after an ischemic period and repetfusion and therefore particularly useful for unforeseen occurrences of ischemic teperfusion such
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): MANNFSSE,
`Maurice [NUNL]; Aloelaan 25, NL-2316 XR Leiden
`(NL). NUIJENS, Johannes Henrlcus [NL/NL]; Zeven-
`huizerlaan 42, NL—1851 M Heiloo (NL). PIEPER, Frank
`[NUNL]; Meindert Hobbemastraat 27, NL-2102 BH
`Heemstede (NL). DE SIMONI, Maria Grazia [IT/IT];
`Head Of The Laboratory Of Inflammation And Nervous
`System Deseases,
`Instituto Mario Negri, Via Eritrea,
`62, I-20157 Milaan (IT). ZIERE, Gljsbertus Johannes
`[NIJNL]; Europasingel 20, NL-2396 EM Koudeke (NL).
`
`H 0
`
`')
`
`bO\bCG
`
`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(43) International Publication Date
`28 June 2007 (28.06.2007)
`
`(10) International Publication Number
`
`WO 2007/073186 A2
`
`W'i |I||||||||I|||||||I|||||||||||||||||||||||||||||||||||||I||||||||||||||||||||||||||||||
`
`(51) International Patent Clawification:
`A61K 38/57 (2006.01)
`A611” 41/00 (2006.01)
`A61P 9/10 (2006.01)
`
`(21) International Application Number:
`PCT/NL2006/050321
`
`(22) International Filing Date:
`19 December 2006 (19.12.2006)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Prlority Data:
`051 12630.8
`60/760,944
`
`21 December 2005 (21.12.2005)
`23 January 2006 (23.01.2006)
`
`EP
`US
`
`(71) Applicant (forall designated States except US): PHARM-
`ING INTELLECTUAL PROPERTY BV [NUNL]; Dar-
`winweg 24, NL—2333 CR Leiden (NL).
`
`(74) Agent: VAN WESTENBRUGGE, Andrics; Postbus
`29720, NL-2502 LS Den Haag (NL).
`
`(81)
`
`States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY. BZ, CA, CH, CN.
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI.
`GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS,
`JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS,
`LT, LU, LV, LY, MA, MD, MG, MK, MN, MW MX, MY,
`MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, 171‘, RO, RS,
`RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84) Dwignated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl,
`FR, GB, GR, l-IU, IE, IS, IT, LT, LU, LV, MC, NL, PL, PI‘,
`RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`without international search report and to be republished
`upon receipt of that report
`with sequence listing part of description published sepa-
`rater in electronic form and available upon request from
`the International Bureau
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes andAbbreviations" appearing at the begin-
`ning ofeach regular issue of the PCT Gazette.
`
`(54) Title: USE OF Cl INHIBITOR FOR THE PREVENTION OF ISCHEMIA-REPERFUSION INJURY
`
`N < \
`
`9
`0e
`
`N (57) 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
`
`CSL EXHIBIT 1007
`CSL v. Shire
`
`Page 1 of 47
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`1
`
`U se of C 1 inhibitor for the prevention of ischemia-reperfusion injury
`
`Field of the invention
`
`5
`
`The present invention relates to the therapeutic and prophylactic use of C 1
`
`inhibitor for preventing, reducing and treating ischemia-reperfusion injury, particularly
`
`cerebral ischemia-reperfusion injury that may occur as a result of a stroke.
`
`Background of the invention
`
`10
`
`Ischemia-reperfusion injury is a well known occuring pathologic condition. It
`
`may either represent a foreseen pathologic condition or an unforeseen pathologic
`
`condition. Stroke is one ofthe most common types ofunforeseen ischemia-reperfusion
`
`injury. Stroke is the third cause of death and the leading cause of long-term disability in
`
`industrialized countries. Stroke is a type of cardiovascular disease that affects the
`
`15
`
`arteries leading to and within the brain. A stroke occurs when such arteries are blocked
`
`by a clot or bursts and results in ischemia of the cerebral tissues that are served by the
`
`blocked artery. Direct darnage to the brain is caused by the interruption of the blood
`
`flow, mainly due to loss of oxygenation to the viable tissue, ultimately leading to
`
`infarction if not reversed. However if the insult is reversed (either physiologically or
`
`20
`
`therapeutically) then reperfusion ofthe ischemic tissue may paradoxically cause further
`
`indirect damage. When there is a long duration of ischemia, the "direct" darnage
`
`resulting from hypoxia alone is the predominant mechanism. For shorter duration's of
`
`ischemia, the indirect or reperfusion mediated darnage becomes increasingly more
`
`important to the final outcome.
`
`25
`
`Cl inhibitor (CIINH), the inhibitor of complement Cl, has been reported to
`
`display neuro-protective action by reducing ischemia-reperfusion injury in rodent
`
`models for 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 neuro-protective
`
`action ofCIINH on brain ischemia-reperfusion injury does not require Clq (De Simoni
`
`30
`
`et al., 2004, Am J Pathol. 164: 1857-63). More recently Storini et al. (2005, Neurobiol
`Dis. 12.: 10-7) reported that CIINH exerts an anti-inflammatory and anti-apoptotic
`action on ischemia-reperfusion injury through inhibition of inflammation and cell
`
`recruitment from the vasculature to the ischemic site. However, the window in time
`
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`2
`
`around the stroke during which administration of C 1 INH is therapeutically effective is
`
`rather narrow. It is therefore an object of the present invention to provide for CliNH
`
`with a broader window in terms oftime of administration.
`
`5
`
`Description of the invention
`
`The present invention is based on the surprising finding that where naturally
`
`occurring plasma derived Cl inhibitor (CIINH), has lost most of its ability to reduce
`
`ischemia reperfusion injury in a mouse model for transient cerebral focal ischemia
`
`when administered after ischemia, a recombinant preparation of CliNH is still able to
`
`10
`
`exert its neuroprotective effects also when injected at least 1 hour after ischemia and/or
`
`reperfusion. Surprisingly, a neuroprotective effect can still be reached when the CliNH
`
`is injected 18 hours after ischemia and/or reperfusion. The difference between the
`
`naturally occurring plasma derived CIINH and the recombinant preparation of CIINH
`
`is that the first has a plasma half life of at least 24 hours and is fully sialylated
`
`15
`
`glycoprotein, and the latter has a reduced plasma half life and has a different
`
`glycosylation as compared to the plasma derived product.
`
`A difference known between the naturally occurring plasma derived C IINH and
`
`the recombinant preparation of CIINH is the extent and type of glycosylation. The
`
`recombinant glycoprotein contains a broad array of different N-glycans, comprising
`
`20
`
`oligomannose-, hybrid-, and complex-type structures, whereas the N-glycans of plasma
`
`derived CIINH are mainly composed of fully sialylated complex-type structures. As a
`
`result of the differences in glycosylation, the plasma derived glycoprotein has a plasma
`
`half life of at least 24 hours and the recombinant CliNH has a reduced plasmahalf life.
`
`In one aspect the present invention therefore relates to a method for the
`
`25
`
`prevention, reduction or treatment of at least one of ischemia and reperfusion injury,
`
`whereby the Cl inhibitor is administered after the ischemia and/or after the reperfusion.
`
`The method preferably comprises the step of administering an effective amount of a
`
`CIINH having a plasmahalf life that less than the plasmahalf life of a plasma derived
`
`CIINH. Altematively, the method preferably comprises the step of administering an
`
`30
`
`effective amount of a CliNH that has a different glycosylation as compared to the
`
`plasma derived CIINH. This method relates to a therapeutic and/or prophylactic use of
`
`Cl inhibitor for preventing, reducing and/or treating any type of ischemia-reperfusion
`
`lllJUfY.
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`A Cl inhibitor, also referred to as Cl esterase inhibitor is herein defined, as an
`
`inhibitor of complement C 1. C IINH belongs to the superfamily of serine proteinase
`
`inhibitors and is the only inhibitor of C 1 r and C 1 s of the complement system and is the
`
`major inhibitor of factor XIIa and kallikrein of the contact system. In addition C IINH
`
`5
`
`also inhibits other serine proteases of the coagulation and fibrinolytic systems like
`
`factor XI, tissue type plasminogen activator and plasmin (Schapira et al. 1985,
`
`Complement 2,: 111; Davis, 1988, Ann. Rev. Immunol. .Q: 595). Human CIINH is a
`
`protein of 500 amino acids, including a 22 amino acid signal sequence (Carter et al.
`
`1988, Euro. J. Biochem. 173; 163). Plasma CIINH is a glycoprotein of approximately
`
`10
`
`76 kDa and is heavily glycosylated, up to 26% of its molecular mass consists of
`
`carbohydrate (Perkins et al., 1990, J. Mol. Biol. 214 , 751). A CIINH for use in the
`
`methods of the present invention preferably is a protein with an amino acid sequence
`
`that has at least 65, 67, 68, 69, 70, 75, 80, 85, 90, 95, 98 or 99% identity with the amino
`
`acid sequence ofthe maturehuman CIINH as depicted in SEQ ID NO: I.
`
`15
`
`For the purpose of the present invention, the degree of identity between two
`
`amino acid sequences refers to the percentage of amino acids that are identical between
`
`the two sequences. First, homologaus polypeptide sequences are searched using the
`
`Basic Local Alignment Search Tool (BLAST) algorithm, which is described in
`
`Altschul, et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST
`
`20
`
`analyses
`
`is publicly available
`
`through the National Center for Biotechnology
`
`Information (http://w-vvvv.ncbi.nlm.nih.gov/). The BLAST algorithm parameters W, B,
`
`and E determine the sensitivity and speed of the alignment. The BLAST program uses
`
`as defaults a word length (W) of 3, the BLOSUM62 scoring matrix (see Henikoff &
`
`Henikoff, Proc. Natl. Acad. Sei. USA 89: 10915 (1989)) alignments (B) of 50,
`
`25
`
`expectation (E) of 10, M=5, N=-4. Next, the degree of identity (as defined above) of
`
`homologaus 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.
`
`The CIINH preferably has CIINH activity as may e.g. be assayed as described
`
`30
`
`by Drouet et al. (1988, Clin Chim Acta. 174:121-30). More preferably, the CIINH is a
`
`human C IINH (hC IINH) which is understood to mean that the C IINH has an amino
`
`acid sequence that naturally occurs in man (as e.g. SEQ ID NO:l or CAA30314) but
`
`does not mean that the CIINH is produced in and obtained from e.g. human plasma.
`
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`According to one aspect of the invention the C liNH for use in the methods of the
`
`invention preferably has a reduced plasmahalf life as compared to the plasmahalf life
`
`of plasma derived CliNH, more preferably the plasmahalf life of the CliNH of the
`
`invention is less than the plasma halflife ofCliNH derived from human plasma. By "a
`
`5
`
`reduced plasma half life" is meant the negative change in circulating half life of a
`
`C liNH of the invention relative to the circulating half life of a plasma derived C liNH.
`
`In this context, a plasma derived CliNH refers to naturally occurring C1INH which is
`
`typically derived from plasma and which may be purified from plasma but is not
`
`modified in chemically or enzymatically.
`
`10
`
`Plasma half life is measured by taking blood samples at various time points after
`
`administration ofthe CliNH, and determining the concentration ofthe CliNH in each
`
`sample. Correlation of the serum concentration with time allows calculation of the
`
`plasma half life. The reduction of plasma half life of a C liNH of the invention relative
`
`to the circulating half life of a plasma derived C1INH preferably is at least about two-
`
`15
`
`fold, at least about three-fold, at least about four-fold, at least about six-fold, more
`
`preferably at least about eight-fold, and most preferably at least about ten-fold. In other
`
`words, plasmahalf life of a CliNH of the invention preferably is less than 60, 50, 40,
`
`30, 25, 20, 15, 12.5 or 10% ofthe plasmahalf life of a plasma derived CliNH, i.e. its
`
`naturally occurring counterpart.
`
`20
`
`E.g. the plasma half life of the CliNH of the invention that is used in the
`
`Examples herein, which is obtained from the milk of transgenic rabbits, exhibits a
`
`plasma half life in humans of about 3 hours, which about four- to eight-fold less than
`
`the average plasma half life of a plasma derived C liNH in man. It is understood that
`
`the determination of the reduction of plasma half life of a CliNH of the invention as
`
`25
`
`compared tothat ofplasma derived CliNH is preferably performed under similar ifnot
`
`identical conditions, i.e. preferably at corresponding dosages, sampling regimes, in the
`
`same organism, which may be a laboratory animal such as a mouse or human subjects,
`
`and in about the same number of test subjects. Furthermore, it is understood that the
`
`average plasma half lives of both CliNH preparation are compared as may be
`
`30
`
`determined by standard method of statistical analysis.
`
`A C liNH with shorter half life, be it a naturally occurring or a recombinantly
`
`produced C1INH, may be prepared by any convenient method. It may for example be
`
`prepared in vivo in a recombinant host cell or organism that results in a C liNH with a
`
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`
`modified carbohydrate structure ( as compared to the plasma derived C IINH) or the
`
`carbohydrate structure of a naturally occurring CIINH may be chemically or
`
`enzymatically modified in vitro. Preferably, the CIINH of the invention is modified
`
`compared to the plasma derived C IINH the following way: removal of a carbohydrate
`
`5 moiety (from a naturally occurring variant or recombinantly expressed variant of the
`
`glycoprotein), preferably the removal of sialic acid and/or galactose from a N-linked
`
`carbohydrate chain and/or the removal of a carbohydrate chain resulting in exposure of
`
`mannose, galactose, N-acetylglucosamine and/or fucose residues.
`
`According to another aspect ofthe invention the CIINH for use in the methods of
`
`10
`
`the invention preferably has a different glycosylation as compared to the plasma
`
`derived CIINH. Modifications to the carbohydrate structure of a CIINH of the
`
`invention include modifications which lead to underglycosylation, overglycosylation,
`
`to the asialio form of CIINH, or any other modifications which lead to a different
`
`glycosylation pattem.
`
`15
`
`In vitro, underglycosylation may be the result of a deletion of a carbohydrate
`
`moiety or ofa complete carbohydrate chain ofCIINH. Modifications may involve both
`
`N- or 0-link:ed carbohydrate chains, or only one type of chain. It may involve all the
`
`chains, or only some of the chains. Overglycosylation may for instance be the result of
`
`the addition of an extra carbohydrate moiety or a complete carbohydrate chain to the
`
`20
`
`CIINH molecule. An asialo-form of CIINH or a form having a reduced level of
`
`terminal sialic acid residues may 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 N- and 0-link:ed carbohydrate
`
`groups. In general, maximal half life of a glycoprotein requires that its N- and 0-link:ed
`
`25
`
`carbohydrate groups 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 carbohydrate moieties of glycoproteins results in enhanced plasma clearance by the
`
`asialoglycoprotein receptor in the liver. Thus in a preferred embodiment, CIINH for
`
`30
`
`use in the methods of the present invention preferably has a reduced level of terminal
`
`sialic acid residues as compared to plasma derived human Cl inhibitor. Sialic acid may
`
`be removed in several ways. For instance, it may be removed chemically or
`
`enzymatically, for example, by treatment with sialidase. Suitable sialidases for this
`
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`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 e.g. be obtained from V-labs, Inc. (Covington,
`
`Louisiana, USA). In a further preferred embodiment, CIINH for use in the methods of
`
`the present
`
`invention preferably has exposed mannose, N-acetylglucosamine
`
`5
`
`phosphomannose, galactose and/or N-acetylgalactosamine residues. An exposed sugar
`
`residue will usually be a terminal sugar residue on a glycan branch or at least a sugar
`
`residue that is accessible for interactions with a moiety having affinity for the residue
`
`(such as a carbohydrate binding domain). A CIINH with exposed galactose, N(cid:173)
`
`acetylgalactosamine, N-acetylglucosamine, mannose, fucose or phosphomannose
`
`10
`
`residues may e.g. be obtained by enzymatic treatment with one or more of ß-D-N(cid:173)
`
`acetylhexosaminidase, endo-ß-D-galactosidase, and/ or a-D-N-acetylgalactosaminidase
`
`(also obtainable form e.g. V-labs, Inc., Covington, Louisiana, USA).
`
`In vivo, modifications of carbohydrate chains of C1INH may be introduced by
`
`using recombinant production systems. Both prokaryotic and eukaryotic cell cultures
`
`15 may 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, optimal, natural or complete glycosylation is often
`
`difficult to achieve and consequently, recombinantly produced glycoproteins in general
`
`20
`
`have a different glycosylation pattem than their natural Counterparts. Usually this
`
`different glycosylation pattem is incomplete (as compared to the natural Counterparts)
`
`having exposed galactose, N-acetylglucosamine and/or mannose residues. Likewise,
`
`production of C1INH in eukaryotic microorganisms like yeasts or fungi will result in
`
`C IINH with exposed mannose residues.
`
`25
`
`C IINH with modified carbohydrate structures may also be prepared in transgenic
`
`animals, preferably in non-human animals, such as in transgenic rabbits, bovine, mice,
`
`rats, goats and sheep. Preferably, such glycoproteins are expressed in the mammary
`
`glands of these non-human transgenic animals such that the glycoproteins may be
`
`obtained from the milk of the animal. The skilled person will understand that it will
`
`30
`
`depend on the specific glycoprotein to be produced and on the amount which has to be
`
`produced, which transgenic animal is best used for production. A particularly preferred
`
`CIINH for use in the present invention is a CIINH that is obtained from the milk of a
`
`transgenic bovine or an animal of the order Lagomorpha, preferably of the family
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`7
`
`Leporidae, more preferably of the genus Oryctolagus and most preferably a rabbit of
`
`the species Oryctolagus cuniculus.
`
`Different types of modifications to the structure of the carbohydrate chain of the
`
`CIINH protein as compared to its natural plasma-derived counterpart may be obtained
`
`5
`
`from
`
`recombinant
`
`production
`
`systems,
`
`such
`
`as
`
`different
`
`glycosylation,
`
`underglycosylation or overglycosylation may be
`
`introduced separately or
`
`in
`
`combination, simultaneously or consecutively, some types may be introduced to one
`
`part of the molecule, while others are introduced to another part of the molecule.
`
`Preferred combinations of modifications contribute to the therapeutic efficacy of the
`
`10
`
`protein
`
`include exposed galactose, N -acetylgalactosamine, N -acetylglucosamine,
`
`mannose, fucose and/or phosphomannose residues on the C1INH of the invention. The
`
`CIINH of the invention may e.g. have glycans of the oligomannose type or of the
`
`highmannose type. Preferably at least about 5, 10, 15, 20, 40 or 60% of the terminal
`
`residues on
`
`the glycans on
`
`the CIINH are
`
`selected
`
`from galactose, N-
`
`15
`
`acetylgalactosamine, N-acetylglucoseamine, mannose, fucose and phosphomannose
`
`residues. E.g. a preferred CIINH for use in the present invention contains about 2, 4, 5,
`
`6-fold less sialic acid as compared to its natural counterpart and/or at least about 5, 10,
`
`15, 20, 40 or 60% of its N-linked glycans are neutral carrying terminal hexoses with
`
`equatorial 3- and 4-0H groups, such as mannose and N-acetylglucosamine. In contrast,
`
`20
`
`plasma derived CIINH has no oligomannose type glycosylation. A preferred CIINH
`
`for use in the present invention e.g. is a recombinant human C1INH produced in the
`
`mammary glands of rabbits which has 5-6 fold less sialic acid as compared to its
`
`natural counterpart and about 15% of its N-linked glycans areneutral carrying terminal
`
`mannose residues.
`
`25
`
`In a preferred embodiment, the different glycosylation of the C IINH for use in
`
`the present invention results in a higher affinity for a mannose binding protein as
`
`compared to its plasma derived counterpart. The mannose binding protein (MBP) is
`
`also referred to as mannan-binding protein, mannose-binding lectin (MBL), mannan(cid:173)
`
`binding lectin, or bactericidal Ra-reactive factor. MBP is a collectin that belongs to a
`group of soluble Ca2+-dependent (C-type) lectins. MBP is an activator of complement
`
`30
`
`via the lectin pathway (that differs from the classical and alternative pathways of
`
`complement activation). The complement system is an important component of the
`
`innate immune defense and is activated by three pathways: the classical pathway, the
`
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`
`alternative pathway, and the recently discovered lectin or Mannose binding lectin
`
`(MBL) pathway.
`
`The activation of the classical pathway begins when the catalytic domains C Ir and C 1 s
`
`bind to immune complexes via the recognition protein Clq (see figure 11).
`
`5
`
`The alternative pathway is continuously turning over at a slow rate in an antibody(cid:173)
`
`independent manner and will attack particles that are not specifically protected agairrst
`
`complement.
`
`The lectin or MBL pathway is initiated or activated upon binding of MBL to
`
`carbohydrate structures present on various pathogens or other cellular structures. Two
`
`10
`
`serine proteases: mannan-binding lectin associated serine protease (MASP)-1 and -2
`
`(see figure 11) are associated with MBL and show striking similarities with the serine
`
`proteases Cis and Clr. The complex has C4- and C3-activating capacities upon binding
`
`to mannan. The complex contains two serine proteases MASP-1 and MASP-2 linked by
`
`a disulfide bond. In this form, MASP is capable of cleaving C4 and C3 resulting in
`
`15
`
`their activation. The CIINH of the invention preferably has a higher affinity for a
`
`human MBP as compared to its plasma derived counterpart.
`
`MBP recognizes exposed hexoses with equatorial 3- and 4-0H groups, such as
`
`mannose and N-acetylglucosamine and/or N-acetyl-hexosamines. A preferred CIINH
`
`of the invention therefore carries such terminal hexoses. The high er affinity for MBP,
`
`20
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`preferably human MBP of the C IINH of the invention preferably is such that it allows
`
`a more efficient targeting, binding and/or inhibition of MBP as compared to its natural
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`plasma derived counterpart that lacks exposed mannose and N-acetylglucosamine
`
`residues. Human MBP is herein understood to refer to the protein characterized by
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`Kawasaki et al. (1983, J. Bioehern 94:937-47), having an amino acid sequence as
`
`25
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`described by Taylor et al. (1989, Biochem. J. 262 (3), 763-771; NCBI accession no.
`
`CAA34079). The structure of rat MBP complexed with an oligosaccharide is described
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`by Weis et al. (1992, Nature. 360:127-34). Fora further description ofhuman MBP see
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`e.g US 6,846,649 and references cited therein.
`
`30 All of these pathways (classical, alternative and lectin or MBL) generate a crucial
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`enzymatic activity that eventually leads to the assembly of the membrane attack
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`complex (MAC or C5b-C9) (see Figure 11). Under physiological conditions, activation
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`of the complement system is effectively controlled by the coordinated action of soluble
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`and membrane-associated regulatory proteins. One of these proteins is C 1 inhibitor
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`(CIINH), a serine protease inhibitor that binds to Cis and Clr and currently the only
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`known physiological inhibitor of the classical pathway. In addition, CIINH is able to
`
`inactivate MBL-mediated complement activation by binding and inhibiting MASP-1
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`5
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`and MASP-2.
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`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
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`commercially available enzyme immuno assay, specific for the detection of each of the
`
`10
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`three complement pathways with deposition of C5b-C9 as a common read-out. Briefly,
`
`wells of microtitre strips are coated with specific activators of each of the three
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`complement pathways. Human serum is diluted in diluent containing specific blocker
`
`to ensure that only the respective pathway is activated. C IINH of the invention or its
`
`plasma-derived counterpart is further added in a concentration ranged between 0 and 75
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`15
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`11mol, 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,
`
`complement is activated by the specific coating. The wells are then washed and C5b(cid:173)
`
`C9 formed is detected with a specific alkaline phosphatase labelled anti C5b-C9
`
`antibody. After a further washing step, detection of specific antibodies is obtained by
`
`20
`
`incubation with alkaline phosphatase substrate solution. The amount of complement
`
`activation correlates with the colour intensity and is measured in terms of absorbance
`
`(optical density OD). Using this kit, both recombinant human CIINH (rhCIINH) ofthe
`
`invention and plasma-derived CIINH (pdCIINH) were found to have similar inhibiting
`
`capacities for the classical pathway. However, the CIINH of the inventionwas found to
`
`25
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`have approximatively 20% more inhibiting capacitiy for the MBL pathway than
`
`plasma-derived CIINH (see example 3).
`
`Therefore accordingly, in this preferred embodiment, the different glycosylation ofthe
`
`CIINH for use in the present invention results in a higher affinity for a MBP as
`
`compared to its plasma derived counterpart, which results in a more efficient inhibition
`
`30
`
`ofMBP, leading to a more efficient inhibition ofthe lectin pathway. More efficient
`
`inhibition ofthe lectin pathway preferably means 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
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`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
`
`5 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 by the Wielisa
`
`kit as described above.
`
`10
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`The method of the invention may be applied to prevent, reduce or treat any type of
`
`ischemia and reperfusion injury. Preferably, the method of the invention is applied
`
`wherein the ischemia and reperfusion injury is known to arise at least in part, more
`
`preferably mostly via the lectin pathway. For myocardial ischemia and reperfusion
`
`injury (J Immunology 2005, 175: 541-546), renal ischemia-reperfusion injury (Am J
`
`15
`
`Pathol. 2004 165(5):1677-88), gastrointestinal ischemia reperfusion injury (J Immunol.
`
`2005 15:174(10):6373-80), and for stroke (deSimoni et al, 2004 Am J. Pathol.
`
`164:1857-63) it has been shown that reperfusion injury arises mostly via the lectin
`
`pathway and hardly via the classical pathway. Hence, a CIINH of the invention
`
`preferably is a more potent inhibitor of the lectin pathway as compared to its natural
`
`20
`
`plasma derived counterpart. Preferably a C1INH of the invention is a more potent in
`
`vivo inhibitor of the lectin pathway in man as compared to its natural plasma derived
`
`counterpart.
`
`Unlike the experimental model used in the Examples herein, the occurrence of ischemia
`
`in reallife often is an unforeseen event. Therefore administration ofCIINH prior to the
`
`25
`
`occurrence of ischemia and/or subsequent reperfusion is not generally a feasible option
`
`and inevitably in practice C IINH will have to be administered some time if not several
`
`hours after ischemia and/or subsequent reperfusion. This, however, seriously limits the
`
`therapeutic usefulness of conventional plasma derived CIINH because it is mostly
`
`ineffective when administered subsequent to ischemic reperfusion and only has a very
`
`30
`
`small time window for therapeutic efficacy (see Figure 2 and deSimoni et al, 2004 Am
`
`J. Pathol. 164:1857-63). In contrast, a CIINH for use in the present invention as
`
`defined above, is still able to exert 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
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`the reperfusion. Therefore, in a preferred embodiment of the method for the prevention,
`
`reduction or treatment of at least one of unforeseen or foreseen occurrence of ischemia
`
`and reperfusion injury, the CIINH ofthe invention is administered at least at the end or
`
`after the ischemic period, i.e. when the ischemic tissue is reperfused. More preferably,
`
`5
`
`the CIINH of the invention is administered at least 10, 15, 20, 30, 45, 60, 90 or 120
`
`minutes after the ischemic period or after the start of reperfusion. Preferably, the
`
`C1INH 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 C 1 inhibitor is administered at least 3 hours after ischemia or after the
`
`10
`
`onset of ischemia and/or reperfusion, preferably at least 6 hours, more preferably at
`
`least 9 hours, even more preferably at least 18 hours.
`
`In one preferred embodiment, the method is applied to prevent, reduce or treat an
`
`unforeseen, sudden or acute occurrence of ischemic reperfusion. Conditions and
`
`15
`
`disorders associated with an unforeseen, sudden or acute occurrence of ischemic
`
`reperfusion injury include but are not limited to ischemic reperfusion injury after acute
`
`myocardial infarction (AMI), after stroke, including perinatal stroke, after hemorrhagic
`
`shock, after
`
`intestinal
`
`ischemia, after emergency coronary surgery for failed
`
`percutaneous transluminal coronary angioplasty (PCTA), after any vascular surgery
`
`20 with blood vessel cross clamping ( e.g. of aorta, leading to skeletal muscle ischemia), or
`
`after pancreatitis after manipulation of pancreatic or bile duct (ERCP). In such
`
`instances the CIINH ofthe invention preferably is administered at least 1, 5, 10, 15, 20,
`
`30, 45, 60, 90 or 120 minutes after the acute myocardial infarction (AMI), after stroke,
`
`including perinatal stroke, after hemorrhagic shock, after