ES2369522T3 - USE OF A C1 INHIBITOR FOR THE PREVENTION OF INJURIES BY ISCHEMIA-REPERFUSION. - Google Patents

Abstract

Use of a C1 inhibitor that has a reduced level of terminal sialic acid residues compared to the plasma derived human C1 inhibitor, where the reduced level of terminal sialic acid residues results in a plasma half-life of less than 6 hours, in the preparation of a pharmaceutical composition for the prevention, reduction or treatment of at least one ischemia and reperfusion injury, where the C1 inhibitor is administered at least 10 minutes after the ischemia period and / or the onset of reperfusion.

Description

Use of a C1 inhibitor for the prevention of ischemia-reperfusion injury

Field of the Invention

[0001] The present invention relates to the prophylactic and therapeutic use of the C1 inhibitor to prevent, reduce and treat an ischemia-reperfusion injury, in particular a cerebral ischemia-reperfusion injury that may occur as a result of a stroke.

Background of the invention

[0002] Ischemia reperfusion injury is a well known pathological condition. It may represent an expected pathological condition or an unforeseen pathological condition. Stroke is one of the most common types of unforeseen ischemia-reperfusion injury. Stroke is the third leading cause of death and the leading cause of long-term disability in industrialized countries. Stroke is a type of cardiovascular disease that affects the arteries that go to and from the brain. A stroke occurs when such arteries are blocked by a clot or rupture and result in ischemia of the brain tissues that are treated by the blocked artery. Direct damage to the brain is caused by disruption of blood flow, mainly due to the loss of oxygenation to viable tissue, ultimately leading to heart attack if it is not reversed. However, if the lesion is reversed (either physiologically or therapeutically), then reperfusion of the ischemic tissue can paradoxically cause more indirect damage. When ischemia is long lasting, the damage "direct" resulting from hypoxia alone is the predominant mechanism. For ischemia of shorter duration, indirect or reperfusion-mediated damage becomes increasingly important for the final outcome.

[0003] The C1 inhibitor (INH C1), the C1 complement inhibitor, has shown neuroprotective action by reducing ischemia-reperfusion injury in rodent models with cerebral ischemia-reperfusion (De Simoni et al., 2003, J Cereb Blood Flow Metab. 23: 232-9; Akita et al., 2003, Neurosurgery 52: 395-400). The neuroprotective action of INH C1 in cerebral ischemia reperfusion injury does not require C1q (De Simoni et al., 2004, be J Pathol. 164: 185763). More recently Storini et al. (2005, Neurobiol Dis. 19: 10-7) reported that INH C1 exerts an antiapoptotic and anti-inflammatory action in ischemia-reperfusion injury through the inhibition of inflammation and cell recruitment from the vasculature to the ischemic site. However, the time span in a stroke in which the administration of INH C1 is therapeutically effective is rather limited. Therefore, it is an objective of the present invention to provide the INH C1 with a longer time window in terms of administration time.

Description of the invention

[0004] The present invention is based on the surprising finding that when the naturally-derived plasma-derived C1 (INH C1) inhibitor has lost most of its ability to reduce ischemiarefusion injury in a mouse model with ischemia Transient focal brain when administered after ischemia, a recombinant preparation of INH C1 remains capable of exerting its neuroprotective effects also when injected at least 1 hour after ischemia and / or reperfusion. Surprisingly, a neuroprotective effect can still be achieved when INH C1 is injected 18 hours after ischemia and / or reperfusion. The difference between the naturally-derived plasma-derived INH C1 and the recombinant INH C1 preparation is that the former has a plasma half-life of at least 24 hours and is completely sialylated glycoprotein, and the latter has a reduced plasma half-life and has a different glycosylation compared to the plasma derived product.

[0005] A known difference between naturally derived plasma-derived INH C1 and the recombinant INH C1 preparation is the extent and type of glycosylation. The recombinant glycoprotein contains a wide array of different N-glycans, comprising oligomanous, hybrid and complex type structures, while plasma derived INH C1 Nglycans are mainly composed of completely sialylated complex type structures. As a result of the differences in glycosylation, plasma-derived glycoprotein has a plasma half-life of at least 24 hours and recombinant INH C1 has a reduced plasma half-life.

[0006] Therefore, in one aspect the present invention relates to a method for the prevention, reduction or treatment of at least one ischemia-reperfusion injury, where the C1 inhibitor is administered at least after the period of ischemia and / or the onset of reperfusion and where the C1 inhibitor has a reduced level of terminal sialic acid residues compared to the human C1 plasma-derived inhibitor. The method preferably comprises the step of administering an effective amount of a C1 INH with a plasma half-life less than the plasma half-life of a plasma-derived INH C1. Alternatively, the method preferably comprises the step of administering an effective amount of a C1 INH having a different glycosylation compared to the plasma derived INH C1. This method refers to a prophylactic and / or therapeutic use of a C1 inhibitor for the prevention, reduction and / or treatment of any type of ischemia-reperfusion injury.

[0007] A C1 inhibitor, also referred to as C1 esterase inhibitor is defined herein, as a C1 complement inhibitor. The INH C1 belongs to the serine proteinase inhibitor superfamily and is the only C1r and C1s inhibitor of the complement system and is the major inhibitor of factor XIIa and kalikrein of the contact system. In addition, C1 INH also inhibits other coagulation serine proteases and fibrinolytic systems such as factor XI, tissue plasminogen activator and plasmin (Schapira et al. 1985, Complement 2: 111; Davis, 1988, Ana. Rev. Immunol. 6: 595). Human C1 INH is a 500 amino acid protein, including a signal sequence of 22 amino acids (Carter et al. 1988, Euro. J. Biochem. 173, 163). The plasma INH C1 is a glycoprotein of approximately 76 kDa and is strongly glycosylated, up to 26% of its molecular mass is carbohydrate (Perkins et al., 1990, J. Mol. Biol. 214, 751). A C1 INH for use in the methods of the present invention is preferably a protein with an amino acid sequence having at least 65, 67, 68, 69, 70, 75, 80, 85, 90, 95, 98 or 99 % identity with the amino acid sequence of mature human INH C1 as depicted in SEQ ID NO: 1.

[0008] On the occasion of the present invention, the degree of identity between two amino acid sequences refers to the percentage of amino acids that are identical in the two sequences. First, homologous polypeptide sequences are searched using the BLAST (Basic Local Alignment Search Tool) algorithm, which is described in Altschul, et al., J. Mol. Biol. 215: 403-410 (1990). The software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The parameters W, B and E of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLAST program uses a word length (W) of 3 as default values, the BLOSUM62 score matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, hope (E) of 10, M = 5; N = -4. Then, the degree of identity (as defined above) of homologous sequences is determined using the CLUSTALW alignment algorithm (Higgins D. Et al (1994). Nucleic Acids Res. 22: 4673-4680) using the following parameters; Gap size: 5, Gap open: 11, Gap extension: 1, Mismatch: -15, Word size: 3.

[0009] The INH C1 preferably has INH C1 activity as can, for example, be evaluated as described by Drouet et al. (1988, Clin Chim Acta. 174: 121-30). More preferably, the INH C1 is a human INH C1 (INH C1h) which is understood to mean that a INH C1 has an amino acid sequence that occurs naturally in man (eg, SEQ ID NO: 1 or CAA30314) , but it does not mean that the INH C1 is produced and obtained from, for example, human plasma.

[0010] According to one aspect of the invention, the INH C1 for use in the methods of the invention preferably has a reduced plasma half-life compared to the derived plasma half-life of INHC1, more preferably the plasma half-life of INH C1 of the invention is shorter than the plasma half-life of INH C1 derived from human plasma. For "reduced plasma half-life" the negative change in the circulating half-life of an INH C1 of the invention is understood in relation to the circulating half-life of a plasma-derived INH C1. In this context, a plasma-derived INH C1 refers to a naturally occurring INH C1 that is typically plasma-derived and that can be purified from plasma, but is not chemically or enzymatically modified.

[0011] The plasma half-life is measured by taking blood samples at various time points after administration of the INH C1 and determining the concentration of the INH C1 in each sample. The correlation of serum concentration over time allows the plasma half-life to be calculated. The reduction of the plasma half-life of INH C1 of the invention in relation to the circulating plasma half-life of a plasma-derived INH C1 is preferably at least about double, at least about triple, at least about quadruple, at least about sixfold, more preferably at least about octuple and most preferably at least about tenfold. In other words, the plasma half-life of a C1 INH of the invention is preferably less than 60, 50, 40, 30, 25, 20, 15, 12.5 or 10% of the plasma half-life of a C1 INH derivative, that is, its natural counterpart.

[0012] e.g. The plasma half-life of the INH C1 of the invention used in the examples herein, which is obtained from the milk of transgenic rabbits, shows a plasma half-life in humans of approximately 3 hours, which is four to eight times shorter than the plasma half-life of a C1 INH derived from human plasma. It is understood that the determination of the plasma half-life reduction of an INH C1 of the invention compared to the plasma-derived INH C1 is preferably performed under similar, if not identical conditions, that is, preferably in corresponding dosages, regimens Sampling, in the same organism, which can be a laboratory animal such as a mouse or human subjects, and on the same number of test subjects. In addition, it is understood that the plasma half-life of both INH C1 preparations are compared as can be determined by standard statistical analysis method.

[0013] A INH C1 with a shorter half-life, can be of natural origin or a recombinantly produced INH C1, can be prepared by any convenient method. It can for example be prepared in vivo in a recombinant host cell or organism that results in a C1 INH with a modified carbohydrate structure (as compared to the plasma derived INH C1) or the carbohydrate structure of a naturally occurring C1 INH can be chemically or enzymatically modified in vitro. Preferably, the INH C1 of the invention is modified in comparison with the plasma-derived INH C1 as follows: removal of a carbohydrate fraction (from a naturally occurring variant or recombinantly expressed variant of the glycoprotein), preferably the removal of Sialic acid and / or galactose from a carbohydrate chain linked by N-glucosidic bond and / or the elimination of a carbohydrate chain resulting in exposure of mannose, galactose, N-acetylglucosamine and / or fucose residues.

[0014] According to another aspect of the invention, the INH C1 for use in the methods of the invention preferably has a different glycosylation compared to the plasma-derived INH C1. Modifications of the carbohydrate structure of a C1 INH of the invention include modifications that lead to infraglycosylation.

[0015] In vitro, infraglycosylation may be the result of a deletion of a carbohydrate fraction or of a complete C1INH carbohydrate chain. Modifications may involve both carbohydrate chains linked by N-glucosidic or O-glucosidic bonds, or only one type of chain. It can involve all the chains, or just some of the chains. Overglycosylation can for example be the result of the addition of an extra carbohydrate fraction or a complete carbohydrate chain to the INH C1 molecule. An Asian form of INH C1

or a form with a reduced level of terminal sialic acid residues can typically be obtained by removal of a sialic acid group. It is well known that the half-life of a glycoprotein in the blood is highly dependent on the composition and structure of its carbohydrate groups linked by N-and Oglycoside bonds. In general, the maximum half-life of a glycoprotein requires that its carbohydrate groups linked by N- and O-glycosidic bonds have a terminal sialic acid. If this terminal sialic acid is not present, the glycoprotein is rapidly cleared from the blood due to the exposure of galactose residues. It is well established that the presence of terminal galactose residues in glycoprotein carbohydrate fractions results in plasma space enhanced by the asialoglycoprotein receptor in the liver. Thus, the INH C1 for use in the methods of the present invention has a reduced level of terminal sialic acid residues compared to the C1 inhibitor derived from human plasma. Sialic acid can be removed in different ways. For example, it can be removed chemically or enzymatically, for example, by treatment with sialidase. Sialidases suitable for this purpose are described by Chou et al. (1996, J Biol Chem. 271 (32): 19219-24; and 1994, J Biol Chem. 269 (29): 18821-6) and may p. ex. be obtained from V-labs, Inc. (covingtoNPn, Louisiana, USA). In another preferred embodiment, INH C1 for use in the methods of the present invention has preferably exhibited mannose, N-acetylglucosamine phosphamose, galactose and / or N-acetylgalactosamine residues. An exposed sugar residue will usually be a terminal sugar residue in a glycan branch or at least one sugar residue that is accessible for interactions with a fraction with affinity for the residue (such as a carbohydrate binding domain). A C1 INH with exposed galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose or phosphomanose residues may e.g. ex. be obtained by enzymatic treatment with one or more of 1-D-Nacethylhexosaminidase, endo-1-d-galactosidase, and / or aDN-acetylgalactosaminidase (also obtainable from, e.g., Vlabs, Inc., Covington, Louisiana, USA. ).

[0016] In vivo, carbohydrate chain modifications of INH C1 can be introduced using recombinant production systems. Both cultures of eukaryotic and prokaryotic cells can be used, such as yeast cells, fungal cells, insect cells and mammalian cells. For example, COS cells and CHO cells are suitable mammalian production systems. Although mammalian cell culture systems have the capacity to produce glycoproteins with sialylated carbohydrate groups, complete or optimal natural glycosylation is often difficult to achieve and consequently, glycoproteins produced from recombination in general have a different glycosylation model than their natural duplicates. . Normally this different glycosylation model is incomplete (compared to natural duplicates) having exposed galactose, Nacetylglucosamine and / or mannose residues. Likewise, the production of INH C1 in eukaryotic microorganisms such as yeasts or fungi will result in INH C1 with exposed mannose residues.

[0017] INH C1 with modified carbohydrate structures can also be prepared in transgenic animals, preferably non-human animals, such as rabbits, cattle, mice, rats, goats and transgenic sheep. Preferably, such glycoproteins are expressed in the mammary glands of these transgenic nonhuman animals so that glycoproteins can be obtained from the animal's milk. The person skilled in the art will understand that this will depend on the specific glycoprotein to be produced and on the amount it has to be produced, which transgenic animal is better to use for production. A particularly preferred INH C1 for use in the present invention is a C1 INH which is obtained from the milk of a transgenic bovine or an animal of the order Lagomorfa, preferably from the Leporidae family, more preferably from the orictolagus genus and most preferably a Rabbit of the Orictolagus cuniculus species.

[0018] Different types of modifications to the structure of the carbohydrate chain of the INH C1 protein compared to its natural plasma-derived duplicate can be obtained from recombinant production systems, such as different glycosylation, infraglycosylation can be introduced separately or in combination, simultaneously or consecutively, some types can be introduced to one part of the molecule, while others are introduced to another part of the molecule. Preferred combinations of modifications that contribute to the therapeutic efficacy of the protein include exposed galactose, N-acetylgalactosamine, Macethylglucosamine, mannose, fucose and / or phosphomanose residues in the INH C1 of the invention. The INH C1 of the invention may e.g. ex. have oligomanosa glycans or altamanosa type. Preferably at least about 5, 10, 15, 20, 40 or 60% of the terminal residues in the glycans in the INH C1 are selected from galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose and phosphomanose residues. For example, a preferred INH C1 for use in the present invention contains approximately up to 2, 4, 5, 6 times less sialic acid compared to its natural duplicate and / or at least about 5, 10, 15, 20, 40 or 60% of its glycans linked by N-glycosidic linkages are neutral support terminal hexoses with equatorial groups of 3 and 4-OH, such as mannose and N-acetylglucosamine. In contrast, the plasma-derived INH C1 does not have a glycosylation type oligomanose. A preferred INH C1 for use in the present invention, e.g. For example, it is a recombinant human INH C1 produced in the mammary glands of rabbits that have 5-6 times less sialic acid compared to their natural duplicate and approximately 15% of their glycans linked by N-glycosidic bond are terminal mannose residues neutral support.

[0019] In a preferred embodiment, glycosylation other than INH C1 for use in the present invention results in a higher affinity for a mannose binding protein compared to its plasma-derived duplicate. Mannose binding protein (MBP) is also referred to as mannan binding protein, mannose binding lectin (MBL), mannan binding lectin, or bactericidal Ra-reactive factor. MBP is a collection that belongs to a group of Ca2 + -dependent (type-C) soluble lectins. MBP is a complement activator via the lectin pathway (which differs from the alternative and traditional complement activation pathways). The complement system is an important component of the innate immune defense and is activated by three routes: the classical pathway, the alternative pathway, and the newly discovered lectin pathway or mannose binding lectin (MBL).

[0020] Activation of the classical pathway begins when the catalytic domains C1r and C1s are linked to immunological complexes by the C1q recognition protein (see Figure 11).

[0021] The alternative pathway is continuously rotating at a slow rate in an independent manner of antibody and will attack particles that are not specifically protected against complement.

[0022] The lectin or MBL pathway is activated or initiated after MBL binding to carbohydrate structures present in various pathogens or other cellular structures. Two serine proteases: Mannan-binding lectin-associated serine protease (MASP) -1 and -2 (see Figure 11) are associated with MBL and show similarities of shock with C1s and C1r serine proteases. The complex has C4- and C3- activation capabilities after binding to mannan. The complex contains two serine proteases MASP-1 and MASP-2 linked by a disulfide bond. In this way, MASP is able to break C4 and C3 resulting in its activation. The INH C1 of the invention preferably has a higher affinity for a human MBP compared to its plasma-derived duplicate.

[0023] MBP recognizes exposed hexoses with equatorial groups of 3 and 4-OH, such as mannose and Nacethylglucosamine and / or N-acetyl-hexosamines. A preferred INH C1 of the invention therefore carries such terminal hexoses. The highest affinity for MBP, preferably human MBP of the INH C1 of the invention is preferably such that it allows a more efficient objective, binding and / or inhibition of MBP compared to its duplicate derived from natural plasma to which exposed mannose is lacking. and residues of N-acetylglucosamine. Human MBP is here understood to refer to the protein characterized by the fact that Kawasaki et al. (1983,

J. Biochem 94: 937-47), with an amino acid sequence as described by Taylor et al. (1989, Biochem. J. 262 (3), 763-771; NCBI acquisition No. CAA34079). The complex MBP rat structure with an oligosaccharide is described by Weis et al. (1992, Nature. 360: 127-34). For another description of human MBP see p. eg US 6,846,649 and references cited there.

[0024] All of these pathways (classical, alternative and lectin or MBL) generate a crucial enzymatic activity that ultimately leads to the assembly of the membrane incursion complex (MAC or C5b-C9) (see Figure 11). Under physiological conditions, the activation of the complement system is effectively controlled by the coordinated action of membrane-associated and soluble regulatory proteins. One of these proteins is the C1 inhibitor (INH C1), a serine protease inhibitor that binds C1s and C1r and usually the only known physiological inhibitor of the classical pathway. In addition, INH C1 is capable of inactivating the complement of MBL-mediated activation by binding and inhibition of MASP-1 and MASP-2.

[0025] The activation of the different complement pathways is preferably measured in human sera by the Wielisa kit (product No. COMPL 300, Wieslab, Sweeden). This is a commercially available immune enzyme assay, specific for the detection of each of the three complement pathways with CSb-C9 deposition as a common reading. Briefly, wells of the microtiter strips are coated with specific activators of each of the three complement pathways. Human serum is diluted in diluent containing specific blocker to ensure that only the respective route is activated. The INH C1 of the invention or its plasma-derived duplicate is subsequently added at a concentration between 0 and 75 µmol, incubated for 30 minutes at room temperature and added to the wells. During a subsequent incubation of the diluted human serum in the well for 60 minutes at 37 ° C, the complement is activated by the specific coating. The wells are then washed and C5b-C9 formed are detected with a specific alkaline phosphatase labeled anti C5b-C9 antibody. After another washing step, the detection of specific antibodies is obtained by incubation with alkaline phosphatase substrate solution. The amount of complement activation correlates with color intensity and is measured in terms of absorbency (DO optical density). Using this equipment, both human recombinant INH C1 (rhC1-INH) of the invention and plasma-derived INH C1 (PdC1-INH) were shown to have similar inhibition capabilities for the classical pathway. However, the INH C1 of the invention proved to have approximately 20% more inhibitory capacity for the MBL pathway than the plasma-derived INH C1 (see example 3).

[0026] Therefore, in this preferred embodiment, glycosylation other than INH C1 for use in the present invention results in a higher affinity for an MBP compared to its plasma-derived duplicate, which results in further inhibition. effective MBP, leading to a more effective inhibition of the lectin pathway. The most effective inhibition of the lectin pathway preferably includes at least 5% more inhibition, even more preferably at least 10% more inhibition, even more preferably at least 15% more inhibition even more preferably at least 20% even more preferably at least 25 % even more preferably at least 30% even more preferably at least 35% even more preferably at least 40% even more preferably at least 45% even more preferably at least 50% even more preferably at least 55% even more preferably at least 60% even more preferably at least 65% even more preferably at least 70% even more preferably at least 75% even more preferably at least 80% even more preferably at least 85% even more preferably at least 90% even more preferably at least 95% and most preferably at least 98% more inhibition. The activation of the lectin pathway is preferably measured with the Wielisa kit as described above.

[0027] The method of the invention can be applied to prevent, reduce or treat any type of ischemia and reperfusion injury. Preferably, the method of the invention is applied where ischemia and reperfusion injury is known to arise at least in part, more preferably primarily through the lectin pathway. For myocardial ischemia and reperfusion injury (J Immunology 2005, 175: 541-546), reperfusion injury of renal ischemia (Sen J Pathol. 2004 165 (5): 1677-88), reperfusion injury of gastrointestinal ischemia (J Inmmnol. 2005 15: 174 (10): 6373-80), and for stroke (deSimoni et al, 2004 Ser J. Pathol. 164: 1857-63) has shown that reperfusion injury arises primarily through the lectin pathway and hardly through the classical way. Therefore, an INH C1 of the invention is preferably a more potent inhibitor of the lectin pathway compared to its duplicate derived from natural plasma. Preferably a C1 INH of the invention is a more potent in vivo inhibitor of the lectin pathway in man compared to its duplicate derived from natural plasma.

[0028] Unlike the experimental model used in the examples here, the incidence of real-life ischemia is often an unforeseen event. Therefore the administration of INH C1 before the incidence of ischemia and / or subsequent reperfusion is not generally a workable option and inevitably in practice INH C1 will have to be administered some time but several hours after the ischemia and / or subsequent reperfusion . This, however, severely limits the therapeutic utility of conventional plasma-derived INH C1 because it is mainly ineffective when administered after ischemic reperfusion and only has a very small time frame for therapeutic efficacy (see Figure 2 and de Simoni et al. , 2004 Ser J. Pathol. 164: 1857-63). In contrast, an INH C1 for use in the present invention as defined above, remains capable of exerting its neuroprotective effects also when injected at least 1 hour after ischemia or after the onset of ischemia and / or 30 minutes after the start of reperfusion. Therefore, the INH C1 of the invention is administered at least at the end or after the ischemic period, that is when the ischemic tissue undergoes reperfusion. More preferably, the INH C1 of the invention is administered at least 10, 15, 20, 30, 45, 60, 90 or 120 minutes after the ischemic period or after the onset of reperfusion. Preferably, the INH C1 of the invention is administered no more than 24, 12, 6, 4 or 3 hours after ischemia or after the onset of ischemia and / or reperfusion. In another preferred embodiment, the C1 inhibitor is administered at least 3 hours after ischemia or after the onset of ischemia and / or reperfusion, preferably at least 6 hours, more preferably at least 9 hours, even more preferably at minus 18 hours

[0029] In a preferred embodiment, the method is applied to prevent, reduce or treat an unexpected, sudden or acute incidence of ischemic reperfusion. Conditions and disorders associated with an unforeseen, sudden or acute incidence of ischemic reperfusion include but not limited to ischemic reperfusion injury after an acute myocardial infarction (IMA), after a stroke, including perinatal stroke, after hemorrhagic shock, after intestinal ischemia, after emergency coronary surgery for percutaneous transluminal coronary angioplasty (PTCA), after any vascular surgery with transverse blood vessel impingement (eg aorta, leading to skeletal muscle ischemia), or after pancreatitis after manipulation of the pancreatic or bile duct (ERCP). In such examples the INH C1 of the invention is preferably administered at least 1, 5, 10, 15, 20, 30, 45, 60, 90 or 120 minutes after acute myocardial infarction (IMA), after a stroke, including perinatal stroke, after hemorrhagic shock, after intestinal ischemia, after emergency coronary surgery for percutaneous transluminal coronary angioplasty (PTCA), after any vascular surgery with transverse blood vessel impingement (e.g. aorta, leading to skeletal muscle ischemia), or after pancreatitis after manipulation of the pancreatic or bile duct (ERCP). Alternatively, the time of administering the INH C1 of the invention can be defined as preferably at least 1, 5, 10, 15, 20, 30, 45, 60, 90 or 120 minutes after the start of reperfusion.

[0030] In addition, ischemic reperfusion injury is preferably defined as an ischemic reperfusion injury where therapy or surgery induces reperfusion but not ischemia. Such therapy or surgery includes but is not limited to:

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 Pharmacological thrombolysis, including intravascular and intravenous stroke therapies, acute coronary syndromes, peripheral arterial occlusion, pulmonary embolism, renal artery occlusion,

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 mechanical thrombolysis, for example, percutaneous coronary intervention, peripheral arterial angioplasty, visceral arterial angioplasty,

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 aortocoronary bypass graft,

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 carotid endarterectomy,

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 mesenteric ischemia,

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 shock including hemorrhagic, cardiogenic, neurogenic, analphylactic type,

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 flap failure, for example plastic surgery,

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 reimplantation of fingers and arms,

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 strangulated intestine

[0031] Alternatively, in another preferred embodiment, the method is applied to prevent, reduce or treat an expected incidence of ischemic reperfusion. An expected incidence of ischemia reperfusion injury preferably includes an adjustment in which a therapy or surgery induces ischemia and subsequently reperfusion. A non-limiting list is given following therapy or surgery where there is a temporary induced period of low or no blood flow, that is, ischemia or hypoxia, followed by reperfusion:

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 cardiopulmonary bypass,

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 aneurysm repair, including aortic or cerebral,

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 carotid endarterectomy in which a staple is used during surgery,

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 circulatory arrest with deep hypothermia,

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 use of tourniquet, that is, in trauma settings,

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 solid organ transplant,

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 any other yatrogenic blood flow rupture.

[0032] In addition, conditions and disorders associated with an expected incidence of ischemic reperfusion injury include, but are not limited to, ischemic reperfusion injury after an organ transplant (lung, liver, kidney, heart), after any surgery. vascular with transverse blood vessel impingement (e.g. aorta, leading to skeletal muscle ischemia), or after pancreatitis after manipulation of a pancreatic or bile duct (ERCP), after or during extra-bodily circulation (ECC) .

[0033] In a preferred embodiment of the method for the prevention, reduction or treatment of at least one expected incidence of ischemia and reperfusion injury, the INH C1 of the invention is administered at least at the end or after the ischemic period, it is say when ischemic tissue undergoes reperfusion. More preferably, the INH C1 of the invention is administered at least 10, 15, 20, 30, 45, 60, 90 or 120 minutes after the ischemic period or after the onset of reperfusion. Preferably, the INH C1 of the invention is administered no more than 24, 12, 6, 4 or 3 hours after ischemia or after the onset of ischemia and / or reperfusion. In another preferred embodiment, the C1 inhibitor is administered at least one hour after ischemia or after the onset of ischemia and / or reperfusion, 3 hours after ischemia or after the onset of ischemia and / or reperfusion, preferably at least 6 hours, more preferably at least 9 hours, even more preferably at least 18 hours.

[0034] According to a preferred embodiment, the INH C1 of the invention is continuously administered to a subject in need and / or in case of an organ transplant to the organ to be transplanted. The organ to be transplanted is preferably preserved in a composition with a suitable medium and adequate amount of INH C1.

[0035] In a more preferred embodiment, the method is applied in an unforeseen incidence of ischemic reperfusion. Even more preferably, an incidence of ischemic reperfusion injury after a stroke or perinatal stroke. In these types of unforeseen incidence of ischemic reperfusion, we demonstrate that the C1 inhibitor of the invention exerts a neuroprotective effect in ischemic penumbra. Ischemic penumbra preferably refers to the hippocampus and / or cortex. A neuroprotective effect preferably means that neurodegeneration is counteracted in the hippocampus and / or cortex after treatment with the C1 inhibitor of the invention up to 3 hours after the appearance of ischemia in the hippocampus and up to 9 hours after the appearance of ischemia in The cortex More preferably, neurodegeneration is counteracted up to 4, 5, 6 hours or more in the hippocampus and up to 10, 11, 12 hours or more in ischemia. Neurodegeneration is preferably evaluated as in Example 2: brain sections are stained with a specific marker for neuronal degeneration, preferably Jade (Schmued LC, et al, reference 4) and analyzed with fluorescent microscopy. Using this method, the neurodegeneration counteract assumes that at least 2% less colored cells are visualized in the treated sample compared to the untreated sample. Preferably, the neurodegeneration counteract is at least 5% less colored cells, at least 7%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or more.

[0036] Alternatively or in combination with the above-mentioned embodiment, the use of the C1 inhibitor exerts a reduction in the injury induced by ischemia and / or reperfusion. More preferably, when the ischemic reperfusion injury occurred after a stroke or perinatal stroke, the use of the C1 inhibitor of the invention exerts a reduction in the size of the infarction. Even more preferably, the size of the infarction is quantified as presented in Example 2. Even more preferably, using this method of quantification, at least 3 hours after the onset of ischemia, a reduction of at least 10% of the size of the infarction is achieved, even more preferably at least 20%, even more preferably at least 40%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90%

[0037] An INH C1 for use in the methods of the invention may be part of pharmaceutical compositions of the prior art. These pharmaceutical compositions typically comprise INH C1 and a pharmaceutically acceptable carrier or excipient. These pharmaceutical compositions can be administered in various ways depending on whether systemic or local treatment is desired, the area to be treated and the stability of the active compound. Appropriate formulations will depend on the method of administration. The pharmaceutical composition is preferably administered by parenteral administration, such as for example by intravenous, intra-arterial, intramuscular or subcutaneous intraperitoneal injection or infusion; or by intracranial or intrathecal administration. In a preferred embodiment, it is administered by intravenous infusion. Formulations suitable for parenteral administration are known in the art and are typically liquid formulations. INH C1 preparations for parental administration must be sterile. Sterilization is performed by filtration through sterile filtration membranes, before or following lyophilization and reconstitution. INH C1 preparations can be administered continuously by infusion or by bolus injection. The liquid formulations of INH C1 can for example be administered by an infusion pump. A typical composition for intravenous infusion could contain 100 to 500 ml of 0.9% sterile NaCl or 5% glucose optionally supplemented with 20% albumin solution and 100 to 500 mg of the INH C1. A typical pharmaceutical composition for intramuscular injection would contain, for example, 1-10 ml of sterile buffered water and 1 to 250 mg of the INH C1 of the present invention. Methods for preparing parenterally administrable compositions are well known in the art and are described in more detail in various sources, including, for example, Remington's Pharmaceutical Science (15th ed., Mack Publishing, Easton, PA, 1980) (incorporated by reference in its totality for all objectives).

[0038] The effective dose, ie effective concentration and frequency, of the INH C1 when used in the methods of the invention will depend on the specific pharmaceutical composition used, the severity of the disease and the general state of the patient's health. In general, the effective dose of a pharmaceutical composition that is based on a C1 INH for use in the methods of the invention can be found by routine optimization. A suitable starting point is the dose that is used for the pharmaceutical equivalent composition that is based on the plasma-derived INH C1. A great advantage of a pharmaceutical composition of the invention is that a high initial dose can be used in the treatment, which enhances the probability of successful treatment. This high initial dose is possible because the INH C1 in the pharmaceutical composition of the invention shows a space faster than its natural duplicate. In particular for the treatment of acute cases, a high initial dose of the INH C1 of the invention may be advantageous. This high initial dose may be at least 1.5, at least 2, 3 or 4 times the dose of the natural duplicate that would be administered.

[0039] In a preferred embodiment, the INH C1 of the invention is administered intravenously at a dose greater than 50, 100, 200, 400, 600, 800, or 1000 U / kg body mass of the individual, preferably in the range of 50-2000, 100-1000, 200-800, 400-700 or 500-700 U / kg body mass of the individual. One unit (U) of INH C1 is the amount of INH C1 present in 1 milliliter of human blood. One unit corresponds to approximately 275 micrograms of plasma-derived INH C1. Assuming a molecular weight of 110,000 dalton, the concentration in human plasma of INH C1 is 2.5 micromoles per liter (Nuijens et al. (1989), J. Clin. Invest. 84: 443).

[0040] In another preferred embodiment of the method of the invention the pharmaceutical composition contains a thrombolytic agent or is for use in combination with a thrombolytic agent or after further treatment with such an agent. A thrombolytic agent is understood herein as an agent (drug) that is capable of dissolving a blood clot (thrombus) and reopens an artery or vein. The thrombolytic agents are normally serine proteases and convert plasminogen to plasmin that breaks fibrinogen and fibrin and dissolves the clot. Preferred thrombolytic agents include reteplase (r-PA or Retavase), alteplase (t-PA or Activase), urokinase (Abbokinase), prourokinase, anisolated purified streptokinase activator complex (APSAC), and streptokinase.

[0041] In another aspect, the invention pertains to the use of an INH C1 of the invention as defined above for the production of a medicament for the prevention, reduction or treatment of reperfusion injury according to any of the methods defined above. .

[0042] In this document and in its claims, the verb "understand" and their conjugations are used in their non-limiting sense to mean that units that follow the verb are included, but units not specifically mentioned are not excluded. In addition, the reference to an item by the indefinite article " a " or "a" It does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article " a " or "a" normally means "at least one".

Description of the figures

[0043]

Figure 1 Evaluation of infarct size forty-eight hours after ischemia in mice treated with saline or with 15U RhC1-INH (recombinant human INH C1, see example 1.2) per mouse before and after one hour of ischemia.

Figure 2 Evaluation of infarct size twenty-four hours after ischemia in mice treated with saline solution or with 15U of plasma-derived INH C1 per mouse 1 hour before and after ischemia.

Figure 3 Infarct volume evaluated 48h after ischemia in mice receiving saline or 15U / mouse of rabbit RhC1-INH at different time points from the beginning of ischemia. Data are expressed as follows ± SEM (n = 6 mice per group). * P <0.05; ** P <0.01 against saline solution, one-way ANOVA and Dunnett as post-hoc test.

Figure 4 Semi-quantitative evaluation of Fluoro-Jade staining.- = no positivity, + = low positivity, ++ = intermediate positivity, +++ = high positivity.

Figure 5 Representative images of neurodegeneration by Fluoro-Jade staining in the striatum of ischemic mice receiving saline or 15U / mouse of rabbit RhC1-INH at different time points of the appearance of ischemia. Bar: 100μm

Figure 6 Representative images of neurodegeneration by Fluoro-Jade staining in the dentate gyrus of ischemic mice receiving saline or 15U / mouse of rabbit INH C1 at different time points of the appearance of ischemia. Bar: 100μm

Figure 7 Representative images of neurodegeneration by Fluoro-Jade staining in the cortex of ischemic mice receiving saline or 15U / mouse of rabbit INH C1 at different time points of the appearance of ischemia. Bar: 100μm

Figure 8 Infarct volume evaluated 48h after ischemia in mice receiving saline or 5, 10, 15U / mouse of rabbit RhC1-INH 3 hours after the onset of ischemia. Data are expressed as follows + SEM (n = 6 mice per group). ** P <0.01 against saline solution, one-way ANOVA and Dunnett as post-hoc test.

Figure 9 Infarct volume evaluated 48h after ischemia in mice receiving saline or 15U / mouse PdC1-INH or INH C1 from cow or rabbit three hours after the onset of ischemia. Data are expressed as follows ± SEM (n = 6 mice per group). * P <0.05; ** P <0.01 against saline solution, one-way ANOVA and Dunnett as post-hoc test.

Figure 10 General (upper panel 10a) and focal (lower panel 10b) deficits evaluated 48h after ischemia in mice receiving saline solution or 15U / mouse of PdC1-INH or INH C1 of cow or rabbit three hours after onset of ischemia. (n = 6 mice per group). ** P <0.01 against saline solution, one-way ANOVA and Kruskal-Wallis as post-hoc test.

Figure 11 Overview of the different complement activation pathways.

Figures 12, 13 Effect of RhC1-INH and PdC1-INH when activating the traditional complement pathway. Increasing dose of RhC1-INH or PdC1-INH (x axis) was added to two different samples of normal human serum (sample 1 upper panel. Sample 2 lower panel). As a control, the buffer in which RhC1-INH is dissolved (20 mM citrate, 0.19 M sucrose pH 6.8, 0.22 μm filtered) was taken along the same dilutions as INH C1hr. The reading was deposition of CSb-9, the normal serum control in the assay defining 100% (v axis). The data are the mean and SD (n = 3).

Figures 14, 15 Effect of RhC1-INH and PdC1-INH when activating the MBL complement pathway. Increasing doses of RhC1-INH or PdC1-INH (x-axis) were added to two different samples of normal human serum (sample 1 upper panel. Sample 2 lower panel). As a control, the buffer in which RhC1-INH is dissolved (20 mM citrate, 0.19 M sucrose pH 6.8, 0.22 μm filtered) was taken along the same dilutions as INH C1hr. The reading was deposition of C5b-9, the normal serum control in the assay defining 100% (y axis). The data are the mean and SD (n = 3).

Figure 16 Effect of INH rhc and PdC1-INH when activating the classical pathway and MBL activation complement. Increasing doses of RhC1-INH or PdC1-INH were added to two different samples of normal human serum. As a control, the buffer in which RhC1-INH dissolves (20 mM citrate, 0.19 M sucrose pH 6.8, 0.22 μm filtered) was taken along the same dilutions as INH C1hr. The reading was deposition of CSb-9 and percentage complement activation was calculated by measurement with this formula: (Sample-NC) / (PC-NC ') x100. PC is set at 100%. The results shown are the mean SD of 3 independent dilutions in each concentration evaluated.

Examples

Example 1

[0044] Previous experiments showed that a single dose of RhC1-INH (15U / mouse) administered at the beginning of the ischemic period significantly reduces ischemic volume, as assessed 48 hours after ischemia in our mouse model with focal cerebral ischemia in a manner very similar to the plasma-derived INH C1. In this example we have explored the time slot of efficacy for the neuroprotective activity of RhC1-INH in ischemic volume and functional deficits. We have also studied the effect of RhC1-INH as a result of 7 days by evaluation of the glial response and neurodegeneration.

1. Methods

1.1 Transient focal cerebral ischemia

[0045] Ischemia occurred due to occlusion of the middle cerebral artery (MCAO) as described above (De Simoni et al., 2003 and 2004, supra). Anesthesia was induced by 5% isoflurane in N2O / O2 (70/30%) mixed and maintained by 1.5 2% isoflurane in the same mixture. To confirm the adequacy of vascular occlusion in each animal, blood flow was measured with flowmetry Doppler laser (Transonic BLF-21) using a 0.5 mm flexible fiber optic probe (Transonic, type M, 0.5 mm diameter) located on the surface of the brain and fixed with impression material in the skull at the following coordinates: AP = .- 1mm; L = -3.5mm. Briefly, the common adequate carotid artery was exposed and a siliconized filament (7-0) was introduced into the internal carotid artery through an incision made in the common carotid artery and advanced to the cerebral anterior artery to block its bifurcation in the Anterior cerebral artery and monoclonal antibody. The filament was advanced until a blood flow reduction of> 70%, compared to the preischemic baseline, was observed. After 30 min of ischemia, blood flow was restored by carefully removing the nylon filament.

1.2 Drug treatment

[0046] The mice received a single iv injection of RhC1-INH at the dose of 15U / mouse in 150 µl or the same volume of saline at different times of ischemia:

-

 at the beginning of the ischemic period (pre RhC1-INH).

-

 at the end of the ischemic period (post RhC1-INH).

-

 one hour after the beginning of the ischemic period (1h-post RhC1-INH). RhC1-INH used in this study was produced in transgenic rabbits that expressed human INH C1 in their mammary glands and purified the milk obtained from these animals as described in WO 01/57079.

1.3 Evaluation of neurological deficits

[0047] Forty-eight hours after ischemia, each mouse was estimated on two neurological function scales unique to the mouse, by a trained researcher attached to the experimental conditions. For mice of general deficits they were marked from 0 to 28 in each of the following categories: hair, ears, eyes, spontaneous activity, epileptic behavior. For mice with focal deficits, they were marked from 0 to 28 in each of the following categories: body symmetry, ascent, anterior arm symmetry, mandatory turn, sensory response. Data are expressed as mean and percentiles from 25th to 75th.

1.4 Quantification of infarct size

[0048] Forty-eight hours after ischemia, the mice were deeply anesthetized with Equitensin (120 µl / mice, ip) and transcardially perfused with 30ml of 0.1mol / l PBS, pH 7.4, followed by 60ml of cooled paraformaldheide (4 %) in PBS. After carefully removing the brains from the skull, they were transferred to 30% sucrose in PBS at 4 ° C overnight for cryoprotection. The brains were then quickly frozen by immersion in isopentane at -45 ° C for 3 min, being sealed in vials before and stored at -70 ° C until use. To determine the size of the lesion, 20 μm brain crown sections were cut in series at 240 μm intervals and stained with neutral red (Neutral Red Gurr Certistain, BDH, England). In each stratum, the infarct areas were blindly evaluated and delineated by the relative paleness of histological coloration. The area of infarction was determined by subtracting the area of healthy tissue in the ipsilateral hemisphere from the area of the contralateral hemisphere in each section. The infarct volumes were calculated by the integration of infarct areas in each brain stratum as quantified with computer-assisted image analyzer and calculated by analytical imaging system.

1.5 Open field test

[0049] Seven days later, the behavior of the mouse with ischemia was evaluated by the open field test. This test can be useful for detecting anxiety and exploring locomotor behavior and activity in long-term ischemic mice. The open field consisted of a plastic box (41 x 41 x 41cm) containing 4 different objects. The open field area was divided into a central area of 28 x 28cm and the surrounding confinement zone. The mice were individually placed in the center of the open field and their behavior was observed for 5 minutes by experts outside the experimental conditions. The number of internal crossings (mainly related to anxiety behavior), external crossings (mainly related to motor activity), rear crossings (mainly related to exploratory behavior) and contacts with objects (mainly related to sensory / motor activity) were counted.

1.6 Neural Account

[0050] Seven days after ischemia, mice were perfused transcardially as described above. To determine the neuronal count, 20μm coronary sections of the brain were cut in series at 640 μm intervals and stained with cresyl violet (Cresyl Violet acetate, Sigma, St. Louis, MO). Three sections of ipsi- and collateral hemispheres of 20 μm were selected for the neuronal count. The first section was in the stereotactic coordinates anteroposterior to +0.86 of bregma. The amount of neuronal loss was calculated by combining the number of viable neurons in three sections of both hemispheres and expressed as a percentage of controlateral hemisphere. An Olimpus BX61 microscope, bounded with a video camera with Soft Imaging System Colorview and AnalySIS software was used. The quantitative analysis was performed in a 40X magnification by researchers outside the treatment.

1.7

 Immunohistochemistry for astrocytes and microglia

[0051] Seven days after ischemia, 20μm thick coronary brain sections of ischemic mice were prepared and used to evaluate the immunocoloration of astrocytes and microglia / macrophages. Briefly, the sections were rinsed for 30 minutes in 0.4% Triton 100 X-100 in 0.1 mol / L PBS followed by 15 minutes in 0.1% Triton X-100 and 3% normal goat serum (NGS) in PBS. Sections were then incubated overnight with antibodies to astrocytes and microglia (anti-GFAP 1: 1500, Chemicon; anti-CD11b 1: 250, kindly donated by Dr. A. Doni, Mario Negri institute). The next day, the sections were washed in PBS and incubated with biotinylated secondary antibodies for 1 hour, washed and incubated with avidin-biotinperoxidase. After reacting with 3 -3-diaminobenzidine tetrahydrochloride the sections were washed, dried, dehydrated through classified alcohols, fixed in xylene and covered using upright DPX before optical microscopy analysis.

2.

 Results

2.1 Time window of effectiveness

2.1.1 Evaluation of neurological deficits

[0052] Neurological deficits were evaluated in ischemic mice receiving RhC1-INH or saline solution 48h after ischemia. A slight, though not significant, reduction in each group of mice treated with RhC1-INH was observed compared to ischemic mice treated in saline (pre RhC1-INH: 9 and 12 post RhC1-INH: 7 and 11; post- 1h RhC1-INH: 9 and 13, saline: 10 and 12.5, average of focal and general deficits, respectively) (data not shown).

2.1.2 Evaluation of infarct size

[0053] Forty-eight hours after ischemia, mice treated with RhC1-INH showed a marked reduction in ischemic volume, at 15U / mouse pre, post and 1 hour post dose (13.67 ± 2.59mm3, 9.06 ± 0.77mm3 and 8.24 ± 1.00 mm3, respectively), compared to mice treated with saline solution (41.51 ± 7.01mm3) (Figure 1, the data are expressed as mean ± SEM).

2.2 Results after seven days

2.2.1 Open field test

[0054] Ischemia induced a significant reduction in the number of offspring compared to untreated animals while in the group treated with RhC1-INH this parameter was not different from non-ischemic mice. The other parameters evaluated showed no difference between the three groups.

2.2.2 Neural Account

[0055] To assess whether the protective effect of RhC1-INH is long-lasting, we evaluate the loss of neurons seven days after the induction of ischemia and drug treatment. The results show that the protection effect of INH C1 is still present at that time: 14% ± 2.18% against 4% ± 1.24% of saline and mice treated with RhC1-INH, respectively (data not shown).

2.2.3

 Immunohistochemistry for microglia / macrophages and astrocytes

[0056] Seven days after ischemia a large amount of activated microglia and infiltrated macrophages were observed in the injured and striated hippocampus of ischemic mice receiving saline (data not shown). Fifteen units of pre-RhC1-INH were able to counteract this activation and infiltration in the areas considered (data not shown). The ipsilateral hippocampus of ischemic mice treated with saline showed a slight astrocytosis that was not different from that observed in RhC1-INH ischemic mice (data not shown). Other areas of the brain showed no relevant astrocytosis activation in any group.

3.

 Conclusions

[0057] The present data show that RhC1-INH at the dose of 15U / mouse is similarly effective in reducing ischemic volume when administered at the beginning (pre) or at the end of the ischemic period (ie, after reperfusion ). More importantly, the inhibitor is able to exert its neuroprotective effects also when it is injected 1 hour after the onset of ischemia (post 1h). In addition, the protective action of RhC1-INH is still present 7 days after ischemia. These results are in contrast to plasma-derived INH C1h that when injected 1 hours after ischemia has practically lost the ability to exert neuroprotective effects (see Figure 2).

[0058] The main results of this study are the following:

one.

 The half-life of RhC1-INH in mouse plasma is approximately 3 hours (at a dose of 15U / mouse). The good correlation between antigen and functional activity indicates that the recombinant protein circulates in plasma in its active form only; It is possible that tissue distribution contributes to the reduction of plasma levels.

2.

 RhC1-INH, at the dose of 15U / mouse -pre is very effective in reducing ischemic volume (69% reduction).

3.

 RhC1-INH at the dose of 15U / mouse is able to clearly reduce the number of degeneration neurons in the hippocampus as assessed by Fluoro-Jade staining, thus indicating that the reduction in ischemic volume is due to the preservation of neurons.

Four.

 RhC1-INH is similarly effective in reducing ischemic volume when administered at the beginning (pre), at the end of the ischemic period (i.e., post reperfusion) or 1 hour after the onset of ischemia (post 1h, i.e. 30 min from the reperfusion principle). Thus RhC1-INH has a broader time window of efficacy than PdC1-INH (which is no more effective when administered 1h after ischemia).

5.

The neuroprotective effect of the pre-RhC1-INH dose is long-lasting, as shown in the neuronal count performed 7 days after the onset of ischemia.

6.

 RhC1-INH, induced a slight improvement of focal and general deficits evaluated 48 hours after ischemia. This finding is similar to that observed with PdC1-INH. To assess the effect of RhC1-INH on long-term behavioral outcomes, we analyzed mouse behavior with open field tests. Seven days after ischemia, the behavior of the offspring shows a significantly lower score in the ischemic compared to untreated mice. This reduction is not present in mice treated with RhC1-INH whose score is not different from control mice.

7.

 RhC1-INH is able to counteract the activation / recruitment of microglia / macrophages in the brain of ischemic mice as both have been evaluated at early (48h) and late (7 days) time points. These cells are an index of the inflammatory response of brain tissue.

8.

 The strong astrocytic response caused by ischemia at 48h is moistened with RhC1-INH. Astrocytic activation decreases markedly at 7 days in both experimental groups and no difference between mice treated with saline and those treated with RhC1-INH can be observed.

Example 2: study on the neuroprotective action of RhC1-INH in mouse models with focal cerebral ischemia

[0059] We have previously shown that 15U of RhC1-INH have a marked neuroprotective action in a murine cerebral ischemia / reperfusion model also when administered 1 hour after the onset of ischemia / reperfusion, in variation with PdC1-INH which, At that time of post-treatment, it is no longer effective. This neuroprotection is long lasting, in fact seven days after ischemia and treatment, ischemic brains of mice treated with RhC1-INH still show a decreased infarct size. In the following experiments we have determined the time window of efficacy (beyond 1 hour later) and the dose response of the neuroprotective activity of RhC1-INH in the ischemic volume. We have also made a direct comparison between PdC1-INH, and RhC1-INH of rabbit and cow (in the most effective dose and points for RhC1-INH of rabbit) using the same protocol.

METHODS

Animals

[0060] The procedures involving animals and their care were carried out in accordance with institutional guidelines in accordance with national laws (DL n.116, GU suppl. 40, 18 February 1992) and international laws and policies (EEC Board of Directors 86 / 609, OJ L 358.1 Dec. 12,1987; NIH Guide for the Care and Use of Laboratory Animals, US National Research Council USA 1996). C57B1 / 6 male mice (26-28 g, Charles River, Calco, Italy) were housed in cages, 5 in each, and kept at constant temperature (21 ± 1 ° C) and relative humidity (60%) with regular hours light / dark (7 am-7 μm). Food (Altromin granules for mice) and water available ad libitum.

Transient focal cerebral ischemia

[0061] Ischemia was achieved by middle cerebral arterial occlusion (MCAO) as previously described in 1 "3. Anesthesia was induced by 5% isoflurane in NaO / Oa (70/30%) mixture and maintained by 1.5-2% isoflurane in the same mixture. To confirm the adequacy of vascular occlusion in each animal, blood flow was measured by flowmeter doppler laser (Transonic BLF-21) using a 0.5mm fiber optic probe, (Transonic M type, 0.5mm diameter) located at the surface of the brain and fixed with impression material in the skull at the following coordinates: AP = -1mm; L = -3.5mm. Briefly, the common adequate carotid artery was exposed and a siliconized filament (7-0) was introduced into the internal carotid artery through an incision made in the common carotid artery and advanced to the cerebral anterior artery to block its bifurcation in the artery Anterior cerebral and MCA. The filament was advanced until a> 70% reduction in blood flow, compared to preischemic baseline, was observed. After 30 min of ischemia, blood flow was restored by carefully removing the nylon filament.

Medication treatment

[0062] Mice received a single iv injection of INH C1 (rabbit RhC1-INH, cow RhC1-INH or PdC1-INH) at different doses at different times of ischemia. Control mice received the same volume of saline.

Evaluation of neurological deficits.

[0063] Forty-eight hours after ischemia, each mouse was estimated on two neurological function scales unique to the mouse, by expert researchers outside the experimental conditions. For general deficits the mice were marked from 0 to 28 in each of the following categories: hair, ears, eyes, spontaneous activity, epileptic behavior. For focal deficits the mice were marked from 0 to 28 in each of the following categories: body symmetry, movement, climbing, anterior arm symmetry, mandatory turning, sensory response. Data are expressed in average and percentiles.

Quantification of infarct size

[0064] Forty-eight hours after ischemia, the mice were deeply anesthetized with Equitensin (120 jil / mice, ip) and transcardially perfused with 30ml of PBS O.lmol / 1, pH 7.4, followed by 60ml of paraformaldheide cooling (4 %) in PBS. After carefully removing the brains from the skull, they were transferred to 30% sucrose in PBS at 4 ° C overnight for cryoprotection. The brains were then quickly frozen by immersion in isopentane at -45 ° C for 3 min being sealed in vials before and stored at -70 ° C until use. To determine the size of the lesion, 20fjm coronary brain sections were cut in series at 240 (j, m intervals and stained with neutral red (Neutral Red Gurr Certistain, BDH, England). In each section, the affected areas were blindly evaluated and delineated by the relative paleness of the histological coloration.The affected area was determined by subtracting the area of healthy tissue in the ipsilateral hemisphere from the area of the contralateral hemisphere in each section.The infarction volumes were calculated by the integration of affected areas in each Brain section as quantified with computer-assisted image analyzer and calculated by analytical imaging system.

Neurodegeneration evaluation

[0065] The presence of neurodegeneration was evaluated in 20 thick sections of obstruction by coloration with Fluoro-Jade4, a label for neuronal degeneration. Briefly, the sections were dried and rehydrated in ethanol (100% - 75%) and distilled water. Then, they were incubated in 0.06% potassium permanganate for 15 minutes, washed in distilled water and transferred to 0.001% Fluro-Jade staining solution for 30 minutes. After coloring, the sections were rinsed in distilled water, dried, dipped in xylene and covered using DPX mountant (BDH, Poole, UK) before fluorescent microscopy analysis.

RESULTS

EFFECTIVENESS TIME WINDOW IN TRANSITIONAL ISCHEMIA

[0066] To evaluate the efficacy time window, 15U of rabbit RhC1-INH or saline solution were given at 3, 6, 9, 18 and 24 hours from the beginning of ischemia. Forty-eight hours later, ischemic mice treated with rabbit RhC1-INH 3 and 6 hours after the onset of ischemia showed a marked reduction in ischemic volume (11.71 ± 0.63mm3 and 20.38 ± 2.37mm3, respectively ) compared to ischemic mice treated in saline solution (44.43 ± 5.94mm3). Also when administered 9 and 18 hours after ischemia, RhC1-INH was still effective, although to a lesser extent (23.63 ± 4.11mm3 and 27.13 ± 2.58mm3 respectively). Twenty-four hours after ischemia the inhibitor lost its beneficial action (41.92 ± 2.76mm3) (Fig. 3). In mice treated in saline solution, Fluoro-Jade staining showed that neurodegeneration was present in striatum cortex and hippocampus. When administered at early time points, RhC1-INH was able to counteract neurodegeneration in the hippocampus (up to 3 hours) and in the cortex (up to 9 hours). When mice were treated with this inhibitor 6 and 9 hours after ischemia, some degeneration neurons were observed in the hippocampus. At later treatment points (18 and 24 hours), when the ischemic volume was larger, Fluoro-Jade staining showed the presence of neurodegeneration in the cortex. At all the time points considered, striatum showed intensive neurodegeneration, both in those treated with saline and with animals treated with RhC1-INH (Fig. 5, 6, 7). A semi-quantitative evaluation of Fluoro-Jade staining for each animal was performed by researchers outside the experimental conditions (Figure 4).

DOSE RESPONSE IN TRANSITIONAL ISCHEMIA

[0067] Since the dose of INH C1 used in humans for hereditary angioedema is lower than that used in mice for the treatment of stroke, lower doses were used in our ischemic model. Based on the results of the previous experiment we chose post 3 hour treatment to experience the dose response. Different doses of rabbit RhC1-INH (5 and 10 units) were administered 3 hours after the onset of ischemia and reperfusion. The dose of IOU / mouse was still effective in reducing ischemic volume (22.10 ± 3.65mm3), while 5U of rabbit INH C1 did not change the extent of brain damage (47.39 ± 4.08mm3). These data show that rabbit INH C1 is able to modify the ischemic lesion in a dose-dependent manner (Fig. 8).

[0068] In mice treated with 10U of RhC1-INH some neurodegenerative neurons, such as Fluoro-Jade staining, were observed in striatum but not in hippocampus and cortex, while 5U of treated ischemic mice showed neurodegeneration in the striatum cortex ( not shown).

[0069] Focal and general neurological deficits showed no significant variation either in time window of efficacy or in dose response experiment (not shown).

[0070] Comparison between the effect of PdC1-INH and RhC1-INH (of rabbits and cows). Our previous data in PdC1-INH were obtained with a different model of transient cerebral ischemia. To directly compare PdC1-INH, cow RhC1-INH and rabbit RhC1-INH, these compounds were given to mice in which ischemia was induced with the same experimental protocol (silicone coated filament). The inhibitors were administered at the dose of 15U / mouse 3 hours after the onset of ischemia.

[0071] As planned, the PdC1-INH was not able to exert a neuroprotective action at that point in time (47.39 ± 4.08mm3 à). In contrast, ischemic mice treated with cow RhC1-INH showed a significant reduction in ischemic volume compared to mice treated in saline, although to a lesser extent than mice treated with RhC1-INH (Fig. 9). Surprisingly, both focal and general deficits were significantly improved by cow RhC1-INH (Fig. 10).

[0072] Staining with Fluoro-Jade showed a great neurodegeneration in the brain of ischemic mice treated with PdC1-INH in all the areas considered (cortex, striatum and hippocampus). The brain staining of mice treated with cow RhC1-INH showed a variable degree of neurodegeneration in the cortex and hippocampus since in 3 out of every 6 mice a marked neurodegeneration was observed in both areas, while in the other three mice the neurodegeneration was Present in a small amount. Striatum showed extensive Fluoro-Jade staining in 6 out of 6 mice.

OBSERVATIONS

[0073] The most relevant data of this work is the window of efficacy time of rabbit RhC1-INH. The dose of 15U / mouse of rabbit RhC1-INH was able to significantly reduce ischemic volume up to 18 hours after the onset of ischemia, unlike with PdC1-INH that 3 hours after ischemia has already lost its neuroprotective effect . This surprising feature makes RhC1-INH a possible candidate as stroke therapy in humans. The different efficacy of RhC1-INH and PdC1-INH, could be due to the different glycosylation of the two molecules resulting in a higher affinity for a RhC1-INH mannose binding protein (MBP) compared to the derivative of plasma. Joining MBP, RhC1-INH causes inhibition of the complement lectin pathway, implicated in the pathogenesis of ischemia / reperfusion damage in the heart, kidney and gastrointestinal 7 &9; 9. The function of this poorly characterized pathway remains unknown in cerebral ischemia and more experiments are required for the clarity of the RhC1-INH neuroprotection mechanism.

[0074] The superior neuroprotective effect of RhC1-INH on PdC1-INH in the time window after the onset of ischemia can be further explained by a more effective target of the recombinant molecule to the site of tissue damage well through binding to cell surface antigens and / or more effective tissue penetration. Further research is necessary to fully elucidate the exact molecular mechanism highlighting the observation described in this invention.

[0075] Staining with Fluoro-Jade gives indirect evidence of how the lesion evolves over time. Early treatment with RhC1-INH provides a complete rescue of ischemic penumbra (hippocampus and cortex). The later the treatment is administered, the more neurons degenerate into the penumbra. These conclusions confirm that RhC1-INH exerts its neuroprotective action in ischemic penumbra. Rabbit RhC1-INH is able to reduce ischemic volume in a dose dependently. The most effective dose of RhC1-INH (15U / mouse, corresponding to approximately 600U / kg), used for the efficacy time window experiment, is much higher than that used in humans for hereditary angioedema (approximately 25-100U / kg) To verify if a lower dose was still effective in reducing ischemic infarction and neurodegeneration, a dose response experiment was carried out. The results showed that 400U / kg (10 U / mouse) of RhC1-INH were still able to significantly counteract the ischemic attack, although to a lesser extent. A dose of 8 folds higher than that used for HAE (5U / mouse; 200U / kg) was not effective. These conclusions are in line with the evidence that shows that large doses of INH C1 are required for therapeutic use in various inflammation settings5. In particular, such doses are necessary to achieve an important inhibitory effect on the adhesion of endotial molecules6, a mechanism involved in the pathogenesis of ischemia / reperfusion brain damage. Finally, cow RhC1-INH provided neuroprotection, when administered 3 hours after ischemia at the dose of 15U / mouse, although less markedly than rabbit RhC1-INH. The cow inhibitor was also able to improve neurological deficits compared to mice treated with saline. These conclusions indicate that this molecule is capable of improving the general conditions of ischemic mice.

Example 3: Comparison of the ability of plasma-derived RhC1-INH and INH C1 to inhibit the activation of classical and MBL pathways.

Materials and methods

[0076] The effect of RhC1-INH and PdC1-INH (Cetor, Sanquin, Amsterdam, The Netherlands) on the function of the lectin and traditional pathway was examined in the Wieslab TM complement system screen (Euro-Diagnostica, Malmø , Sweden) using two different sources of serum. A source of serum is included in the kit, where it is used as a positive control (hereafter referred to as serum sample 1). The other serum sample was obtained from a commercially available cluster of human serum (cluster of 25 different donors; Kordia, Leiden, The Netherlands), hereinafter referred to as serum sample 2. Both serum samples were incubated in independent triples. with 0, 15, 30 and 75 μmol of RhC1-INH or PdC1-INH for 30 min at room temperature. Therefore, stock solutions of PdC1-INH and RhC1-INH were diluted in water at appropriate concentrations. Corresponding volumes with 15, 30 and 75 μmol of RhC1-INH or PdC1-INH were taken and adjusted to 15 μl with water. The buffer in which RhC1-INH is dissolved (20 mM citrate, 0.19 M sucrose pH 6.8.0.22 μm filtered) was taken along at the same dilutions as RhC1-INH to control for interference with the Wieslab complement system. The positive (PC) and negative (NP) controls of the traditional route and MBL (provided with the equipment), and both serum samples were diluted 1/101 in CP diluent for the classical route and MP diluent for the MBL route according to instructions manufacturer. Of these diluted sera, 127.5 μl was supplemented with 22.5 μl water, PdC1-INH, RhC1-INH or buffer and incubated for 30 minutes at RT. Then, 100 μl / well of PC, NC, CP or MP diluent (preliminary forms) and samples were pipetted into the appropriate plate and incubated for 1 hour at 37 ° C. After incubation the wells were washed 3 times with 300 μl / well water solution and subsequently incubated for 30 minutes at room temperature with 100 μl / well of conjugate. After another wash, the wells were incubated with 100 μl / well of substrate and again incubated for 30 minutes at room temperature. The reaction was stopped by the addition of 100 µl / well 5 mM EDTA and the absorbency was detected at 405 nm.

[0077] For the calculation of the results, the absorbency of the preliminary forms (diluent CP or MP) was subtracted from the PC, NC and samples. The percentage complement activation was calculated by measurement with this formula: (Sample-NC) / (PC-NC) x100. This means that the PC is always set to 100%. For each condition the standard deviation, mean and CV% was calculated.

Results

Effect of RhC1-INH and PdC1-INH on the classical pathway examined by Wielisa.

[0078] The inhibitory properties of both RhC1-INH and PdC1-INH in classical pathway activation were analyzed in two different serum samples. As shown in Figures 12, 13 and 16, both doses of RhC1-INH and PdC1-INH dependently reduced the classical C5b-9 mediated deposition pathway in both serum samples. While RhC1-INH - at a concentration of 75 μM - appears to inhibit the activation of the traditional route in serum 1 slightly stronger than PdC1-INH, this effect was not seen in serum sample 2. In the other concentrations evaluated no difference In inhibitory properties it was observed between RhC1-INH and PdC1-INH. Therefore, it was concluded that both RhC1-INH and PdC1-INH are equally effective in activating the classical inhibition pathway in human sera.

Effect of RhC1-INH and PdC1-INH on the MBL pathway examined by Wielisa.

[0079] In the same set of experiments, also the inhibitory properties of both RhC1-INH and PdC1-INH in the activation of the MBL pathway were analyzed. As shown in Figures 14, 15 and 16, both RhC1-INH and PdC1-INH also in dependent doses reduced the activation of the MBL pathway. However, unlike the classic pathway where none of the differences were seen, RhC1-INH proved to be a more potent inhibitor of the MBL pathway when compared to PdC1-INH. In all 3 concentrations evaluated and in both serum samples, the inhibition mediated RhC1-INH of the MBL pathway is - 20% higher compared to PdC1-INH. Therefore, it was concluded that RhC1-INH is a more effective inhibitor of the MBL pathway than PdC1-INH.

conclusion

[0080] The results show that both RhC1-INH and PdC1-INH are equally effective in inhibiting the classical pathway, but RhC1-INH is a more potent inhibitor of the MBL pathway. At all concentrations evaluated, inhibition of the MBL pathway by RhC1-INH ~ 20% stronger compared to PdC1-INH.

REFERENCES

one.

 From Simoni, M. G. et al. Neuroprotection by complement (C1) inhibitor in mouse transient brain ischemia. J Cereb Blood Flow Met 23, 232-239 (2003).

2.

 From Simoni, M. G. et al. The powerful neuroprotective action of C1-inhibitor on brain ischemia-reperfusion injury does not require C1q. Am JPathol 164, 1857-63 (2004).

3.

 Storini, C. et al. C1 inhibitor protects against brain ischemia-reperfusion injury via inhibition of cell recruitment and inflammation. Neurobiol Disease 19,10-17 (2005).

Four.

 Schmued, L. C. &amp; Hopkins, K. J. Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res 874, 123-30. (2000).

5.

 Caliezi, C. et al. C1 esterase inhibitor: an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema. Pharmacol Rev 52.91-112 (2000).

 6.

 Cai, S. et al. A direct role for C1 inhibitor in regulation of leukocyte adhesion. J Immunol 174, 6462-6 (2005).

7.

 Walsh, M. C. et al. Mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury. J Immmunol 175, 541-6 (2005).

8.

 Moller-Kristensen, M. et al. Mannan-binding lectin recognizes structures on ischaemic reperfused mouse kidneys and is implicated in tissue injury. Scand JImmunol 61,426-34 (2005).

9.

 Hart, M. L. et al. Gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving C1q. J Immunol 174, 6373-80 (2005).

SEQUENCE LIST

[0082]

<110> Pharming Intellectual Property B.V.

<120> Use of C1 inhibitor for the prevention of reperfusion ischemia lesions

<130> P6005723PCT

<140> P6005723PCT <141> 2006-12-19

<150>

05112630.8 <151> 2005-12-21

<150>

60 / 760,944 <151> 2006-01-23

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 478

<212> PRT

<213> Homo sapiens

<400>

Claims (15)

one.

Use of a C1 inhibitor that has a reduced level of terminal sialic acid residues compared to the plasma derived human C1 inhibitor, where the reduced level of terminal sialic acid residues results in a plasma half-life of less than 6 hours, in the preparation of a pharmaceutical composition for the prevention, reduction or treatment of at least one ischemia and reperfusion injury, where the C1 inhibitor is administered at least 10 minutes after the ischemia period and / or the onset of reperfusion.

2.

Use according to claim 1, wherein the C1 inhibitor is administered between at least 10 minutes and 24 hours after the ischemia period and / or the onset of reperfusion.

3.

Use according to claim 1 or 2, wherein the C1 inhibitor comprises a glycan having a terminal residue selected from galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose and fucose.

Four.

Use according to any of claims 1-3, wherein the C1 inhibitor has an amino acid sequence that has at least 65% identity with the amino acid sequence of SEQ ID NO: 1.

5.

Use according to any of claims 1-4, wherein the C1 inhibitor is obtained from a genetically modified cell or organism.

6.

Use according to claim 5, wherein said organism is a non-human transgenic animal.

7.

Use according to claim 6, wherein the C1 inhibitor is obtained from the milk of said non-human transgenic animal.

8.

Use according to claim 6 or 7, wherein the non-human transgenic animal is a bovine or an animal of the order Lagomorpha.

9.

Use according to claim 8, wherein the non-human transgenic animal is a rabbit.

10.

Use according to any of claims 1-9, wherein the C1 inhibitor is used in an amount in the range of 50-2000 units per kg of body weight.

eleven.

Use according to any of claims 1-10, wherein the pharmaceutical composition also contains a thrombolytic agent, or is for use in combination with a thrombolytic agent or is for use after use with such agent.

12.

Use according to any of claims 1-11, wherein the pharmaceutical composition is for the prevention, reduction or treatment of an unexpected or acute occurrence of ischemia and reperfusion injury.

13.

Use according to claim 12, wherein the unforeseen or acute occurrence of ischemia and reperfusion injury occurs after a stroke or perinatal stroke.

14.

Use according to any of claims 1-11, wherein the pharmaceutical composition is for the prevention, reduction or treatment of an expected occurrence of ischemia and reperfusion injury.

fifteen.

Use according to claim 14, wherein the expected occurrence of ischemia and reperfusion injury occurs after an organ transplant.