CN101365478A - Use of c1 inhibitor for the prevention of ischemia-reperfusion injury - Google Patents
Use of c1 inhibitor for the prevention of ischemia-reperfusion injury Download PDFInfo
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- CN101365478A CN101365478A CNA2006800482879A CN200680048287A CN101365478A CN 101365478 A CN101365478 A CN 101365478A CN A2006800482879 A CNA2006800482879 A CN A2006800482879A CN 200680048287 A CN200680048287 A CN 200680048287A CN 101365478 A CN101365478 A CN 101365478A
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- reperfusion injury
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Abstract
The present invention relates to the therapeutic and prophylactic use of Cl inhibitor for preventing, reducing and treating ischemia and reperfusion injury. The C 1 inhibitor of the present invention is still therapeutically effective when administered after an ischemic period and reperfusion and therefore particularly useful for unforeseen occurrences of ischemic reperfusion such as e.g. a stroke.
Description
Invention field
The present invention relates to treatment and the prophylactic use of C1 inhibitor in prevention, reduction and treatment ischemia reperfusion injury, special application in cerebral ischemia-reperfusion injury that the result owing to apoplexy takes place.
Background of invention
Ischemia reperfusion injury is (occuring) pathological condition in a kind of generation of knowing.It can be the pathological condition of prediction or the pathological condition that can't predict.Apoplexy is a kind of modal ischemia reperfusion injury that can't predict.Apoplexy is to cause dead the third-largest factor in the industrialized country, causes prolonged disability.Apoplexy is a kind of typical cardiovascular disease, and the tremulous pulse in brain or the brain is led in influence.When this tremulous pulse by thrombus obstruction or when breaking apoplexy takes place, cause the cerebral tissue ischemia of supporting by the tremulous pulse of described obstruction.Direct infringement to brain is owing to due to the blood flow interruption, mainly be because the Oxidation of tissue survival is kept in forfeiture, causes due to the infarction at last if can not reverse.Yet if described infringement is reversible (reversing naturally or through treatment), the perfusion again of ischemic tissue can cause further consequential damages contradictoryly.When having long-term ischemia, deriving from anoxybiotic separately " directly " infringement is main mechanism.For the ischemia of short period, pour into the infringement that mediates indirectly or again becomes the prior factor for final result interestingly.
It is reported that inhibitor-C1 inhibitor (C1INH) of complement C1 shows neuroprotective (De Simoni et al by reducing ischemia reperfusion injury in rodentine cerebral ischemia-re-perfusion model, 2003, J Cereb Blood Flow Metab.23:232-9; Akita et al., 2003, Neurosurgery52:395-400).C1INH does not need CIq (De Simoni et al., 2004, Am J Pathol.164:1857-63) for the neuroprotective of cerebral ischemia-reperfusion injury.Recently, Storini etc. (2005, Neurobiol Dis.19:10-7) have reported that C1INH raises to ishemic part and to ischemia reperfusion injury performance antiinflammatory and anti-apoptotic effect by inflammation-inhibiting and cell from vascular system.Yet it is narrower to give the medicable apoplexy time-of-week of C1INH window.Therefore, an object of the present invention is to provide C1INH with broad administration time window.
Invention is described
The present invention is based on wonderful discovery; promptly in the of short duration focal cerebral ischemia model of mice; lose its most of ability that reduces ischemia reperfusion injury when the deutero-C1 inhibitor of naturally occurring blood plasma (C1INH) gives after ischemia, and the C1INH prepared product of reorganization ischemia and/or again perfusion after at least 1 hour injection still can bring into play its neuroprotective.Astoundingly, when at ischemia and/or pour into again back 18 hours whens injection, still can reach neuroprotective.The difference of deutero-C1INH of naturally occurring blood plasma and recombinant C 1INH prepared product is that the former plasma half-life is at least 24 hours and is complete sialylated glycoprotein that the latter has the plasma half-life of reduction and has the glycosylation different with the blood plasma product.
Known difference between deutero-C1INH of naturally occurring blood plasma and the recombinant C 1INH prepared product is glycosylated degree and type.The glycoprotein of reorganization contains a large amount of different N-polysaccharide, comprises few mannose type, heterozygous and composite structured, and the N-polysaccharide of the deutero-C1INH of blood plasma is mainly by complete sialylated composite structured the composition.Because glycosylated difference, so the plasma half-life of the deutero-glycoprotein of blood plasma is at least 24 hours, and recombinant C 1INH has the plasma half-life of reduction.
Therefore, an aspect of of the present present invention relates to prevention, reduces or treats the method for at least a ischemia and reperfusion injury, and wherein the C1 inhibitor is being given at ischemia and/or after pouring into again.Described method preferably includes the step of the plasma half-life that the gives effective dose C1INH shorter than the half-life of the deutero-C1INH of blood plasma.Perhaps, described method preferably includes the step that gives having of the effective dose glycosylated C1INH different with the deutero-C1INH of blood plasma.This method relates to the treating and/or preventing property application of C1 inhibitor in the ischemia reperfusion injury of prevention, reduction and/or treatment any kind.
The C1 inhibitor is also referred to as the C1 esterase inhibitor, is meant the inhibitor of complement C1 at this paper.C1INH belongs to serpin superfamily, is the Cir of complement system and unique inhibitor of CIs, and is the factor XI, plasma thromboplastin antecedent Ia of contact system and the main inhibitor of kallikrein.In addition, C1INH is other serine protease of anticoagulant and fibrinolytic system also, as factor XI, plasma thromboplastin antecedent, organize plasminogen activator and fibrinolysin (Schapira et al.1985, Complement 2:111; Davis, 1988, Ann.Rev.Immunol.6:595).The protein that people C1INH is made up of 500 aminoacid comprises signal sequence (Carter et al.1988, Euro.J.Biochem.173 that 22 aminoacid is formed; 163).Plasma C 1INH is that molecular weight is about 76kDa and a large amount of glycosylated glycoprotein, forms (Perkins et al., 1990, J.MoI.Biol.214,751) near 26% by carbohydrate in its molecular weight.The C1INH that is used for the inventive method preferably has the protein that has the aminoacid sequence of at least 65,67,68,69,70,75,80,85,90,95,98 or 99% homogeny with the aminoacid sequence that becomes acquaintance C1INH shown in the SEQ ID NO:1.
For the present invention, the homogeny degree between two aminoacid sequences is meant amino acid whose percentage ratio identical between two sequences.At first, use et al., J.MoI.Biol.215:403-410 (1990) described Basic Local Alignment Search Tool (BLAST) algorithm retrieval homeopeptide sequence as Altschul.Carry out software that BLAST analyzes can openly derive from American National biotechnology information centre (
Http:// www.ncbi.nlm.nih.gov/).BLAST algorithm parameter W, B and E have determined correlated susceptiveness of series arrangement and speed.Blast program uses default parameters: word length (W)=3, and BLOSUM62 rating matrix (scoring matrix) (is seen Henikoff ﹠amp; Henikoff, Proc.Natl.Acad.Sci.USA89:10915 (1989)) sequence contrast (B)=50, expected value (E)=10, M=5, N=-4.Then, use C1USTALW to arrange contrast algorithm (Higgins D.et al (1994) .NucleicAcids Res.22:4673-4680), utilize following parameter to determine the homogeny degree of homologous sequence (as above-mentioned): breach size: 5, breach open (Gap open): 11, breach extends (Gap extension): 1, mispairing :-15, word size: 3.
C1INH preferably have can be for example by the C1INH activity of the described mensuration of Drouet et al. (1988, Clin Chim Acta.174:121-30).More preferably, C1INH is people C1INH (hCHNH), mean that described C1INH has naturally occurring aminoacid sequence in human body (for example shown in SEQ ID NO:1 or the CAA30314), but do not mean that described C1INH is for example producing or deriving from human plasma in the human plasma.
According to an aspect of the present invention, the C1INH that uses in the inventive method preferably has the plasma half-life shorter than the plasma half-life of the deutero-C1INH of blood plasma, and C1INH plasma half-life more preferably of the present invention is lower than the plasma half-life of the C1INH that derives from human plasma." plasma half-life of reduction " is meant the negative sense variation of the circulating half-life of C1INH of the present invention with respect to the circulating half-life of the deutero-C1INH of blood plasma.In this article, the deutero-C1INH of blood plasma is meant naturally occurring C1INH, its typical case derive from blood plasma and can be from blood plasma purification, but without chemistry or enzyme modification.
Plasma half-life is by giving different time points blood sampling after the C1INH, and determines the concentration of C1INH in each sample and measure.The dependency of serum-concentration and time also can be used to calculate plasma half-life.The plasma half-life of C1INH of the present invention preferably reduces at least 2 times, 3 times, 4 times, 6 times than the circulating half-life of the deutero-C1INH of blood plasma, more preferably reduces about at least 8 times, most preferably reduces about at least 10 times.In other words, the plasma half-life of C1INH of the present invention preferably be lower than the deutero-C1INH of blood plasma (being its naturally occurring homologue) plasma half- life 60,50,40,30,25,20,15,12.5 or 10%.
For example, the plasma half-life of the C1INH that uses in the embodiments of the invention (deriving from the milk of transgene rabbit) is about 3 hours in human body, than the deutero-C1INH of blood plasma at low about 4-8 of the intravital average blood plasma of people half-life doubly.Be understood that C1INH of the present invention preferably carries out than the mensuration that the plasma half-life of the deutero-C1INH of blood plasma reduces under condition of similarity (if not the same terms), promptly preferably under the condition of the tested object of corresponding dosage, sample, identical organism (can be laboratory animal such as mice or people's object) and equal number, carry out.In addition, can contrast the average blood plasma half-life of these two kinds of C1INH prepared products by canonical statistics credit analysis method.
No matter can have more short-decayed C1INH by any conventional method preparation, be C1INH natural or that reorganization produces.For example can in recombinant host cell or organism, prepare in the body, acquisition has the C1INH (deutero-C1INH compares with blood plasma) of the carbohydrate structure of modification, and perhaps the carbohydrate structure of naturally occurring C1INH can be external through chemistry or enzyme modification.Preferably, comparing C1INH of the present invention with the deutero-C1INH of blood plasma modifies in the following way: (from the naturally occurring variant of glycoprotein or recombinant expressed variant) removes carbohydrate component, preferably from the carbohydrate chain that N-connects, remove asialoglycoprotein and/or galactose, thereby and/or remove carbohydrate chain and expose mannose, galactose, N-acetyl-glucosamine and/or fucosyl residues.
According to a further aspect in the invention, the C1INH that uses in the inventive method preferably has the different glycosylation with the deutero-C1INH of blood plasma.The modification that the carbohydrate structure of C1INH of the present invention is carried out comprises and causes low glycosylation, crosses the modification of glycosylation to asialo (asialio) form A 1INH, perhaps causes any other modification of different glycosylation pattern.
External low glycosylation can be the result of carbohydrate component or the whole carbohydrate chains of disappearance C1INH.Modification can comprise carbohydrate chain that N-or O-are connected or only one type chain modify.Can comprise to all chains or only some chains modify.Cross glycosylation and can for example be and in the C1INH molecule, add the extra carbohydrate component or the result of carbohydrate chain completely.The C1INH that the C1INH of asialo form or terminal sialic acid residues level reduce form can the typical case obtain by removing sialic acids groups.The half-life of glycoprotein highly depends on the composition and the structure of the carbohydrate group that its N-is connected with O-in the well known blood.Normally, the carbohydrate group that requires its N-to be connected with O-has terminal sialic acid the maximum half-life of glycoprotein.If this terminal sialic acid does not exist, then described glycoprotein exposes owing to galactose residue and is removed from blood rapidly.The existence of fully having determined terminal galactose residues in the carbohydrate component of glycoprotein causes asialoglycoprotein receptor in the liver that its plasma clearance is strengthened.Therefore, in preferred embodiments, the C1INH that uses in the inventive method preferably has the terminal sialic acid residues than the deutero-people C1 of blood plasma inhibitor reduction level.Sialic acid can be removed by some modes.For example, can remove, for example remove by handling with sialidase by chemistry or method of enzymatically treating.The sialidase that is suitable for this purpose by Chou et al. (1996, J Biol Chem.271. (32): 19219-24; And1994, J Biol Chem.269 (29): 18821-6) describe, can for example derive from V-labs, Inc. (Covington, Louisiana, USA).In another preferred embodiment, the C1INH that uses in the inventive method preferably has mannose, N-acetyl-glucosamine phosphomannose, galactose and/or the N-acetylgalactosamine residue of exposure.The saccharide residue that exposes normally is positioned at the terminal saccharide residue in the polysaccharide branch or is can be easy to and the saccharide residue (as the carbohydrate binding structural domain) that described residue is had the interaction between component of affinity at least.C1INH with galactose, N-acetylgalactosamine, N-acetyl-glucosamine, mannose, fucose or phosphomannose residue of exposure can for example obtain by carry out the enzyme processing with following one or more enzyme: β-D-N-acetylhexosamine enzyme, interior-beta-D-galactosidase and/or α-D-N-acetamino galactosidase enzyme (also can derive from for example V-labs, Inc., Covington, Louisiana, USA).
Can be to modifying in the body of the carbohydrate chain of C1INH by using the recombinant production system carry out.Can use prokaryotic cell and eukaryotic cell culture, as yeast cells, fungal cell, insect cell and mammalian cell.For example, COS cell and Chinese hamster ovary celI are suitable mammal production systems.Although mammalian cell culture system has the ability to produce the glycoprotein with sialylated carbohydrate group, but be difficult to the natural or glycosylation completely of realizing ideal usually, therefore the glycoprotein that produces of reorganization has the glycosylation pattern different with its natural homologue usually.Usually this different glycosylation pattern is incomplete (comparing with natural homologue), has galactose, N-acetyl-glucosamine and/or the mannose residue of exposure.In addition, in eukaryotic microorganisms such as yeast or fungus, produce the C1INH that C1INH has acquisition the mannose residue of exposure.
C1INH with carbohydrate structure of modification also can prepare in transgenic animal, preferably prepares in the non-human animal, as in transgene rabbit, cattle, mice, rat, goat and sheep.Preferred this glycoprotein is expressed in these non-human transgenic animals' mammary gland, and described thus glycoprotein can derive from the milk of described animal.Those skilled in the art known this will depend on the specificity glycoprotein of generation and generation amount, be best suited for the transgenic animal of production.Being particularly preferred for C1 INH of the present invention is the C1INH that derives from the milk of transgenic cow or rabbit (Lagomorpha) order animal; the preferred Leporidae of described rabbit order animal section; being more preferably Oryctolagus and belonging to, most preferably is Oryctolagus cuniculus kind.
Compare with the deutero-homologue of its natural blood plasma, the dissimilar modification that the structure of the proteic carbohydrate chain of C1INH is carried out can derive from the recombinant production system, for example can be simultaneously or continuously separately or combination import different glycosylations, low glycosylation or cross glycosylation, some modifications can import in the part of described molecule, and other modifications can import in another part of described molecule.Help the preferred modification of described proteinic therapeutic efficiency to make up galactose, N-acetylgalactosamine, N-acetyl-glucosamine, mannose, fucose and/or the phosphomannose residue that comprises the expression on the C1INH of the present invention.C1INH of the present invention can for example have few mannose type polysaccharide or high mannose type polysaccharide.At least about terminal residue of 5,10,15,20,40 or 60% of the polysaccharide on the preferred C1INH is selected from galactose, N-acetylgalactosamine, N-acetyl-glucosamine, mannose, fucose and phosphomannose residue.For example, the preferred C1INH that is used for the present invention contains than its natural homologue and lacks about 2,4,5,6-sialic acid doubly, and/or the polysaccharide that its about at least N of 5,10,15,20,40 or 60% connects is neutral, carry terminal hexose, as mannose and N-acetyl-glucosamine with calm 3-and 4-OH group.On the contrary, the deutero-C1INH of blood plasma does not have the glycosylation of few mannose type.The preferred C1INH that is used for the present invention for example is the recombined human C1INH that produces in rabbit mammary gland, and it lacks 5-6 sialic acid doubly than natural homologue, and the polysaccharide that its N-of about 15% connects is neutral, carries terminal mannose residue.
In a preferred embodiment, the different glycosylation that is used for the present invention's C1INH causes mannose-binding protein is had than the higher affinity of the deutero-homologue of its blood plasma.Mannose-binding protein (MBP) also is known as maltose-binding protein, mannose-binding lectin (MBL), mannan-binding lectin, and the Ra-response factor perhaps sterilizes.MBP belongs to a kind of soluble Ca
2+The collectin of dependency (C type) agglutinin family.MBP passes through (different with the classical pathway and the alternative pathway of complement activation) lectin pathway as the complement activation thing.Complement system is the important component of innate immunity defence, is activated by following three approach: classical pathway, alternative pathway and the agglutinin or mannose binding lectin (MBL) approach that disclose recently.
As catalyst structure domain C1r and C1s during by Recognition Protein C1q bind immune complex, classical pathway begins to activate (seeing Figure 11).
Alternative pathway continues to carry out (turning over) with antibody dependent/non-dependent mode low speed, attacks not at complement and the granule protected by specificity.
Agglutinin or MBL approach are based on the combining of carbohydrate structure that exists on MBL and various pathogen or other cellularity and initial or activate.Serine protease (MASP)-I and-2 (seeing Figure 11) that two kinds of serine protease-mannan-binding lectins are relevant is relevant with MBL, demonstrates the remarkable similarity with serine protease C1s and C1r.Described complex is based on have C4 and C3 activation capability with combining of mannan.Described complex contains two kinds of serine protease MASP-I and the MASP-2 that is connected by disulfide bond.In this form, MASP energy c4 cleavage and C3 cause its inactivation.Compare C1INH of the present invention with the deutero-homologue of its blood plasma and preferably have higher affinity for people MBP.
MBP identification has the hexose of the exposure of calm 3-and 4-OH group, as mannose and N-acetyl-glucosamine and/or N-acetyl-hexosamine.Therefore the preferred C1INH of the present invention has this terminal hexose.C1INH of the present invention makes more effectively targeting, combination and/or inhibition MBP than its deutero-homologue of native plasma that does not have the mannose that exposes and a N-acetyl-glucosamine residue for the more high-affinity of MBP, preferred people MBP.People MBP is meant the protein of being identified by Kawasaki et al. (1983, J.Biochem 94:937-47) at this paper, have Taylor et al. (1989, Biochem.J.262 (3), 763-771; NCBI registration number CAA34079) described aminoacid sequence.With the structure of the compound rat MBP of oligosaccharide by Weis et al. (1992, Nature.360:127-34) describe.See for example US6 about further describing of people MBP, 846,649 and the list of references wherein quoted described.
All these approach (classical pathway, alternative pathway and agglutinin or MBL approach) all produce vital enzymatic activity, cause the assembling (seeing Figure 11) of membrane attack complex (MAC or C5b-C9) at last.Under physiological conditions, the activation of complement system is effectively control by the proteic synergism of soluble and membrane-bound adjusting.One of these protein are C1 inhibitor (C1INH), and this is a kind of serpin in conjunction with C1s and C1r, are the physiologic depressors of present unique known classical pathway.In addition, C1INH by in conjunction with and suppress MASP-1 and MASP-2 and the complement activation of inactivation MBL-mediation.
Preferably (product no.COMPL300, Wieslab Sweeden) measure by the Wielisa test kit in human serum in the activation of different complement pathways.This is a kind of commercially available enzyme immunoassay (EIA), and the deposition of utilizing C5b-C9 is as conventional reading (common read-out) and each approach of three kinds of complement pathways of specific detection.In brief, with the hole of microtitre bar specificity activator bag quilt with each approach of three kinds of complement pathways.Human serum dilutes in the diluent that contains the specificity sealer, to guarantee only to activate approach separately.Further add of the present invention C1INH or its blood plasma deutero-homologue of concentration in 0-75 μ mol scope,, add in the described hole room temperature insulation 30 minutes.The human serum of described dilution subsequently in described hole in 37 ℃ of insulations during 60 minutes, complement is activated by the specificity bag.Wash described hole then, the C5b-C9 of the formation anti-C5b-C9 antibody test of specificity alkali phosphatase enzyme mark.After further washing, by specific antibody being detected with the insulation of alkaline phosphatase substrate solution.Make the complement activation amount relevant with intensity of colour, and as absorbance measuring (optical density OD).Use this test kit, find that recombined human C1INH of the present invention (rhCHNH) and the deutero-C1INH of blood plasma (pdCHNH) have similar inhibition ability for classical pathway.Yet, find that CIINH of the present invention is than inhibition ability high about 20% (see embodiment 3) of the deutero-C1INH of blood plasma for the MBL approach.
Therefore, in this embodiment preferred, the different glycosylation that is used for C1INH of the present invention causes having than the higher affinity of the deutero-homologue of its blood plasma for MBP, and this causes the more effective inhibition to MBP, and making can more effective inhibition lectin pathway.More effective inhibition to lectin pathway preferably is meant high at least 5% inhibition, also more preferably high at least 10% inhibition, also more preferably high at least 15% inhibition, also more preferably high at least 20% inhibition, also more preferably high at least 25% inhibition, also more preferably high at least 30% inhibition, also more preferably high at least 35% inhibition, also more preferably high at least 40% inhibition, also more preferably high at least 45% inhibition, also more preferably high at least 50% inhibition, also more preferably high at least 55% inhibition, also more preferably high at least 60% inhibition, also more preferably high at least 65% inhibition, also more preferably high at least 70% inhibition, also more preferably high at least 75% inhibition, also more preferably high at least 80% inhibition, also more preferably high at least 85% inhibition, also more preferably high at least 90% inhibition, also more preferably high at least 95% inhibition and most preferably high at least 98% inhibition.The activation of lectin pathway is preferably measured by above-mentioned Wielisa test kit.
Method of the present invention can be used for preventing, reducing or treat the ischemia and the reperfusion injury of any kind.Preferably, method of the present invention be applied to wherein ischemia and reperfusion injury known to small part by in, the situation about more preferably mainly producing by lectin pathway.For myocardial ischemia and reperfusion injury (J Immunology 2005,175:541-546), renal ischaemia-reperfusion injury (Am J Pathol.2004165 (5): 1677-88), gastrointestinal tract ischemia reperfusion injury (J Immunol.200515:174 (10): 6373-80) and apoplexy (deSimoni et al, 2004 Am J.Pathol.164:1857-63), it mainly is to produce by lectin pathway that reperfusion injury has been shown, and seldom produces by classical pathway.Therefore, the preferably stronger lectin pathway inhibitor of C1INH of the present invention than the deutero-homologue of its native plasma.Preferred C1INH of the present invention is the inhibitor of the lectin pathway stronger than the deutero-homologue of its native plasma in human body.
The incident different with the experimental model that uses in the embodiment of the invention, that the generation of ischemia normally can't be predicted in the actual life.Therefore it is normally unpractical to give C1INH before ischemia and/or the generation of perfusion more subsequently, and C1INH will have in reality, and several hrs gives after ischemia and/or perfusion more subsequently.Yet, this has seriously limited the therapeutic efficiency of the deutero-C1INH of normal plasma, because its most of inefficacy when after ischemia-reperfusion, giving, and only has minimum therapeutic efficiency time window (see Fig. 2 and deSimoni et al, 2004Am J.Pathol.164:1857-63 is described).On the contrary, ischemia or after ischemic episode at least 1 hour and/or injected C1INH of the present invention in 30 minutes after the perfusion again in beginning, it still can bring into play its neuroprotective when also.Therefore, be used for preventing, reduce or treat at least a that can't predict or the ischemia of prediction and a preferred embodiment of the method that reperfusion injury takes place of the present invention, C1INH of the present invention at least ischemia finish or afterwards (promptly when the tissue of ischemia is poured into again) give.More preferably, C1INH of the present invention gave after the perfusion after ischemia or in beginning more at least in 10,15,20,30,45,60,90 or 120 minutes.Preferably, C1INH of the present invention is no more than in 24,12,6,4 or 3 hour time after ischemia or at ischemia and/or after perfusion takes place again and gives.In another preferred embodiment, the C1 inhibitor after ischemia or at ischemia and/or after perfusion takes place again at least 3 hours, preferably at least 6 hours, more preferably at least 9 hours, more preferably gave at least 18 hours.
In a preferred embodiment, described method is used to prevent, reduce or treat the ischemia-reperfusion of that can't predict, unexpected or acute generation.Pathological changes relevant with ischemia reperfusion injury that can't predict, unexpected or acute generation or disease comprise but non-ly are limited to emergency treatment coronary artery surgery after acute myocardial infarction (AMI), apoplexy comprise perinatal stage apoplexy, hemorrhagic shock, intestinal ischemia, percutaneous tranluminal coronary angioplasty (PCTA) failure, have blood vessel pancreatitis processing (ERCP) ischemia reperfusion injury afterwards after clamping any vascular surgery (for example large artery trunks clamps, and causes the skeletal muscle ischemia) of (blood vessel crossclamping) or handling ductus pancreaticus or bile duct.In this case, the C1INH of the present invention preferably emergency treatment coronary artery surgery after acute myocardial infarction (AMI), apoplexy comprise perinatal stage apoplexy, hemorrhagic shock, intestinal ischemia, percutaneous tranluminal coronary angioplasty (PCTA) failure, the pancreatitis processing (ERCP) after having any vascular surgery (for example large artery trunks clamps, and causes the skeletal muscle ischemia) that blood vessel clamps or handling ductus pancreaticus or bile duct gave at least 1,5,10,15,20,30,45,60,90 or 120 minute afterwards.Perhaps, the time that gives C1INH of the present invention is preferably beginning after the perfusion at least 1,5,10,15,20,30,45,60,90 or 120 minute again.
In addition, the ischemia reperfusion injury that can't predict preferably is meant it wherein is the situation that perfusion again rather than ischemia are induced in treatment or operation.This treatment or operation comprise but non-being limited to:
-medicine thrombolytic comprises intravenous and endovascular method that apoplexy, acute coronary syndrome, periphery artery occlusion, pulmonary infarction, renal artery obturation are carried out;
-mechanical thrombolytic, for example percutaneous coronary intervention (pci), peripheral arterial angioplasty, internal organs artery angioplasty;
-bypass operation of coronary artery;
-carotid artery intima divests art;
-mesentery ischemia;
-shock comprises hemorrhagic shock, cardiogenic shock, neurogenic shock, anaphylactic shock;
-flap necrosis (flap-failure), for example plastic operation;
Replanting of-finger or toes and limbs;
-strangulated intestinal obstruction.
Perhaps, in another preferred embodiment, described method is used to prevent, reduce or treat the ischemia-reperfusion generation of prediction.The ischemia-reperfusion of prediction preferably include wherein treatment or ischemia and dabbling again situation are subsequently induced in operation.Hereinafter listed wherein treatment or operation and induced temporary transient no blood or blood flow to reduce, be i.e. ischemia or anoxia, the dabbling again situation of appearance then:
-cardiopulmonary bypass;
-aneurysm repair comprises the reparation of large aneurysm, cerebral aneurysm;
-carotid artery intima divests art, wherein uses clip at intra-operative;
-Deep Hypothermia Circulatory Arrest art;
The application of-tourniquet is used tourniquet during promptly put the injury outside
-solid organ transplantation;
-any other iatrogenic blood flow interrupts.
In addition, pathological changes relevant or disease with the generation of ischemia reperfusion injury of prediction comprise but non-be limited to organ transplantation (lung, liver, kidney, heart) afterwards, have any vascular surgery (for example large artery trunks clamps, and causes the skeletal muscle ischemia) that blood vessel clamps ischemia reperfusion injury of generation afterwards or after ductus pancreaticus or bile duct are handled (ERCP) pancreatitis afterwards, during the extracorporeal circulation (ECC) or afterwards.
In prevention, reduce or treat in the preferred embodiment of the method that the ischemia of at least a prediction and reperfusion injury take place, C1INH of the present invention promptly gives when ischemic tissue pours into again at least in the ischemia end of term or give afterwards.More preferably, C1INH of the present invention gave after the perfusion after ischemic stage or in beginning more at least in 10,15,20,30,45,60,90 or 120 minutes.Preferably, C1INH of the present invention is after ischemia or at ischemia and/or be no more than 24,12,6,4 or 3 hours after perfusion takes place again and give.In another preferred embodiment, the C1 inhibitor after ischemia or at ischemia and/or after perfusion takes place again at least 1 hour, 3 hours, preferably at least 6 hours, 9 hours, more preferably gave at least 18 hours.
Perhaps, the present invention provides a kind of prevention, reduction on the other hand or has treated the ischemia of at least a prediction and method that reperfusion injury takes place, and C1INH wherein of the present invention is at ischemia with before pouring into again or give during this period.Those skilled in the art known according to the present invention the plasma half-life of C1INH, can adjust give C1INH of the present invention possible time point the earliest to obtain optimum.
According to an embodiment preferred, C1INH of the present invention need to continue its object and/or continue to treat transplanted organ in the organ transplantation situation.Treat that transplanted organ preferably preserves in the compositions with suitable culture medium and The addition of C 1INH.
Perhaps, or with the combination of aforementioned embodiment preferred, before the ischemia of prediction and reperfusion injury take place, preferably be meant before the ischemia of at least a prediction and reperfusion injury take place 3 hours at the most, preferred 2 hours, 1 hour, more preferably gave C1INH of the present invention at the most in 30 minutes.
The ischemia that needs the object of C1INH of the present invention to be meant to predict and the object of reperfusion injury.The ischemia of prediction and the generation of reperfusion injury are described at this paper.
Preferably before the ischemia of prediction and reperfusion injury take place, give C1INH because its can with after ischemia and reperfusion injury take place, give the generation that the identical or better mode of C1INH prevents the great majority relevant with reperfusion injury with ischemia (if not all) to damage.
In a preferred embodiment, described method is used for the generation of the ischemia-reperfusion that can't predict.More preferably, ischemia reperfusion injury occurs in after apoplexy or the perinatal stage apoplexy.In the ischemia-reperfusion that can't predict of these types took place, we had confirmed the neuroprotective of C1 inhibitor of the present invention in ischemic penumbra (pneumbra).Ischemic penumbra preferably is meant Hippocampus and/or cortex.Neuroprotective is meant preferably that in Hippocampus taking place after the ischemia at the most 3 hours and took place in cortex after the ischemia at the most to handle the back with C1 inhibitor of the present invention in 9 hours reduces neural degeneration in Hippocampus and/or the cortex.More preferably, at Hippocampus ischemia at the most 4,5,6 or more hours and at cortex ischemia at the most 10,11,12 or reduced neural degeneration in more hours.Neural degeneration is preferably as evaluation as described in the embodiment 2: with brain tissue slice with the preferred Jade of label (Schmued LC etc., the list of references 4) dyeing that is specific to neuronal degeneration, by the fluorescence microscope analysis.Make in this way, reduce neural degeneration and observe painted cell in being meant in the sample of handling than untreated sample and be reduced by at least 2%.Preferably, neurodegenerative minimizing is meant that painted cell is reduced by at least 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or more.
Perhaps, or with previous embodiments combination, use the C1 inhibitor and can reduce ischemia and/or the damage that causes of perfusion again.More preferably, when ischemia reperfusion injury takes place after apoplexy or perinatal stage apoplexy, use C1 inhibitor of the present invention and can reduce infarct size.More preferably, infarct size is as quantification as described in the embodiment 2.More preferably, use this quantization method, after ischemia took place at least 3 hours, infarct size reduced at least 10%, preferably at least 20%, 40%, 60%, 70%, 80%, and most preferably infarct size reduces 90%.
The C1INH that is used for the inventive method can be used as this area part of pharmaceutical compositions or with this area drug regimen applied in any combination.These pharmaceutical compositions typical case comprises described C1INH and materia medica acceptable carrier or excipient.According to the part of hope or the stability of systematic treating scheme, area for treatment and reactive compound, can give these materia medica compositionss in several ways.Determine suitable prescription according to the method for giving.Described pharmaceutical composition preferably gives by the outer method of intestinal, for example gives by intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion mode; Perhaps give method afford by intrathecal injection or intracranial.In a preferred embodiment, by the intravenous infusion method afford.The suitable prescription that gives outward by intestinal is known in the art, and the typical case is a liquid formulations.The C1INH prepared product that intestinal gives outward must be aseptic.Sterilization is easy to realize by the aseptic filtration membrane filtration before or after can and rebuilding in lyophilizing.The C1INH prepared product can continue to give by infusion or the method for injecting.Liquid C1INH prescription can for example give by infusion pump.The compositions of typical intravenous infusion can contain 0.9% aseptic NaCl of 100-500ml or 5% glucose solution, the optional C1INH that adds 20% albumin solution and 100-500mg.The pharmaceutical composition that is used for intravenous injection contains for example aseptic buffered water of 1-10ml and 1-250mg C1INH of the present invention.The method for compositions that preparation can give outside intestinal is known in the art, in a plurality of data, describe in detail, for example incorporating Remington ' s Pharmaceutical Science (15th ed. for referencial use into its full content, Mack Publishing, Easton, PA, 1980) the middle description.
The effective dose of the C1INH that uses in the inventive method is certain drug compositions, the seriousness of pathological changes and patient's the general situation that valid density and frequency depend on use.Normally, can determine effective dose by conventional optimal seeking method based on the pharmaceutical composition of the C1INH that uses in the inventive method.Suitable starting point is based on the using dosage of the pharmaceutical composition of equal value of the deutero-C1INH of blood plasma.The advantage of pharmaceutical composition of the present invention is can use high predose when treatment, improves the successfully probability of treatment.This high predose is possible, because the C1INH in the pharmaceutical composition of the present invention illustrates clearance rate faster than its natural homologue.Particularly for the treatment of acute case, the C1INH of high predose of the present invention can be favourable.This high predose can be at least 1.5,2,3 or 4 times of naturally occurring homologue dosage.
In a preferred embodiment, C1INH of the present invention gives through intravenous, dosage is greater than 50,100,200,400,600,800 or the 1000U/kg whose body weight, is preferably 50-2000,100-1000,200-800,400-700 or 500-700U/kg whose body weight.The C1 INH of 1 unit (U) is the amount of the C1INH that exists in the 1ml human blood.Such unit is corresponding to the deutero-C1INH of about 275 μ g blood plasma.The putative molecule amount is 110,000 dalton, in the human plasma concentration of C1INH be 2.5 μ mol/L (Nuijens et al. (1989), J.Clin.Invest.84:443).
In another preferred embodiment of the inventive method, described pharmaceutical composition further contains thrombolytic or uses with the thrombolytic applied in any combination or after treating subsequently with this thrombolytic.Thrombolytic described herein is meant energy dissolved blood clot (thrombosis) and reopens tremulous pulse or venous medicament (medicine).Thrombolytic is serine protease normally, changes plasminogen into fibrinolysin, makes Fibrinogen and fibrin decompose and thromboembolism.Preferred thrombolytic comprises the streptokinase activator complex (APSAC) of the purification of reteplase (r-PA or Retavase), alteplase (t-PA or Activase), urokinase (Abbokinase), prourokinase, Fructus Foeniculi acidylate, and streptokinase.
On the other hand, particularly the authority except the U.S. the present invention relates to C1INH of the present invention according to the application of above-mentioned any method in the medicine of producing prevention, reduce or treating reperfusion injury.
In this paper and claims thereof, verb " comprises " with its non-limiting implication to be used, and is meant the project that comprises this word back, does not get rid of unspecified project.In addition, " one ", " a kind of " etc. do not get rid of and have more than one factor in the literary composition, unless to need it in the literary composition be one and have only a factor.Therefore, " one " typically refers to " at least one ".
Accompanying drawing is described
Fig. 1: before the ischemia, afterwards reach 1 hour after, the evaluation of the mice 48 hours infarct sizes behind ischemia after handling with saline or every mice 15U rhC1INH (recombined human C1INH sees embodiment 1.2).
Fig. 2: before the ischemia, afterwards reach 1 hour after, the evaluation of the mice 24 hours infarct sizes behind ischemia after handling with saline or the deutero-hC1INH of every mice 15U blood plasma.
Fig. 3: begin the back from ischemia and accept the mice of saline or every mice 15U rabbit rhC1-INH in different time points in ischemia long-pending evaluation of infarction stove after 48 hours.Data are represented (every group of 6 mices) with meansigma methods ± SEM.
*P＜0.05,
*P＜0.01, with respect to saline, unidirectional ANOVA and Dunnett check as post-hoc.
Fig. 4: the painted sxemiquantitative assessment of Fluoro-Jade.-=do not have is positive, +=low positive, ++=medium the positive, ++ +=high positive.
Fig. 5: at ischemia the back different time points taking place accepts the neural degeneration presentation graphics that shows by Fluoro-Jade dyeing in the striatum of ischemia mice of saline or every mice 15U rabbit rhC1-INH.Bar bar: 100 μ m.
Fig. 6: at ischemia the back different time points taking place accepts in the hippocampal dentate of ischemia mice of saline or every mice 15U rabbit rhC1-INH by the neural degeneration presentation graphics shown in the Fluoro-Jade dyeing.Bar bar: 100 μ m.
Fig. 7: at ischemia the back different time points taking place accepts in the cortex of ischemia mice of saline or every mice 15U rabbit rhC1-INH by the neural degeneration presentation graphics shown in the Fluoro-Jade dyeing.Bar bar: 100 μ m.
Fig. 8: the mice that saline or every mice 5,10,15U rabbit rhC1-INH take place to accept in back 3 hours at ischemia is estimated in that the infarction stove of ischemia after 48 hours is long-pending.Data are represented (every group of 6 mices) with meansigma methods ± SEM.
*P＜0.01, with respect to saline, unidirectional ANOVA and Dunnett check as post-hoc.
Fig. 9: the mice that saline or every mice 15U pdC1-INH or cattle or rabbit rhC1-INH take place to accept in back 3 hours at ischemia is estimated in that the infarction stove of ischemia after 48 hours is long-pending.Data are represented (every group of 6 mices) with meansigma methods ± SEM.
*P＜0.05,
*P＜0.01, with respect to saline, unidirectional ANOVA and Dunnett check as post-hoc.
Figure 10: overall neurologic impairment (top 10a) and the focal neurologic impairment (below 10b) (every group 6 mices) of the mice of saline or every mice 15U pdC1-INH or cattle or rabbit rhC1-INH in ischemia post-evaluation in 48 hours takes place to accept in back 3 hours at ischemia.
*P＜0.01, with respect to saline, unidirectional ANOVA and Kruskal-Wallis check as post-hoc.
Figure 11: the different approaches general introduction of complement activation.
Figure 12,13: rhC1INH and pdC1INH are to the activated effect of CCP.In two different samples (top is a sample 1, and the below is a sample 2) of normal human serum, add rhC1INH or the pdC1INH (x-axle) that increases dosage.In contrast, utilize buffer (20mM citrate, the 0.19M sucrose pH6.8 that wherein is dissolved with the same rhC1INH that dilutes of rhC1INH; Through 0.22 μ m membrane filtration).Reading is the deposition of C5b-9, and the normal serum contrast is defined as 100% (y-axle) in the mensuration.Data are represented (n=3) with meansigma methods and SD.
Figure 14,15: rhC1INH and pdC1INH are to the activated effect of MBL complement pathway.In two different samples (top is a sample 1, and the below is a sample 2) of normal human serum, add rhC1INH or the pdC1INH (x-axle) that increases dosage.In contrast, utilize buffer (20mM citrate, the 0.19M sucrose pH6.8 that wherein is dissolved with the same rhC1INH that dilutes of rhC1INH; Through 0.22 μ m membrane filtration).Reading is the deposition of C5b-9, and the normal serum contrast is defined as 100% (y-axle) in this mensuration.Data are represented (n=3) with meansigma methods and SD.
Figure 16: rhC1INH and pdC1INH are to CCP and the activated effect of MBL complement pathway.In two different samples (top is a sample 1, and the below is a sample 2) of normal human serum, add rhC1INH or the pdC1INH (x-axle) that increases dosage.In contrast, utilize buffer (20mM citrate, the 0.19M sucrose pH6.8 that wherein is dissolved with the same rhC1INH that dilutes of rhC1INH; Through 0.22 μ m membrane filtration), reading is the deposition of C5b-9, each complement activation percentage ratio of measuring calculates with following formula: (sample-NC)/(PC-NC) * 100.PC is made as 100%.The result represents with the meansigma methods SD at 3 independent verdunning of each test concentrations.
Embodiment
Previous experiment is illustrated in the brain focal ischemia mouse model behind ischemia 48 hours to be estimated and finds, gives single dose rhC1INH (15U/ mice) significantly to reduce the ischemia volume with the closely similar mode of the deutero-C1INH of blood plasma when ischemia begins.In this embodiment, we have studied the effect time window of rhC1INH neuroprotective activity for ischemia volume and functional impairment.We also by estimate neural degeneration and neuroglia response studies rhC1INH for seven days the result (seven-days outcome) effect.
1. method
1.1 the focal cerebral ischemia of a property crossed
As discussed previouslyly realize ischemia (De Simoni et al, 2003 and 2004, see above) by middle cerebral artery thromboembolism (MCAO).By at N
2O/O
2(70/30%) 5% isoflurane induced anesthesia in the mixture, the 1.5-2% isoflurane that is used in the equal mixture is kept.In order to confirm the appropriate degree of every animal medium vessels obturation, use laser Doppler flowmetry (Transonic BLF-21) utilization to be positioned on the brain surface and be fixed on softish 0.5mm fibre-optical probe (Transonic on the skull with impression material, Type M, 0.5mm diameter) measure blood flow, coordinate is AP=-1mm, L=-3,5mm.In brief, expose right common carotid artery, import silicon silk (7-0) in the internal carotid artery and march to anterior cerebral artery, enter anterior cerebral artery and MCA to block its branch by the otch of on common carotid artery, doing.Described silicon silk is advanced to be compared blood flow and reduces until observing with baseline before the ischemia〉70%., carefully remove described nylon yarn and recover blood flow after 30 minutes at ischemia.
1.2 Drug therapy
Different time gives the 15U/ mice rhC1INH of mice 150 μ l or the saline of equal volume through the intravenous single injection behind ischemia:
-when ischemic stage, begin (before the rhC1INH-),
-when ischemic stage, finish (behind the rhC1INH-),
-begin back 1 hour (behind the rhC1INH1h-) in ischemic stage.
The rhC1INH that uses in this research produces in the transgene rabbit of expressing human C1INH in mammary gland, and from the milk that derives from these animals purification, as described in WO 01/57079.
1.3 the assessment of neurologic impairment
At ischemia after 48 hours, by the research worker of experienced unknown experiment condition at two function of nervous system's scorings of mice uniqueness and every mice is assessed.Each of the following classification of overall neurologic impairment mice must be divided into 0-28: hair, ear, eye, posture, spontaneous activity, epilepsy behavior.Each of the following classification of focal neurologic impairment mice must be divided into 0-28: body symmetry, gait, climbing, circling behavior, forelimb symmetry, mandatory rotation, feel to reply.Data are with median and the 25th to the 75th percentile value representation.
1.4 the quantification of infarct size
After 48 hours, mice is used Equitensin (120 μ l/ mices, peritoneal injection) deep anaesthesia at ischemia, carefully pour into PBS 0.1mol/l, the pH7.4 of 30ml, be poured in the ice-cold paraformaldehyde of 60ml (4%) among the PBS subsequently.From cranial cavity careful take out brain after, it is moved in 30% sucrose solution in PBS, spend the night at 4 ℃ of low-temperature protections.Then brain is immersed in the isopentane in-45 ℃ of quick-freezings 3 minutes, is sealed in afterwards in the bottle ,-70 ℃ of storages until use.In order to determine damaged area, make the crown section of 20 μ m brains at interval successively with 240 μ m, with dimethyl diaminophenazine chloride (Neutral Red GurrCertistain, BDH, England) dyeing.In each section, infarct size ignorant of the economics formula (blindly) is estimated and is described by the pale relatively degree (relative paleness) of histological stain.The normal structure area that deducts in the homonymy hemisphere the area of infarct size by the offside hemisphere from each section is determined.By using a computer assistant images analyser and quantize by image analysis system (AnalyticalImage System) calculating, the comprehensive infarct size on each brain tissue slice and calculate Infarction volume.
1.5 open field test (open field test)
After ischemia 7 days, by the behavior of open field test assessment mice.This test can be used for detecting anxiety state and exploratory behavior and the activity in the long-term ischemia mice.Form (41 x, 41 x 41cm) by a plastic box that comprises 4 different objects for spacious.Territory, spacious place is divided into center band and the boundary zone on every side of 28x28cm.Mice is placed spacious center separately, the behavior of mice is observed 5 minutes by the research worker of unknown experiment condition.Record inside crossings (main relevant), outsidecrossings (main relevant), back leg with locomotor activity with the anxiety behavior stand (rears) (main relevant) with exploratory behavior reach and contact the number of times of (mainly and sensation/locomotor activity be correlated with) with object.
1.6 neuron count
After ischemia 7 days, as previously mentioned mice is carefully poured into.In order to determine neuron count, cut into the crown section of 20 μ m cerebral tissue at interval successively with 640 μ m, with cresol-purple (CresylViolet acetate, Sigma, St.Louis, MO) dyeing.Select three 20 μ m sections of homonymy and offside hemisphere to carry out neuron count mensuration.First section is the fore-and-aft direction stereotaxis coordinate that is positioned at apart from bregma+0.86.The minimizing of neuronal quantity is calculated by the survived neuronal number in three sections of gathering two hemisphere, represents with the percentage ratio that accounts for offside hemisphere.Use is connected with the Olympus BX61 microscope of Soft ImagingSystem Colorview video camera and AnalySIS software.Research worker by unknown disposition is carried out quantitative analysis at 40 * amplification.
1.7 astrocyte and microglia are carried out immunohistochemical analysis
After ischemia 7 days, preparation thickness is the crown cross-sectional slice of the careful dabbling ischemia mouse brain tissue of 20 μ m, is used to estimate astrocyte and microglia/macrophage immunity dyeing.In brief, with section rinsing 30 minutes in containing the 0.1mol/L PBS solution of 0.4%Triton X-100, rinsing 15 minutes in the PBS solution that contains 0.1%Triton X-100 and 3% normal goats serum (NGS) subsequently.Antibody with section and astrocyte and microglia (resists-GFAP1:1500 Chemicon then; Anti--CD11b 1:250, by Dr.A.Doni, Mario Negri Institute is so kind as to give) incubated overnight.Second day, will cut into slices and in PBS, wash, with biotinylated two anti-insulations 1 hour, wash and be incubated with avidin-biotin-peroxidase.After reacting with 3 '-3-diaminobenzidine, four hydrochlorates, washing slice, drying, fixing in dimethylbenzene by the gradient ethanol dehydration, with the capping of DPX mounting medium, afterwards through the optical microscope analysis.
2. result
2.1 the time window of effect
2.1.1 the assessment of neurologic impairment
Accept assessment neurologic impairment situation in rhC1INH or the brinish ischemia mice after 48 hours at ischemia.Observe with the ischemia mice of saline treatment and compare, every group of mice of handling through rhCHNH-all has the slightly reduction of (although not remarkable) (before the rhC1INH-: 9 and 12; Behind the rhC1INH-: 7 and 11; Behind the rhC1INH 1h-: 9 and 13; Saline: 10 and 12.5, be respectively the damaged median of overall and focal sexual function) (data not shown goes out).
2.1.2 the evaluation of infarct size
At ischemia after 48 hours, with the mice (41.51 ± 7.01mm of saline treatment
3) compare, dosage be 15U/ mice-preceding ,-mice that rhC1INH-behind back and the 1h-handles illustrates the ischemia volume and reduces obviously that (the ischemia volume is respectively 13.67+2.59mm
3, 9.06+0.77mm
3And 8.24+1.00mm
3) (Fig. 1, data are represented with meansigma methods ± SEM).
2.2 seven days results
2.2.1 spacious experiment
Ischemia induces the new line number of times significantly to reduce than the animal of not test (N.T.), this parameter and the mice zero difference of ischemia not in the rhC1INH-processed group.Other parameter not shown any difference in these three groups of assessment.
2.2.2 neuron count
For the protective effect of assessing rhC1INH long-term existence whether, we have estimated 7 days neuronic forfeiture situations after inducing ischemia and healing with medicine.The result illustrates the rhC1INH protective effect and still exists at this moment: neuron forfeiture 14% ± 2.18% average (mice of saline treatment) is with respect to 4% ± 1.24% average (mice that rhC1INH handles) (data not shown goes out).
2.2.3 microglia/macrophage and astrocyte are carried out immunohistochemical analysis
After ischemia 7 days, in the Hippocampus of the damage of accepting brinish ischemia mice and striatum, observe the macrophage (data not shown goes out) of a large amount of activated microglias and infiltration.The rhC1INH-pre of 15 units all can weaken this activation and immersional wetting (data not shown goes out) in these two zones.Homonymy Hippocampus through the ischemia mice of saline treatment illustrates slight astrocyte hypertrophy, this with the ischemia mice of handling at rhCHNH-in the zero difference as a result (data not shown goes out) that observes.The activation of the not shown any relevant astrocyte of other brain area in arbitrary group.
3. conclusion
It is that the rhC1INH of 15U/ mice is when beginning at ischemia (pre) or finish (after, promptly when pouring into) when giving object, in that to reduce aspect the ischemia volume effect similar again that data of the present invention illustrate dosage.The more important thing is that it also can bring into play neuroprotective when (behind the 1h-) injected this inhibitor in back 1 hour when taking place at ischemia.In addition, the protective effect of rhC1INH still existence in 7 days behind ischemia.The deutero-hC1INH of these results and blood plasma forms sharp contrast, and when 1 hour injection latter behind ischemia, it almost completely loses the ability (see figure 2) of performance neuroprotective.
The main result of this research is as follows:
1. the half-life of rhC1INH is about 3 hours (at the dosage of 15U/ mice) in the mice plasma.Good correlation between antigen and the functional activity shows that described recombinant protein only circulates with its activity form in blood plasma; It may be the reduction that tissue distribution helps blood plasma level.
2. the rhC1INH of 15U/ mice-pre dosage reduces ischemia volume (reducing by 69%) very effectively.
3. estimate by dyeing, the rhC1INH of the dosage of 15U/ mice can obviously reduce the degeneration neuron number in the Hippocampus, shows that therefore the reduction of ischemia volume avoids damage owing to neuron.
After beginning (preceding) at ischemia, finishing (back) or ischemia and take place 1 hour (after behind the 1h, i.e. perfusion beginning 30 minutes again) when giving, rhC1INH effect aspect reduction ischemia volume is similar.Therefore, rhC1INH is roomy than the action and efficacy time window of pdC1INH (when it no longer works when ischemia gave pdC1INH after 1 hour).
5.rhC1INH-secular during the neuroprotective of predose, illustrate by begin back neuron count of carrying out in 7 days at ischemia.
6. estimate to find after ischemia that rhCHNH makes the damaged slightly improvement of overall and focal sexual function in 48 hours.This discovery is similar to the result who observes with pdC1INH.In order to assess the influence of rhC1INH for long-term action function performance, we by spacious field experimental analysis the behavior of mice.Rearing behavior in 7 days significantly reduced than the score in the untested mice in the ischemia mice after the result illustrated ischemia.There are not its score and control mice zero difference in the mice that this rhC1INH of being reduced in handles.
7. in early days (48 hours) and late period, (7 days) time point was estimated, and the activation that rhC1INH can weaken microglia/macrophage in the ischemia mouse brain tissue/raise is shown.These cells are signs that brain tissue inflammation is replied.
8. the replying by force by rhC1INH of astrocyte that was excited by ischemia at 48 hours weakens.Astrocyte activates all and obviously reduces in two experimental grouies, can observe zero difference in the mice of saline and rhC1INH-processing.
Embodiment 2: to grinding of the neuroprotective of rhC1-INH in the mouse model of focal cerebral ischemia
Study carefully
The rhC1-INH of we previous verified 15U has tangible neuroprotective in mouse brain ischemia/reperfusion model, also like this when giving in 1 hour after ischemia/reperfusion takes place, and pdC1-ENH this time point after treatment is no longer valid.This neuroprotective is secular, in fact after ischemia and treatment 7 days, with the cerebral tissue of the mice ischemia of rhC1-INH treatment the infarct size reduction is shown still.In following experiment, we have determined effect time window (surpass 1 hour after) and the dose response of the neuroprotective of rhC1-INH to the ischemia volume.In addition, we use same approach directly to contrast the effect (is at most effective dose and time point for rabbit rhC1-INH) of pdC1-INH, rabbit and cattle rhC1-INH
Method
Animal
The program that relates to animal and treatment thereof is according to meeting country (18February 1992 for D.L.n.116, G.U.suppl.40) and international law and policy (EEC Council Directive 86/609, OJ L 358,1; Dec.12,1987; NIH Guide for the Care and Use of Laboratory Animals, U.S.National Research Council 1996) research institution instructs and carries out.With male C57B1/6 mice (26-28g, Charles River, Calco, Italy) stable breeding, remains on stationary temperature (21 ± 1 ℃) and relative humidity (60%), rule illumination/dark (7am-7pm) by 5/cage.Arbitrarily get food (mice eats the Altromin granule) and water.
One property crossed focal cerebral ischemia
Realize ischemia by middle cerebral artery occlusion (MCAO) as previously mentioned
1,3Utilization contains NaO/Oa (70/30%) the mixture induced anesthesia of 5% isoflurane, and the 1.5-2% isoflurane that is used in the equal mixture keeps narcotism.In order to confirm the well-formedness of every animal medium vessels obturation, use by laser Doppler flowmetry (Transonic BLF-21) and to be positioned at the brain surface and to be fixed on softish 0.5mm fibre-optical probe (Transonic on the skull with die material, Type M, 0.5mm diameter) measure blood flow, use following coordinate: AP=-1mm, L=-3,5mm.In brief, expose right common carotid artery, import silicon silk (7-0) in the internal carotid artery and march to anterior cerebral artery, enter anterior cerebral artery and MCA to block its branch by the otch of on common carotid artery, doing.Described silicon silk is advanced to be compared blood flow and reduces until observing with baseline before the ischemia〉70%., carefully remove described nylon yarn and recover blood flow after 30 minutes at ischemia.
Drug therapy
Different time gives mice single dose intravenous injection C1-INH (rabbit rhC1-INH, cattle rhC1-INH or pdC1-INH) with various dose behind ischemia.Control mice is accepted the saline of equal volume.
The neurologic impairment assessment
, at two function of nervous system's scorings of mice uniqueness every mice is assessed after 48 hours at ischemia by the research worker of experienced unknown experiment condition.Each following classification of overall neurologic impairment mice must be divided into 0-28: hair, ear, eye, posture, spontaneous activity, epilepsy behavior.Each following classification of focal neurologic impairment mice must be divided into 0-28: body symmetry, gait, climbing, circling behavior, forelimb symmetry, mandatory rotation, feel to reply.Data are with median and percentile value representation.
The quantification of infarct size
After 48 hours, mice is used Equitensin (120l/ mice, peritoneal injection) deep anaesthesia at ischemia, carefully pour into PBS 0.1mol/l, the pH7.4 of 30ml, be poured in the ice-cold paraformaldehyde of 60ml (4%) among the PBS subsequently.After careful taking-up brain from cranial cavity, it is moved in the PBS solution of 30% sucrose, spend the night at 4 ℃ of low-temperature protections.Then brain is immersed in the isopentane-45 ℃ of quick-freezings 3 minutes, is sealed in afterwards in the bottle ,-70 ℃ of storages until use.In order to determine damaged area, make the crown section of brain that thickness is 20 μ m at interval successively with 240 μ m, with dimethyl diaminophenazine chloride (Neutral RedGurr Certistain, BDH, England) dyeing.In each section, infarct size ignorant of the economics formula is estimated and is described by the pale relatively degree (relative paleness) of histological stain.The normal structure area that deducts in the homonymy hemisphere the area of infarct size by the offside hemisphere from each section is determined.By using a computer assistant images analyser and quantize by image analysis system (Analytical ImageSystem) calculating, the comprehensive infarct size on each brain tissue slice and calculate Infarction volume.
Neurodegenerative evaluation
By using Fluoro-Jade
4Dyeing (this is the mark of neuronal degeneration) is the neurodegenerative existence of assessment in the section of 20 μ m at thickness.In brief, with chip drying, rehydration in ethanol (100%-75%) and distilled water.Then it is incubated 15 minutes in 0.06% potassium permanganate, in distilled water, washs, move in the 0.001%Fluoro-Jade staining solution and dyeed 30 minutes.After dyeing, the rinsing in distilled water of will cutting into slices, drying immerses in the dimethylbenzene, and (the fluorescence microscope analysis is carried out in UK) capping afterwards for BDH, Poole with the DPX mounting medium.
The result
Effect time window in the of short duration ischemia
In order to assess the effect time window, begin rabbit rhC1-INH or the saline that the back gave 15U in 3,6,9,18 and 24 hours from ischemia.After 48 hours, at ischemia the back was used saline treatment in 3 and 6 hours with the ischemia mice ratio of rabbit rhC1-INH3 processing ischemia mice (44.43 ± 5.94mm takes place
3) the ischemia volume is shown obviously reduces and (be respectively 11.71 ± 0.63mm
3With 20.38 ± 2.37mm
3).When giving rabbit rhC1-INH in 9 and 18 hours behind ischemia, it is still effective, (is respectively 23.63 ± 4.11mm but effect is less
3With 27.13 ± 2.58mm
3).When giving in 24 hours behind ischemia, this inhibitor is lost its beneficial effect (41.92 ± 2.76mm
3) (Fig. 3).In the mice of saline treatment, Fluoro-Jade dyeing is illustrated in striatum cortex and the Hippocampus and has neural degeneration.When time point gave in early days, rhC1-INH can reduce the neural degeneration in Hippocampus (until 3 hours) and the cortex (until 9 hours).When behind ischemia, handling mice with this inhibitor in 6 and 9 hours, in Hippocampus, observe some degeneration neurons.At processing time point (18 and 24 hours) after a while, when the ischemia volume was big, Fluoro-Jade dyeing was illustrated in and has neurodegenerative neuron in the cortex.At all testing time points, striatum all illustrates neural degeneration (Fig. 5,6,7) widely in the animal of saline and rhC1-INH processing.Research worker by unknown experiment condition is carried out Fluoro-Jade dyeing sxemiquantitative assessment (Fig. 4) to every animal.
Dose response in the of short duration ischemia
Because the C1-INH dosage that uses is lower than the consumption of the present invention in the treatment of mice apoplexy, therefore in ischemia model of the present invention, use lower dosage in people's hereditary angioedema.Based on previous experimental result, select processing to carry out the dose response experiment in back 3 hours.Ischemia and again perfusion the back rabbit rhC1-INH (5 and 10 unit) that gave various dose in 3 hours takes place.The dosage of 10U/ mice is still effective (22.10 ± 3.65mm aspect reduction ischemia volume
3), and the rabbit rhC1-INH of 5U no longer changes the degree (47.39 ± 4.08mm of brain injury
3).These data illustrate rabbit rhC1-INH can change ischemic injuries (Fig. 8) in the dose dependent mode.
In the mice of handling, observe some neurodegenerative neurons by Fluoro-Jade dyeing at striatum, but in Hippocampus and cortex, do not observe with 10U rhC1-INH; And a large amount of neural degeneration (data not shown goes out) appear in the ischemia mice of handling with 5U in striatum cortex.
Any significant change (data not shown goes out) of all not shown overall and focal neurologic impairment in the experiment of effect time window or dose response.
The contrast of DdC1-INH and rhC1-INH (from rabbit and cattle) effect
Before the data about pdC1-INH derived from a kind of different transient cerebral ischemia model.In order directly to contrast pdC1-INH, cattle rhC1-INH and rabbit rhC1-INH, these chemical compounds are used the inductive ischemia mice of same experimental regime (silk that silicon applies).The described inhibitor that dosage is the 15U/ mice takes place to give in back 3 hours at ischemia.
As desired, pdC1-INH puts at this moment can not bring into play neuroprotective (47.39 ± 4.08mm3).Yet the ischemia mice that cattle rhC1-INH-handles illustrates the ischemia volume of obvious reduction than the mice of saline treatment, but the mice low (Fig. 9) that degree is handled than rabbit rhC1-INH-.Surprisingly overall and focal neurologic impairment is all by cattle rhC1-INH be improved (Figure 10).
Fluoro-Jade dyeing is illustrated in the zone (cortex, striatum and Hippocampus) of all considerations of the ischemia mouse brain tissue of handling with pdC1-INH and all has a large amount of neural degeneration.The mouse brain tissue staining that cattle rhC1-INH handles illustrates in cortex and the Hippocampus neural degeneration in various degree, because there are 3 mices all to observe tangible neural degeneration in these two zones in 6 mices, and the neural degeneration amount of other 3 mices seldom.Striatum Fluoro-Jade dyeing widely all is shown in 6 mices.
Comment
The related data of this work is the effect time window of rabbit rhC1-INH.Dosage is that the rabbit rhC1-INH of 15U/ mice still can significantly reduce the ischemia volume until 18 hours after ischemia takes place, and pdC1-INH has lost its neuroprotective at ischemia after 3 hours.This wonderful feature makes rhC1-INH become a kind of possible material standed for of people's syndrome similar to wind disease treatment.The different efficacies of pd and rhC1-INH is attributable to the different glycosylation of these two kinds of molecules, causes that to compare rhC1-INH higher for the affinity of mannose-binding protein (MBP) with the deutero-C1-INH of blood plasma.By with the combining of MBP, rhC1-MH causes participating in the pathogenetic complement lectin pathway of the heart, kidney and gastrointestinal tract ischemia/reperfusion injury and is suppressed
7,9This is also unknown without the effect of approach in cerebral ischemia of fully identifying, needs further experiment to illustrate the mechanism of rhC1-INH neuroprotective.
The good neuroprotective that rhC1INH takes place in the time window of back at ischemia than pdC1INH can following further explanation, and promptly described recombinant molecule is by more effectively target tissue damage location and/or more effective tissue penetration in conjunction with cell surface antigen.Need more study to throw a flood of light on the accurate molecules mechanism of observed result of the present invention.
Fluoro-Jade dyeing provides the circumstantial evidence of damage along with the time progress.Can save ischemic penumbra (Hippocampus and cortex) fully with the rhC1-INH early time treatment.Processing gives lately more, at ischemic penumbra many more neuronal degenerations is just arranged.These find to confirm that rhC1-INH brings into play neuroprotective for ischemic penumbra.Rabbit rhC1-INH can reduce the ischemia volume in the dose dependent mode.The most effective dosage of rhC1-INH (the 15U/ mice is corresponding to about 600U/kg) that uses in the experiment of effect time window is higher than the using dosage (approximately 25-100U/kg) in people's hereditary angioedema.In order to confirm whether lower dosage is still effective in reducing neural degeneration and ischemic infarct, carry out the dose response experiment.The rhC1-INH that the result illustrates 400U/kg (10U/ mice) still can significantly reduce ischemia injury, but degree reduces.(the 5U/ mice, 200U/kg) 8 times dosage is invalid to be higher than the used dosage of HAE.These are found and the indicated result who needs heavy dose of C1-INH in the treatment of various inflammation
5Consistent.Particularly this dosage is to reach necessary to the important inhibitory action of endothelial adhesion molecule
6, this is to participate in Cerebral Ischemia to damage pathological a kind of mechanism.At last, after ischemia, gave dosage in 3 hours and provide neuroprotective, but significantly be lower than the neuroprotective of rabbit rhC1-INH for the cattle rhC1-INH of 15U/ mice.Compare with the mice of saline treatment, the inhibitor in described cattle source also can improve nerve injury.These discoveries show that this molecular energy improves the general situation of ischemia mice.
The deutero-C1INH of embodiment 3:rhC1INH and blood plasma suppresses swashing of classical pathway and MBL approach
The ability contrast of living
Materials and methods
RhC1INH and pdC1INH (Cetor, Sanquin, Amsterdam, The Netherlands) acts on Wieslab TM complement system Screen (Euro-Diagnostica for classical and lectin pathway function, Malmo, Sweeden) the middle serum of two kinds of separate sources that uses detects.A kind of serum is the serum that comprises in the test kit, as positive control (hereinafter being called blood serum sample 1).Another blood serum sample derives from the commercially available human serum storehouse (Serum Bank of 25 different donors; Kordia, Leiden, Holland), hereinafter be called blood serum sample 2.These two blood serum samples are incubated 30 minutes with 0,15,30 and 75 μ mol rhC1INH or pdC1INH in room temperature separately in triplicate.For this reason, pdC1INH and rhC1INH stock solution are diluted in water be debita spissitudo.Get the volume corresponding to 15,30 and 75 μ molrhC1INH or pdC1INH, water is adjusted to 15 μ l.Get buffer (20mM citrate, 0.19M sucrose pH6.8 with the identical dilution rhC1INH of being dissolved with of rhC1INH; Through 0.22 μ m membrane filtration) in contrast to disturb the Wieslab complement system.Instruct according to manufacturer, it is 1/101 that the positive control (PC) of classical pathway and MBL approach and negative control (NC) (providing in the test kit) and two kinds of blood serum samples are diluted in diluent CP (classical pathway) and diluent MP (MBL approach).The serum that is 127.5 these dilutions of μ l was added 22.5 μ l water, pdC1INH, rhC1INH or buffer, room temperature insulation 30 minutes.Then, PC, NC, diluent CP or the MP (blank) in 100 μ l/ holes and sample are moved liquid to suitable flat board, 37 ℃ of insulations 1 hour.After insulation, with hole washing 3 times, the conjugate in room temperature and 100 μ l/ holes is incubated 30 minutes subsequently with 300 μ l/ wash solutions.After the washing, the substrate in hole and 100 μ l/ holes is incubated 30 minutes in room temperature once more once more.By adding the 5mM EDTA cessation reaction in 100 μ l/ holes, read absorbance at 405nm.
For result of calculation, from PC, NC and sample sets, deduct the absorbance of blank group (diluent CP or MP).The percentage ratio of complement activation utilizes following formula to calculate: (sample-NC)/(PC-NC) * 100.This means that PC always is set at 100%.For every kind of condition, calculating mean value, standard deviation and CV%.
The result
Check rhC1INH and pdC1INH effect by Wielisa to classical pathway
In two different blood serum samples, analyze rhC1INH and pdC1INH to the activated inhibitory action of classical pathway.Shown in Figure 12,13 and 16, rhC1INH and pdC1INH equal dose dependent in these two blood serum samples reduces the C5b-9 deposition of classical pathway mediation.As and if the rhC1INH of 75 μ m concentration compares with pdC1INH and to suppress classical pathway doughtily in blood serum sample 1 part omitted and activate, this acting in the blood serum sample 2 do not observe.In all other test concentrations, between rhC1INH and pdC1INH, do not observe inhibiting difference.Therefore, infer rhC1INH and pdC1INH suppresses the classical pathway activation in human serum aspect effect identical.
Check rhC1INH and pdC1INH effect by Wielisa to the MBL approach
In identical experiment is provided with, analyze rhC1INH and pdC1INH inhibitory action to the MBL pathway activation.Shown in Figure 14,15 and 16, rhC1INH and pdC1INH be all activation of dose dependent reduction MBL approach also.Yet opposite with the classical pathway that does not wherein observe difference, rhC1INH compares with pdC1INH that to present be the stronger inhibitor of MBL approach.In all three test concentrations and in these two blood serum samples, rejection ratio pdC1INH height～20% of the MBL approach of rhC1INH-mediation.Therefore, infer that it is the more effective inhibitor of MBL approach that rhC1INH compares with pdC1INH.
Conclusion
RhC1INH is shown the result and pdC1INH effect in suppressing classical pathway equates, but rhC1INH is the stronger inhibitor of MBL approach.In all test concentrations, rhC1INH suppresses strong～20% than the MBL approach of pdC1NH mediation.
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Claims (20)
1. plasma half-life is used for preventing, reducing or treat the application of the pharmaceutical composition of at least a ischemia and reperfusion injury less than 6 hours C1 inhibitor in preparation, and wherein said C1 inhibitor is at ischemia and give after the perfusion again.
2. the application of claim 1, wherein said C1 inhibitor is compared the terminal sialic acid residues level with reduction with the people C1 inhibitor that derives from blood plasma.
3. claim 1 or 2 application, wherein said C1 inhibitor comprises a kind of polysaccharide, and described polysaccharide has the terminal residue that is selected from galactose, N-acetylgalactosamine, N-acetyl-glucosamine, mannose and fucose.
4. the application of aforementioned each claim, wherein said C1 inhibitor has the aminoacid sequence with at least 65% homogeny of aminoacid sequence shown in the SEQID NO:1.
5. the application of aforementioned each claim, wherein said C1 inhibitor derives from genetically engineered cell or organism.
6. the application of claim 5, wherein said C1 inhibitor derives from transgenic nonhuman animal.
7. the application of claim 6, wherein said C1 inhibitor derives from the milk of transgenic nonhuman animal.
8. claim 6 or 7 application, wherein said transgenic nonhuman animal is cattle or Lagomorpha purpose animal, preferred rabbit.
9. the application of aforementioned each claim, wherein said C1 inhibitor is employed with the amount of 50-2000 unit/kg weight range.
10. the application of aforementioned each claim, wherein said C1 inhibitor ischemia and/or again the perfusion after at least 1 hour, preferably at least 3 hours, more preferably at least 6 hours, more preferably at least 9 hours, more preferably gave at least 18 hours.
11. the application of aforementioned each claim, wherein said pharmaceutical composition also contain thrombolytic or use with the thrombolytic applied in any combination or after with this thrombolytic treatment.
12. the application of aforementioned each claim, wherein said pharmaceutical composition are used to prevent, reduce or treat the unexpected or acute generation of at least a ischemia that can't predict and reperfusion injury.
13. the application of claim 12, wherein said pharmaceutical composition are used to prevent, reduce or treat at least a ischemia and reperfusion injury after apoplexy or perinatal stage apoplexy.
14. each application of claim 1-11, wherein said pharmaceutical composition are used to prevent, reduce or treat the ischemia of at least a prediction and the generation of reperfusion injury, the preferably generation after organ transplantation.
15. be used for preventing, reducing or treat the application of the pharmaceutical composition of at least a ischemia and reperfusion injury as the C1 inhibitor of claim 1-9 in preparation, wherein said C1 inhibitor is before the ischemia of at least a prediction and reperfusion injury take place or gives between the emergence period.
16. the application of claim 15, wherein said C1 inhibitor before the ischemia of at least a prediction and reperfusion injury take place 3 hours at the most, preferably at the most 2 hours, more preferably at the most 1 hour, most preferably gave in 30 minutes at the most, and/or C1 inhibitor wherein of the present invention is continued to treat transplanted organ by the object that need to continue it and/or in the organ transplantation situation.
17. the application of claim 15 or 16, wherein Yu Jian ischemia and reperfusion injury is in the organ transplantation situation.
18. each application of claim 1-17, wherein said pharmaceutical composition are used to prevent, reduce or treat known at least a ischemia and the reperfusion injury that produces by lectin pathway, preferred cardiac muscle, kidney, gastrointestinal tract ischemia and reperfusion injury or apoplexy.
19. the application of claim 13, wherein C1 inhibitor performance neuroprotective is preferably brought into play neuroprotective in Hippocampus and/or cortex.
20. the application of aforementioned each claim, wherein said C1 inhibitor performance reduce by ischemia and/or pour into the effect of inductive damage again.
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| CN105517559A (en) * | 2013-03-15 | 2016-04-20 | 夏尔维洛药品公司 | C1-inh compositions and methods for the prevention and treatment of disorders associated with c1 esterase inhibitor deficency |
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- 2006-12-19 ES ES11172880.4T patent/ES2654816T3/en active Active
- 2006-12-19 SI SI200631138T patent/SI1965831T1/en unknown
- 2006-12-19 DK DK06824316.1T patent/DK1965831T3/en active
- 2006-12-19 PT PT111728804T patent/PT2380587T/en unknown
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104080474A (en) * | 2011-12-22 | 2014-10-01 | 德国杰特贝林生物制品有限公司 | Use of C1-inhibitor for the treatment of secondary edema of the central nervous system |
| CN104080474B (en) * | 2011-12-22 | 2016-04-27 | 德国杰特贝林生物制品有限公司 | The application of C1-inhibitor in the treatment in pivot nervous system Secondary cases edema |
| CN105517559A (en) * | 2013-03-15 | 2016-04-20 | 夏尔维洛药品公司 | C1-inh compositions and methods for the prevention and treatment of disorders associated with c1 esterase inhibitor deficency |
| CN111529708A (en) * | 2013-03-15 | 2020-08-14 | 夏尔维洛药品公司 | C1-INH compositions and methods for the prevention and treatment of disorders associated with a deficiency in C1 esterase inhibitors |
| CN111818938A (en) * | 2018-02-28 | 2020-10-23 | 法明知识产权股份有限公司 | Treatment and prevention of preeclampsia |
Also Published As
| Publication number | Publication date |
|---|---|
| SI1965831T1 (en) | 2011-11-30 |
| PT2380587T (en) | 2018-01-19 |
| PT1965831E (en) | 2011-10-19 |
| ES2654816T3 (en) | 2018-02-15 |
| LT2380587T (en) | 2018-01-25 |
| ES2369522T3 (en) | 2011-12-01 |
| HUE035692T2 (en) | 2018-05-28 |
| CN105641688B (en) | 2020-06-09 |
| CN105641688A (en) | 2016-06-08 |
| DK1965831T3 (en) | 2011-10-24 |
| CN101365478B (en) | 2016-06-22 |
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