load and maintenance dose PAI-1 or PAI-1 related polypeptide, for example about 1mg-about 200mg/ days or for example about 1mg-about 175mg/ days, this depends on the order of severity of experimenter's body weight, disease and disease.
Being combined in external grumeleuse hardness and the fibrinolysis time test of factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide shows synergism.In addition, the aspect that forms stable fibrin grumeleuse, increases the half EGCT, increases clot strength and increase anti-fibrinolysis ability that is combined in of factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide all shows synergism.
Compositions can be the unitary agent form that comprises factor VII or the relevant polypeptide of factor VII and the PAI-1 or the PAI-1 related polypeptide of suitable concentration.Said composition can also and comprise the kit form that second unit dosage forms of PAI-1 or PAI-1 related polypeptide is formed by first unit dosage forms that comprises factor VII or the relevant polypeptide of factor VII.In this case, should be successively, in preferred the about 1-2 of each interval hour, in for example each interval 30 minutes or preferably in 10 minutes or more preferably each interval gives factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide in 5 minutes.Can at first give in two kinds of unit dosage forms any.
Owing to the present invention relates to the prevention or the Therapeutic Method of bleeding episodes, perhaps unite the coagulation therapy that gives various active component (can give separately), make up each independently pharmaceutical composition so the invention still further relates to kit form.This test kit comprises two kinds of independently pharmaceutical compositions at least.This test kit comprises the container apparatus that is used to comprise the independent group compound, such as bottle that separates or the little paper tinsel bag that separates.This test kit generally comprises the description that is used to give each independent element.When preferred give each independent element with different dosage form, when giving each independent element at interval with various dose maybe when opening clinicist according to prescription when needing each composition in the titration combination, the test kit dosage form is particularly advantageous.
Test:
The factor VIIa activity test:
The suitable test of can carry out test factor VIIa activity, selecting suitable factor VIIa variant thus is as simple external pre-stage test:
The extracorporeal hydrolysis test
Can test the specific activity of natural (wild type) factor VIIa and factor VIIa variant (hereinafter being referred to as " factor VIIa ").Can also parallel testing they so that direct its specific activity relatively.(MaxiSorp, Nunc carry out on Denmark) at microtitration plate in this test.With final concentration is that (S-2288, Chromogenix Sweden) join to be dissolved in and contain 0.1M NaCl, 5mMCaCl for the chromogenic substrate D-Ile-Pro-Arg-P-nitroanilide of 1mM
2In the factor VIIa (final concentration 100nM) of the 50mM Hepes (pH 7.4) of 1mg/ml bovine serum albumin.At SpectraMax
TM340 plate readers (Molecular Devices, USA) absorbance at last METHOD FOR CONTINUOUS DETERMINATION 405nm place.The value that will obtain after the absorbance that showed in the insulating process in 20 minutes deducts absorbance in the blank well that does not contain enzyme is used to calculate the ratio of variant and the activity of wild type factor VIIa:
Ratio=(the A of factor VIIa variant
405nmThe A of)/(factor VIIa wild type
405nm).
Based on The above results, can identify to have the active factor VIIa variant equal or higher with natural factor VIIa, such as: the active ratio with natural factor VII (wild type FVII) activity of for example described variant is about the variant about in the of 1.0.
Can also working concentration suit 100-1000nM measure the activity of factor VIIa or factor VIIa variant such as the such physiology substrate of factor X, wherein measure the factor Xa that produces adding suitable chromogenic substrate (for example S-2765) back.In addition, can under physiological temp, carry out activity test.
External Proteolytic enzyme test
Can natural (wild type) factor VIIa of parallel testing and factor VIIa variant (hereinafter being referred to as " factor VIIa ") so that direct its specific activity relatively.(MaxiSorp, Nunc carry out on Denmark) at microtitration plate in this test.Factor VIIa (10nM) and factor X (0.8 μ M) are being contained 0.1M NaCl, 5mMCaCl
2With the middle insulation of 100 μ l 50mM Hepes (pH 7.4) of 1mg/ml bovine serum albumin 15 minutes.Contain 50 μ l 50mM Hepes (pH7.4) termination factor X cracking of 0.1M NaCl, 20mM EDTA and 1mg/ml bovine serum albumin then by interpolation.By adding the chromogenic substrate Z-D-Arg-Gly-Arg-p-nitroanilide that final concentration is 0.5mM (S-2765, Chromogenix, Sweden) amount of the factor Xa of mensuration generation.At SpectraMax
TM340 plate readers (Molecular De-vices, USA) absorbance at last METHOD FOR CONTINUOUS DETERMINATION 405nm place.To be used to calculate the ratio of the proteolytic activity between variant and the wild type factor VIIa in the value after the absorbance that showed in the insulating process in 10 minutes deducts absorbance in the blank well that does not contain FVIIa:
Ratio=(A405nm of factor VIIa variant)/(A405nm of factor VIIa wild type).
Based on The above results, can identify and have the active factor VIIa variant equal or higher with natural factor VIIa, such as: the active ratio with natural factor VII (wild type FVII) activity of for example described variant is about the variant about in the of 1.0.
The thrombin generation test:
Can produce the ability of thrombin (as Monroe etc. at (1997) " Britain hematology magazine " (Brit.J.Haematol.) 99 with the test determination factor VII that comprises inhibitor under all relevant thrombins and the physiological concentration and activated blood platelet or the relevant polypeptide of factor VII or PAI-1 or PAI-1 related polypeptide (for example variant), among the 542-547 the 543rd page the above, the document is incorporated herein by reference).
The PAI-1 activity test:
For example, can be with as " clinical chemistries " such as Chandler (Clinical Chemistry), the described in vitro tests of 35 (5) 787-793 (1989) is tested the PAI-1 activity, is provided the suitable test of the screening mode of the used PAI-1 variant of suitable the present invention thus as simple experiment or other test as known in the art (described " PAI-1 test ").
Further explain the present invention by the following example, but, these embodiment are not used for limiting protection domain.Disclosed feature both can also can become with multi-form realization material of the present invention in its combination in any mode separately in foregoing description and the following examples.
Embodiment
Improve the stability of hemostasis grumeleuse by coupling proconvertin a and PAI-1
Method:
Clot dissolution test: will be with containing Innovin (Dade Behring, dilution 2000-are doubly), rFVIIa (Novo Nordisk A/S, Bagsvaerd, Denmark; Variable concentrations) and t-PA (American Diagnostics, buffer 8nM) (20mM HEPES, 150mM NaCl, 5mMCaCl, pH7.4) dilution 10-human normal plasma doubly joins on the ELISA flat board of 96-hole, and at room temperature measures the turbidity at 650nm place in a period of time.Indicate the place, then comprising purification people PAI-1 (American Diagnostica, variable concentrations).
Rotation thrombelastography (roTEG): lack blood plasma (George King Bio-Medical with citric acid salinization human normal plasma who has added 5nM t-PA or Factor IX, Inc.#0800) carry out every mensuration, and analyze and add 1nM FVIIa separately or add 1nMFVIIa and 30nM PAI-1 effect of Combination.By in 20mM HEPES, 150mMNaCl, pH 7.4 buffer, add Innovin (dilution 2000-final concentration doubly, DadeBehring#526945) and calcium (final concentration 15mM) startup blood coagulation.
The result:
Clot dissolution test: add FVII-a and cause EGCT to prolong (accompanying drawing 1) in the dose dependent mode.This 10nM FVIIa that acts on is best down.Under the situation that has 10nM FVIIa to exist, add PAI-1 and cause EGCT further to prolong (accompanying drawing 2).This effect is a dose dependent, and is issued to the best at 10nM PAI-1.
Thromboelastogram: the combination that the roTEG measured value is used to analyze FVIIa and PAI-1 is to maximum grumeleuse hardness (MCF) and the grumeleuse effect that dissolved toleration produced to the t-PA mediation.Measure with human plasma normal and shortage FVIII.Before adding FVIIa/PAI-1, the MCF in normal and hemophilia blood plasma is respectively 25mm and 4mm, and the half EGCT is respectively 12.3 minutes and 14.3 (accompanying drawing 3 and 4).In the human normal plasma, add PAI-1 (30nM) and can not change MCF, but the half EGCT can be extended to 16.1 minutes in (accompanying drawing 3).In hemophilia blood plasma, when adding PAI-1 separately MCF increased to 5mm and the half EGCT is extended to 32.4 minutes (accompanying drawing 4).Add FVIIa (1nM) and prevented that grumeleuse from the dissolving (half EGCT 16.7min) of t-PA-mediation taking place, and to the MCF in the normal plasma without any appreciable impact (accompanying drawing 3), prevented that grumeleuse from dissolving (half EGCT 21.3min) and the remarkable increase of MCF value (15mm) (accompanying drawing 4) and in hemophilia blood plasma, add FVIIa.Yet the two makes the half EGCT increase to 29.8 minutes and 45 minutes respectively to add FVIIa and PAI-1 in normal and hemophilia blood plasma together, and makes the MCF value increase to 27.4mm and 16.9mm respectively.
Conclusion:
These results show, add FVIIa and PAI-1 and can cooperative mode improve the mechanical strength of grumeleuse and to Fibrinolytic toleration in blood plasma.
Claims (53)
1. pharmaceutical composition is comprising factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide.
2. the compositions of claim 1, wherein said factor VII or the relevant polypeptide of factor VII are the relevant polypeptide of factor VII.
3. the compositions of claim 1, the relevant polypeptide of wherein said factor VII is a factor VII aminoacid sequence variant.
4. claim 2 or 3 compositions, wherein when with as described in this manual " extracorporeal hydrolysis test " mensuration, the ratio of the activity of the activity of the relevant polypeptide of described factor VII and natural human factor VIIa (wild type FVIIa) is at least about 1.25.
5. the compositions of claim 1, wherein said factor VII or the relevant polypeptide of factor VII are factor VII.
6. the compositions of claim 5, wherein said factor VII is a human factor VII.
7. the compositions of claim 6, wherein said factor VII is the reorganization human factor VII.
8. any one compositions among the claim 1-7, wherein said factor VII or the relevant polypeptide of factor VII are its activated form.
9. the compositions of claim 8, wherein said factor VII is the recombined human factor VIIa.
10. any one compositions among the claim 1-9, wherein said PAI-1 or PAI-1 related polypeptide are the PAI-1 related polypeptides.
11. the compositions of claim 10, wherein said PAI-1 related polypeptide are PAI-1 aminoacid sequence variants.
12. the compositions of claim 10 or 11, wherein when measuring with as described in this manual " PAI-1 test ", the ratio of the activity of the activity of described PAI-1 related polypeptide and natural human PAI-1 (wild type PAI-1) is at least about 1.25.
13. any one compositions among the claim 1-9, wherein said PAI-1 or PAI-1 related polypeptide are the PAI-1 polypeptide.
14. the compositions of claim 13, wherein said PAI-1 is people PAI-1.
15. the compositions of claim 14, wherein said PAI-1 is recombined human PAI-1.
16. the compositions of claim 15, wherein said PAI-1 is an activation conformation form.
17. any one compositions among the claim 1-16, the ratio that wherein said factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide exist is about 100 by weight: about 1: 100 of 1-(w/w factor VII:PAI-1).
18. any one compositions among the claim 1-17 wherein further comprises in the said composition being suitable for injecting or infusion, the particularly pharmaceutically acceptable excipient of injection.
19. contain the test kit of the ingredient that is used for the treatment of bleeding episodes, comprising:
F) can accept the preparation of carrier on the factor VII of the effective dose of first unit dosage forms or relevant polypeptide of factor VII and the medicine;
G) can accept the preparation of carrier on the PAI-1 of the effective dose of second unit dosage forms or PAI-1 related polypeptide and the medicine; With
H) be used to comprise the container apparatus of described first and second dosage forms.
20. the test kit of claim 19, wherein said factor VII or the relevant polypeptide of factor VII are the relevant polypeptide of factor VII.
21. the test kit of claim 20, the relevant polypeptide of wherein said factor VII are factor VII aminoacid sequence variants.
22. the test kit of claim 20 or 21, wherein when measuring with as described in this manual " extracorporeal hydrolysis test ", the ratio of the activity of the activity of the relevant polypeptide of described factor VII and natural human factor VIIa (wild type FVIIa) is at least about 1.25.
23. the test kit of claim 19, wherein said factor VII or the relevant polypeptide of factor VII are factor VII.
24. the test kit of claim 23, wherein said factor VII is a human factor VII.
25. the test kit of claim 24, wherein said factor VII polypeptides are the reorganization human factor VIIs.
26. any one test kit among the claim 19-25, wherein said factor VII or the relevant polypeptide of factor VII are its activated form.
27. the test kit of claim 26, wherein said factor VII are the recombined human factor VIIas.
28. any one test kit among the claim 19-27, wherein said PAI-1 or PAI-1 related polypeptide are the PAI-1 related polypeptides.
29. the test kit of claim 28, wherein said PAI-1 related polypeptide are PAI-1 aminoacid sequence variants.
30. the test kit of claim 28 or 29, wherein when measuring with as described in this manual " PAI-1 test ", the ratio of the activity of the activity of described PAI-1 related polypeptide and natural human PAI-1 (wild type PAI-1) is at least about 1.25.
31. any one test kit among the claim 19-27, wherein said PAI-1 or PAI-1 related polypeptide are PAI-1.
32. the test kit of claim 31, wherein said PAI-1 are people PAI-1.
33. the test kit of claim 32, wherein said PAI-1 are recombined human PAI-1.
34. the test kit of claim 33, wherein said PAI-1 are activation conformation forms.
35. any one test kit among the claim 19-34, the ratio that wherein said factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide exist is about 100 by weight: about 1: 100 of 1-(w/w factor VII:PAI-1).
36. the preparation that is combined in of factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide is used for the treatment of application in the medicine of bleeding episodes.
37. any one compositions is used for the treatment of application in the medicine of bleeding episodes in preparation among the claim 1-18.
38. the application of claim 36 or 37, wherein said medicine is used to reduce setting time.
39. the application of claim 36 or 37, wherein said medicine is used to prolong EGCT.
40. the application of claim 36 or 37, wherein said medicine is used to increase clot strength.
41. any one application among the claim 36-40 wherein is mixed with described medicine injection or infusion, particularly injection medicament.
42. any one application among the claim 36-41, wherein said bleeding episodes be because of wound or operation or platelet count or active descend cause.
43. any one application among the claim 36-42, wherein said medicine is single dosage form.
44. any one application among the claim 36-42, wherein described medication preparation is become to contain factor VII or the relevant polypeptide of factor VII preparation first unit dosage forms and contain the form of second unit dosage forms of the preparation of PAI-1 or PAI-1 related polypeptide.
45. the Therapeutic Method of experimenter's internal hemorrhage outbreak, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can be treated hemorrhage together effectively.
46. reduce the method for setting time in the experimenter, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can reduce setting time together effectively.
47. strengthen hemostatic method in the experimenter, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can effectively strengthen hemostasis together.
48. prolong the method for EGCT in the experimenter, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can effectively prolong EGCT together.
49. increase the method for clot strength in the experimenter, this method comprises according to experimenter's needs and gives the factor VII of first consumption or preparation and the PAI-1 of second consumption or the preparation of PAI-1 related polypeptide of the relevant polypeptide of factor VII to it that wherein said first consumption and second consumption can effectively increase clot strength together.
50. any one method among the claim 45-49 wherein gives factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide with single dosage form.
51. any one method among the claim 45-49 wherein gives described factor VII or the relevant polypeptide of factor VII and PAI-1 or PAI-1 related polypeptide with first dosage form of the preparation that contains factor VII or the relevant polypeptide of factor VII and the form of second dosage form of the preparation that contains PAI-1 or PAI-1 related polypeptide.
52. the method for claim 51 wherein gives first dosage form and the interval that gives between second dosage form is no more than 15 minutes.
53. contain the test kit of the treatment articles that is useful on bleeding episodes, comprising:
A) can accept carrier on the PAI-1 of the factor VII of the effective dose of single unit dosage forms or relevant polypeptide of factor VII and effective dose or PAI-1 related polypeptide and the medicine; With
B) be used to comprise the container apparatus of described single unit dosage forms.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DKPA200101671 | 2001-11-09 | ||
| DKPA200101671 | 2001-11-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1596122A true CN1596122A (en) | 2005-03-16 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA028236777A Pending CN1596122A (en) | 2001-11-09 | 2002-11-05 | Pharmaceutical composition comprising factor vii polypeptides and pai-1 polypeptides |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP1446146A1 (en) |
| JP (1) | JP2005510514A (en) |
| KR (1) | KR20050043733A (en) |
| CN (1) | CN1596122A (en) |
| AU (1) | AU2002340778B2 (en) |
| BR (1) | BR0213952A (en) |
| CA (1) | CA2464614A1 (en) |
| CZ (1) | CZ2004535A3 (en) |
| HU (1) | HUP0401959A3 (en) |
| IL (1) | IL161523A0 (en) |
| NO (1) | NO20042379L (en) |
| PL (1) | PL370118A1 (en) |
| RU (1) | RU2304980C2 (en) |
| WO (1) | WO2003039580A1 (en) |
| ZA (1) | ZA200403270B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111205244A (en) * | 2018-11-22 | 2020-05-29 | 上海科技大学 | Thiazolocyclic compound, preparation method, intermediate and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009045412A2 (en) * | 2007-10-01 | 2009-04-09 | American Diagnostica, Inc. | Methods of treatment using modified plasminogen activator inhibitor type-1 molecules |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998058661A1 (en) * | 1997-06-23 | 1998-12-30 | Novo Nordisk A/S | Use of fviia for the treatment of bleedings in patients with a normal blood clotting cascade and normal platelet function |
| AT409335B (en) * | 1998-11-10 | 2002-07-25 | Immuno Ag | PHARMACEUTICAL PREPARATION CONTAINING A RECEPTOR ANTAGONIST FOR THE TREATMENT OF BLOOD COagulation disorders |
| JP4455802B2 (en) * | 2000-05-03 | 2010-04-21 | ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト | Human VII coagulation factor variant |
-
2002
- 2002-11-05 WO PCT/DK2002/000735 patent/WO2003039580A1/en active IP Right Grant
- 2002-11-05 HU HU0401959A patent/HUP0401959A3/en unknown
- 2002-11-05 AU AU2002340778A patent/AU2002340778B2/en not_active Ceased
- 2002-11-05 KR KR1020047006606A patent/KR20050043733A/en not_active Application Discontinuation
- 2002-11-05 CA CA002464614A patent/CA2464614A1/en not_active Abandoned
- 2002-11-05 EP EP02774474A patent/EP1446146A1/en not_active Withdrawn
- 2002-11-05 PL PL02370118A patent/PL370118A1/en not_active Application Discontinuation
- 2002-11-05 RU RU2004117535/15A patent/RU2304980C2/en not_active IP Right Cessation
- 2002-11-05 CZ CZ2004535A patent/CZ2004535A3/en unknown
- 2002-11-05 CN CNA028236777A patent/CN1596122A/en active Pending
- 2002-11-05 BR BR0213952-9A patent/BR0213952A/en not_active IP Right Cessation
- 2002-11-05 JP JP2003541871A patent/JP2005510514A/en not_active Withdrawn
- 2002-11-05 IL IL16152302A patent/IL161523A0/en unknown
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- 2004-04-30 ZA ZA200403270A patent/ZA200403270B/en unknown
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111205244A (en) * | 2018-11-22 | 2020-05-29 | 上海科技大学 | Thiazolocyclic compound, preparation method, intermediate and application thereof |
| CN111205244B (en) * | 2018-11-22 | 2023-08-18 | 上海科技大学 | Thiazolo-ring compound, preparation method, intermediate and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| RU2304980C2 (en) | 2007-08-27 |
| HUP0401959A3 (en) | 2009-03-30 |
| CA2464614A1 (en) | 2003-05-15 |
| BR0213952A (en) | 2004-08-31 |
| AU2002340778B2 (en) | 2007-08-02 |
| KR20050043733A (en) | 2005-05-11 |
| IL161523A0 (en) | 2004-09-27 |
| EP1446146A1 (en) | 2004-08-18 |
| CZ2004535A3 (en) | 2005-01-12 |
| WO2003039580A1 (en) | 2003-05-15 |
| ZA200403270B (en) | 2004-11-08 |
| NO20042379L (en) | 2004-06-08 |
| PL370118A1 (en) | 2005-05-16 |
| JP2005510514A (en) | 2005-04-21 |
| RU2004117535A (en) | 2005-04-10 |
| HUP0401959A2 (en) | 2004-12-28 |
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