WO2014008242A1 - Utilisation de l'interleukine-11 pour la protection contre l'ischémie et une lésion de reperfusion - Google Patents

Utilisation de l'interleukine-11 pour la protection contre l'ischémie et une lésion de reperfusion Download PDF

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WO2014008242A1
WO2014008242A1 PCT/US2013/049039 US2013049039W WO2014008242A1 WO 2014008242 A1 WO2014008242 A1 WO 2014008242A1 US 2013049039 W US2013049039 W US 2013049039W WO 2014008242 A1 WO2014008242 A1 WO 2014008242A1
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administered
injury
renal
ischemia
surgical procedure
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H. Thomas LEE
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The Trustees Of Columbia University In The City Of New York
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2073—IL-11
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Definitions

  • This invention is in the field of treating and preventing injury to organs caused by ischemia followed by reperfusion by the administration of interleukin-11.
  • Ischemia-reperfusion injury is caused by the return of blood supply to a tissue (reperfusion) after a period of lack of oxygen (ischemia). During ischemia, there is an absence of oxygen and nutrients normally supplied by the blood. This creates a condition where the return of circulation results in inflammation and oxidative damage, rather than the expected restoration of normal function.
  • ischemia or hypoxia with subsequent reperfusion injury is a major clinical problem affecting virtually every organ in the body.
  • post-operative organ dysfunction due to ischemia-reperfusion injury is a severe threat affecting almost all patients undergoing major surgical procedures including cardiac surgery, liver transplantation, liver resection, renal transplantation, lung transplantation, aortic surgery, and major vascular repair.
  • injury of one organ due to ischemia or hypoxia with reperfusion is frequently associated with remote organ injury affecting distant organs.
  • patients who develop kidney injury due to renal ischemia and reperfusion frequently develop liver, intestine, and lung dysfunction as well as sepsis leading to extraordinarily high mortality (25-80%).
  • IR injuiy can also result in patients requiring dialysis.
  • Renal ischemia-reperfusion injuiy is especially common, and a frequent cause of acute kidney injury (“AKI”) (Ikeda et al. (2006)).
  • Ischemic AKI is a clinical problem for patients subjected to major surgical procedures not only involving the kidney, but the liver, heart or aorta as well, and often leads to multi-organ dysfunction and systemic inflammation with extremely high mortality (Jones and Lee (2008)).
  • the incidence of renal dysfunction after major surgery in high risk patients has been reported to be about 80% (Hoste and Kellum (2007)).
  • the severity and incidence of AKI has been increasing without any improvements in therapy or patient survival over the past 50 years (Jo et al. (2007)).
  • therapies to treat and prevent injury due to I .
  • Drugs that can prevent or treat IR injury to the kidney and other organs can potentially reduce mortality rates, organ dysfunction, and the need for dialysis in a variety of clinical settings.
  • Interleukin -11 (“IL-l l”) is a 20 kDa multifunctional member of the IL-6-type cytokine family and is a key regulator of megakaryocyte maturation (Goldman et al. (2001)). In addition to its hematopoietic properties, recent studies suggest cytoprotective roles for IL- 11 (Kuenzler et al. (2002)). Specifically, IL-l l administration protects against intestinal, cardiomyocyte and endothelial cell death by producing significant anti-necrotic and anti- apoptotic effects in these cell types (Du and Williams (1997)). IL-l l also attenuates the inflammatory responses in a murine model of LPS -induced sepsis (Trepicchio and Dorner (1998); Trepicchio et al. (1997)).
  • IL-l l Exogenous administration of native or human recombinant (“HR”) IL-l l may be limited by its relatively short half-life of about seven hours due to rapid clearance through urinary excretion, hepatic metabolism and enzymatic degradation (Du and Williams (1997)); Grosfeld et al. (1999)).
  • HR native or human recombinant
  • Chemical modification of IL-l l by conjugation to polyethylene glycol (“PEG”) reduces IL-l l glomerular filtration and hepatic uptake, therefore prolonging the half-life of IL-l l (Takagi et al. (2007)).
  • the present invention is based upon the surprising discovery that the administration of interleukin- 11 (IL-l l) or an IL-l l analog, prior to ischemia/reperfusion injury, mitigates the effects of the injury and protects the organs. Even more surprisingly, IL-11 and IL-11 analog administration after ischemia/reperfusion injury also mitigates the effect of the injury and protects the organs. The latter finding is important in the clinical setting where IR injury cannot always be predicted prior to its occurrence, e.g., before a surgical procedure that may cause an IR injury or before a stroke.
  • one embodiment of the present invention is a method of preventing or treating an injury to an organ caused by ischemia, followed by reperfusion, comprising the administration of a therapeutically effective amount of IL-11 or an IL-11 analog, to a subject in need thereof.
  • Such an injury can be caused by surgery, most usually surgery involving major organs, including but not limited to, the kidney, liver, heart, lung, intestine, and aorta and would include, but is not limited to, coronary bypass, major vascular repair, liver resection, and transplantation of the kidney, liver, and lung.
  • I injury can have other causes including, but not limited to, cardiopulmonary bypass during surgery, stroke, liver ischemia, kidney ischemia, aortic occlusion, myocardial occlusion, cardiac arrest, shock and trauma.
  • the IL-11 or IL-11 analog is administered prior to the occurrence of the ischemia which causes the injury, which in this embodiment is most likely during surgery.
  • the IL-11 protein or IL-11 analog is administered within about one (1) week or less prior to the ischemia or surgery, more preferably within about five (5) days or less prior to the ischemia or surgery, more preferably within about one (1) day or less prior to the ischemia or surgery, more preferably within about six (6) hours or less prior to the ischemia or surgery, more preferably within about sixty (60) minutes or less prior to the ischemia or surgery, more preferably within about thirty (30) minutes or less prior to the ischemia or surgery, and more preferably, within about ten (10) to about fifteen (15) minutes or less prior to ischemia or surgery.
  • the IL-11 or IL-11 analog is administered after surgery or after another incident or event has occurred where IR injury would be suspected, including but not limited to, cardiopulmonary bypass during surgery, stroke, liver ischemia, kidney ischemia, aortic occlusion, myocardial occlusion, cardiac arrest, shock and trauma.
  • the IL-11 protein or IL-11 analog is administered preferably within about one (1) week or less following the surgery or IR injury event, more preferably within about five (5) days or less following surgery or the IR injury incident, more preferably within about one (1) day or less following surgery or the IR injury incident, more preferably within about six (6) hours or less following surgery or the IR injury incident, more preferably within about sixty (60) minutes or less following surgery or the IR injury event, and most preferably within about thirty (30) minutes or less following surgery or the IR injury incident.
  • the IL-11 or IL-11 analog is administered following reperfusion of the ischemic organ.
  • the IL-11 protein or IL-11 analog is administered preferably within about one (1) week or less following reperfusion of the ischemic organ, more preferably within about five (5) days or less following reperfusion of the ischemic organ, more preferably within about one (1) day or less following reperfusion of the ischemic organ, more preferably within about six (6) hours or less following reperfusion of the ischemic organ, more preferably within about sixty (60) minutes or less following reperfusion of the ischemic organ, and most preferably within about thirty (30) minutes or less following reperfusion of the ischemic organ.
  • the IL-11 or IL-11 analog is administered at the start of reperfusion of the ischemic organ.
  • the IL-11 or IL-11 analog is administered during reperfusion of the ischemic organ.
  • the IL-11 or IL-11 analog is administered during surgery to prevent or reduce the severity of acute kidney injury or ischemia-induced injury to other organs or tissues.
  • the IL- 11 for use in the method of the invention can include, but is not limited to, native or wild-type IL-1 1, recombinant IL-11, and a variant of native or recombinant IL-11, wherein the variant of IL-1 1 differs in nucleotide sequence or amino acid sequence or both, from native or recombinant IL-11, but retains its ability to protect organs from injury caused IR.
  • the IL-11 for use in the method of the invention also can include an IL-11 analog that retains its ability to protect organs from injury caused by IR.
  • the IL-11 analog can include, but is not limited to, any form of IL-11 listed herein that is modified by polyethylene glycol, or any other long-acting IL-11 analog.
  • Long-acting IL-11 analogs that can be used in the method of the invention include, but are not limited to, the long-acting, PEGylated IL-11 analogs described herein, depot formulations of the IL-11 proteins, fusion proteins that comprise an IL-11 protein, chemically-modified IL-11 proteins, and polymer-modified IL-11 proteins.
  • Polymer-modified IL-11 proteins can be prepared by attaching one or more polymers to a variety of amino acids, such as cysteine, lysines, N-terminal amino acids containing one or more amine groups, and non-natural amino acids introduced into the protein or added to the amino terminus or carboxy-terminus of the protein.
  • amino acids such as cysteine, lysines, N-terminal amino acids containing one or more amine groups, and non-natural amino acids introduced into the protein or added to the amino terminus or carboxy-terminus of the protein.
  • One example of a polymer-modified IL-11 is described in Takagi et al. (2007).
  • the long-acting IL-11 analogs also may contain sugar, starch, fatty acids or lipid groups attached to the IL-11 protein.
  • the polymer, sugar, starch, fatty acid or lipid used for modifying the IL-11 protein can be any polymer, sugar, starch, fatty acid or lipid that confers a half-life that is longer than the half-life of the non- modified IL-11 protein in animals.
  • Examples of methods for incorporating non-natural amino acids into proteins and subsequently modifying the proteins with polymers are described in U.S. Patent No. 7,332,571, U.S. Patent No. 7,385,028, U.S. Patent No. 7,230,068, U.S. Patent No. 8,263,740, U.S. Patent No. 6,586,207, U.S. Patent No. 7,139,665, U.S. Patent No. 7,632,492, and U.S. Patent No.
  • the long-acting IL-11 analog also may be an IL-11 fusion protein.
  • a long-acting IL-11 fusion protein is a fusion protein comprising IL-11 fused to an immunoglobulin domain (U.S. Patent No. 7,754,855).
  • Other long-acting fusion proteins known in the art have been prepared by fusing a protein of interest to albumin, transferrin, transferrin receptors, elastin and elastin-like proteins, XTEN sequences, chorionic gonadotrophin carboxy terminal peptides or by adding non-native amino acids to the N- terminus or C-terminus of a protein. Examples of methods to create long-acting fusion proteins are described in U.S.
  • the fusion protein used to practice the invention can be any IL-11 fusion protein that confers a half- life that is longer than the half-life of the non- fused IL-11 protein in animals.
  • Peptides that bind and activate cellular receptors for IL-11 can also be used in the method of the present invention, and can be prepared using methods known in the art, such as by using phage display methods, that have been used to prepare peptides that bind and activate other proteins and cellular receptors. Examples of methods for selecting and using peptides that bind other proteins and cellular receptors are described in U.S. Patent No. 5,773,569, U.S. Patent No. 5,986,047, U.S. Patent No. 6,660,843, U.S. Patent No. 7,166,707, U.S. Patent No. 7,169,905, U.S. Patent No. 7,186,810, U.S. Patent No. 7,189,827, and U.S. Patent No. 7,488,590. Other protein agonists, including antibodies and monoclonal antibodies, that bind and activate the IL-11 receptor (Putoczki and Ernst (2010) are also contemplated by the present invention.
  • the IL-11 protein for use in the method of the invention is human IL-11 (Paul et al. (1990)). Human IL-11 is synthesized as a 199 amino acid precursor protein containing an N-terminal 21 amino acid signal sequence, which is cleaved from the mature 178 amino acid protein. The mature IL-11 protein begins with proline at amino acid position 22.
  • the IL-11 protein for use in the method of the invention is a recombinant human desPro IL-11 (IL-11 missing the N-terminal proline residue present in the native protein).
  • the IL-11 protein for use in the method of the invention is a human IL-11 protein that is modified with a chemical moiety including, but not limited to, a polymer, a sugar, a starch, a fatty acid or a lipid.
  • the IL-11 protein for use in the method of the invention is human IL-11 and the protein is modified with polyethylene glycol or PEG.
  • the IL-11 protein for use in the method of the invention comprises a cysteine variant of IL-11, such as any of the IL-11 cysteine variants described in PCT/US98/14497, U.S. Patent No. 7,253,267, U.S. Patent No. 7,495,087, and U.S. Patent No.
  • IL-11 cysteine variants compromising one or more added cysteine residues, wherein the added cysteine residue(s) has been substituted for one of more amino acids in the LL-11 protein sequence, or wherein the added cysteine residue(s) has been added preceding the first amino acid of the mature IL-11 protein or following the last amino acid of the IL-11 protein.
  • the added cysteine residue(s) in the IL-11 cysteine variants may be modified with one or more polymers such as PEG.
  • the IL-11 protein for use in the method of the invention is a human IL-11 variant wherein a cysteine residue is added following the last amino acid of the IL-11 protein.
  • the IL-11 protein for use in the method of the invention comprises an IL-11 variant wherein a cysteine residue is added following the last amino acid of the protein and wherein the protein is further modified with one or more PEGs.
  • the IL-11 cysteine variant is modified with a 40 kDa-PEG at the added cysteine residue.
  • the PEG used to modify the IL-11 protein can be any PEG known in the art, including, but not limited to, a linear PEG and a branched PEG. PEGs are available in different sizes ranging from about 2 kDa to about 80 kDa, and all can be used to create IL-11 analogs for use in practicing the method of the invention.
  • Therapeutically effective doses of the IL-11 proteins for use in the method of the invention range from about 1 nanogram of IL-11 per kilogram of body mass of the subject, to about 10 milligrams of IL-11 per kilogram of body mass, more preferably from about 1 microgram of IL-11 per kilogram of body mass, to about 1 milligram of IL-11 per kilogram of body mass, and even more preferably from about 10 micrograms of IL-11 per kilogram body mass to about 100 micrograms of EL- 11 per kilogram of body mass.
  • All major organs can be protected from IR injury by the administration of IL-11 and would include, but are not limited to, the kidneys, liver, lungs, intestines, heart, and blood vessels.
  • the subject is a mammal and in the most preferred embodiment, the mammal is a human.
  • Figure 1 shows representative photomicrographs for hematoxylin and eosin staining ( Figure 1A, magnification 200X), TIJNEL staining ( Figure 1 , representing apoptotic nuclei, magnification 200X) and immunohistochemistry for neutrophil infiltration (Figure ID, magnification 400X) of kidney sections of mice.
  • Figure ID the black arrows denote neutrophils.
  • Photographs are representative of 3-5 independent experiments.
  • Figure 1C is a graph showing the number of apoptotic cells per 200X field in the kidneys of mice after renal IR (30 minutes of ischemia and 24 hours of reperfusion).
  • Figure IE is a graph showing the number of infiltrated neutrophils per 400X field in the kidneys of mice after renal IR (30 minutes of ischemia and 24 hours of reperfusion). *P ⁇ 0.05 vs. vehicle-treated mice subjected to renal IR. Error bars represent 1 SEM.
  • PARP poly(adenosine diphosphate-ribose) polymerase
  • Figure 4 are graphs showing the lactate dehydrogenase (LDH) released after H 2 0 2 - induced necrosis in IEC-6 cells (expressed as a percentage of total LDH released).
  • Figure 4A show the results of cells pretreated with WT HR IL-11 and then treated with ImM H 2 0 2.
  • Figure 4B shows the results of cells pretreated with WT HR IL-11 or PEGylated IL-11 and then treated with 500 ⁇ H 2 0 2 .
  • LDH lactate dehydrogenase
  • interleukin-11 and "IL-11” will be used interchangeably and would include native, or wild-type IL-11 isolated directly from mammalian cells and tissues by any method known in the art, such as affinity column purification, IL-11 synthesized via chemical means or by recombinant DNA technology, by any method loiown in the art, or IL-11 made by any as yet unknown method.
  • WT HR IL- 11 or "WT IL11” is used in some examples and figures this denotes human recombinant IL- 11 that has no variations in the amino acid sequence from native IL-11, except for the absence of the N-terminal proline residue present in native human IL-11.
  • IL-11 lacking the N-terminal proline residue is referred to as desPro IL-11.
  • IL-11 variant or “variant” is a protein with variations in either DNA sequence or amino acid sequence or both, from native IL-11, that does not affect the ability of the IL-11 to protect organs from injury due to ischemia-reperfusion.
  • One such variation is an IL-11 protein with a cysteine residue added following the last amino acid of the native or recombinant protein.
  • Another such amino acid variant is desPro IL-11.
  • IL-11 analog refers to an IL-11 protein that contains any other chemical modification that causes the protein to be chemically or structurally different from native IL-11, but does not affect the ability of the IL-11 to protect organs from injury due to ischemia-reperfusion.
  • chemical modifications include, but are not limited to, modifications of the IL-11 protein with one or more polymers, sugar, starches, fatty acids, or lipids, modifications of the IL-11 protein with any chemical compound, and modification of the IL-11 protein by addition or deletion of amino acids.
  • PEGylated interleukin-11 PEGylated IL-11
  • PEG IL-11 PEGylated IL-11
  • PEG IL-11 PEG IL-11
  • injury would refer to tissue or organ damage, and includes any alteration in tissue or organ structure, cell viability or function.
  • the terms “injury from ischemia followed by reperfusion”, “injury from IR”, “IR injury” and the like would refer to the tissue damage caused when blood supply returns to a tissue after a period of lack of oxygen.
  • the terms “prevent”, “prevention”, and the like refer to acting prior to the tissue and/or organ damage to minimize the damage, to prevent the damage from developing or to minimize the extent of the damage or to slow its course of development.
  • treat refers to a means to slow down, relieve, ameliorate, or alleviate the damage or injury to the tissues and/or organs or reverse the damage after its onset.
  • protection refers to a means to ameliorate the damage from the injury or stop the injury to the organ and/or tissue from occurring.
  • subject as used in this application means an animal with an immune system such as avians and mammals. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates. Avians include, but are not limited to, fowls, songbirds, and raptors.
  • the invention can be used in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications
  • the term "in need thereof would be a subject known or suspected of having an injury caused by ischemia and reperfusion.
  • a subject in need thereof may have had or will be having surgery, including but not limited to, surgery involving the heart, lungs, liver, kidneys, intestines, or aorta.
  • the subject in need thereof may have suffered a stroke, liver ischemia, kidney ischemia, aortic occlusion, myocardial occlusion, cardiac arrest, shock or trauma, or any other medical condition that results in ischemia to one or more tissues or organs.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in the subject, or delays or minimizes or mitigates one or more symptoms associated with the injury, or results in a desired beneficial change of physiology in the subject.
  • Interleukin-11 fIL-11 Interleukin-11 fIL-11
  • Interleukin-1 1 or IL-11 a member of the IL-6-type cytokine family, was first identified from bone marrow-derived stromal cells. It is a key regulator of hematopoiesis and promotes megakaryocyte maturation (Reynolds (2000)). IL-11, as well as its receptors, is expressed in many tissues and cell types (Du and Williams (1997)). In addition to its hematopoietic properties, recent studies suggest a cytoprotective role for IL-11 (Du and Williams (1997)). In several organs including the heart, intestine and endothelial cells, IL-1 1 administration has been shown to attenuate necrotic as well as apoptotic cell death (Du and Williams (1997)).
  • IL-11 administration reduces inflammatory responses in lipopolysaccharide-treated mice (Sheridan et al (1999); Trepicchio et al. (1996)), macrophage inflammation (Trepicchio et al. (1997)), nephrotoxic nephritis (Lai et al. (2001)), and T-cell mediated liver injury (Bozza et al. (1999)).
  • IL-11 treatment following coronary occlusion partially reduces cardiac fibrosis, cardiac cell death and cardiac remodeling (Kimura et al (2007); Obana et al (2010); Fujio et a/ (2011))
  • Oprelvekin is produced in Escherichia coli (E. coli) by recombinant DNA technology.
  • E. coli Escherichia coli
  • the protein has a molecular mass of approximately 19,000 daltons, and is non-glycosylated.
  • the polypeptide is 177 amino acids in length and differs from the 178 amino acid length of native IL-11 only in the lack of the amino-terminal proline residue (referred to as desPro IL-11). This alteration has not resulted in measurable differences in bioactivity either in vitro or in vivo.
  • Both native or wild-type, and recombinant IL-11 can be used in the current invention to protect kidneys and other organs from IR injury.
  • the recombinant IL-11 can be human.
  • PEGylation may increase the half-life of IL-11, it may also result in substantial decrease in biological activity or potency due to its steric hindrance (Takagi et al. (2007)).
  • the most commonly employed method for PEGylating proteins uses compounds that attach PEG to free amines, typically at lysine residues or at the N-terminal amino acid.
  • a critical limitation of this approach is that proteins typically contain several lysines, in addition to the N-terminal amino acid.
  • the PEG moiety can attach to the protein at any of the available free amines, resulting in a heterogeneous product mixture consisting of mono-, di-, tri-, etc, PEGylated species modified at different lysine residues and the N-terminus.
  • the different PEGylated species often possess different intrinsic biological activities. This can present problems when developing a PEGylated protein therapeutic because predictability of biological activity and manufacturing reproducibility are crucial for regulatory approval.
  • amine-PEGylated proteins are unsuitable for commercial use because of low specific activities. Biological activities of amine-PEGylated proteins often are reduced 10-100-fold relative to the non-modified protein. Inactivation results from covalent modification of one or more amino acids required for biological activity or from covalent attachment of the PEG moiety near the active site or ligand binding site of the protein.
  • Site specific PEGylation allows a protein to be selectively modified with PEG at a unique predetermined site.
  • PEGylated proteins that are homogenously modified and have no significantly loss of biological activity.
  • a novel, long acting IL- 11 analog that has undergone cysteine residue specific chemical modification of the protein with PEG was produced (Example 1A) that has increased potency and half- life of IL-11 (by more than 10 fold to approximately 300-500 minutes in rats).
  • Acute kidney injury is a frequent and disastrous clinical complication with high mortality, morbidity and cost (Faubel (2009); Jones and Lee (2008)).
  • Renal IR injury is a major cause of AKI for patients subjected to surgical procedures involving the kidney, heart, liver or aorta.
  • renal tubular necrosis, apoptosis and inflammation during and after renal IR contribute significantly to the pathogenesis of ischemic AKI (Bonventre and Weinberg (2003)).
  • the basis of this invention is the unexpected discovery that recombinant human TL- 11, as well as a novel PEGylated IL-11, attenuates renal tubular cell death in vivo, as well as in vitro, by reducing necrosis, apoptosis and inflammation, and that IL-11 has protective effects against renal IR injury in mice and humans.
  • IL-11 attenuated renal tubular necrosis (Jablonski renal injury score) as well as apoptosis (TUNEL staining) (Example 3; Figures 1A and 1C).
  • IL-11 treatment reduced the influx of pro-inflammatory neutrophils after renal IR in mouse kidneys (Example 3: Figure ID).
  • IL-11 In human kidney cells, IL-11 also reduced necrosis (LDH release) as well as apoptosis (PARP/caspase 3 fragmentation) (Example 4; Figure 2). Therefore, it was concluded that exogenous administration of IL-11 provides powerful renal protection against ischemic AKI by targeting all three pathways of cell death: necrosis, apoptosis and inflammation.
  • Post-ischemic therapy for AKI is a significant clinical advance as not all ischemic AKI can be anticipated prior to surgical procedures or even arise from surgical procedures, but can arise from unexpected events, such as a stroke, aortic occlusion, myocardial occlusion, cardiac arrest, shock or trauma.
  • IL-11 After IL-11 binds to the IL-11 receptor, the ligand-receptor complex interacts with a common receptor subunit, glycoprotein 130 (gpl30), leading to gpl30-associated kinase- mediated tyrosine phosphorylation (Fujio et al. (2011)).
  • gpl30 glycoprotein 130
  • the cytoprotective mechanisms of IL-11 leading to reductions in necrosis, inflammation as well as apoptosis have been investigated in other cell types.
  • IL-11 reduces injury and fibrosis by Janus Kinase - Signal Transducer and Activator of Transducer 3 (JAK-STAT3) pathway activation (Fujio et al. (2011); Kimura et al.
  • IL-11 protects against oxidant induced necrosis and apoptosis via mechanisms involving ERK MAPK, Akt and/or induction of HSP25 (Naugler et al. (2008); Ropeleski et al. (2003); Waxman et al. (2003)).
  • IL-11 produces renal protection by direct induction of sphingosine kinase-1 (SKI) via nuclear translocation of hypoxia inducible factor la (THF-la).
  • SKI sphingosine kinase-1
  • THF-la hypoxia inducible factor la
  • SK is a multifunctional lipid kinase that phosphorylates sphingosine to form sphingosine 1 -phosphate (SIP).
  • SKI is a cytosolic enzyme that migrates to the plasma membrane or to the nucleus upon activation (Hait et al. (2006); Leclercq et al. (2006)).
  • SKI is a well-known mediator of tissue protection (including protection against TR injury), growth and survival (Liu et al. (2002)). Overexpression of SKI is protective in acute lung injury (Wadgaonkar et al. (2009)).
  • SKI activation protects against cardiomyocyte death and SKI -deficient cardiomyocytes had increased injury after ischemia (Vessey et al. (2005)). It has also been previously demonstrated a renal protective role of SKI as well as SlPi receptor activation (Bonventre and Weinberg (2003); Jo et al. (2009); Kim et al. (2007); Kim et al. (2010)). Overall, activation of SKI produces anti-necrotic, anti-inflammatory and anti-apoptotic effects in several organs and cell types.
  • IL-11 treatment not only induces SKI (Example 5) but that the renal protective effects of IL-11 also are dependent on SKI, as mice deficient in SKI enzyme were not protected against renal IR injury with either HR IL-11 or PEGylated IL-11 treatment (Example 6), Again while not being bound by any theories, it appears that IL-11-mediated SKI induction enhances the synthesis of endogenous SIP in the kidney.
  • SIP is a potent lipid signaling molecule that can activate 5 SIP receptors (S1PR) to regulate cell growth, cell survival and modulation of inflammation (Allende et al. (2004)(1); Chae et al. (2004); Venkataraman et al. (2006)).
  • SIPiR activation in particular has been shown to produce tissue protection by attenuating T-lymphocyte-mediated inflammation. Activation of the SIPiRs on endothelial cells also reduces vascular permeability, hence better preserving the integrity of the vascular endothelial cell barrier function (Allende et al. (2003)). Furthermore, direct renal tubular protective effects of SIPiR activation are mediated by activation of the Akt and ERK pathways (Bajwa et al. (2010)). Therefore, both SK1->S1P]R as well as IL-l l-MLl lR-->gpl30 pathways can activate cytoprotective ERK and Akt signal transduction.
  • HJF-1 is a heterodimeric transcription factor composed of an a and a ⁇ subunit (Rosenberger et al. (2008); Schodel et al. (2009)). Under normoxic conditions, prolyl-hydroxylation and ubiquitination of the oxygen-dependent degradation domain of HIF- ⁇ results in rapid FffF-la degradation. With hypoxia or ischemia, HJF-1 a stabilizes and interacts with FHF- ⁇ forming the EQ -l heterodimer. Nuclear HIF-1 translocation allows binding to the hypoxia-responsive element with subsequent induction of several cytoprotective genes. Consistent with this proposed pathway, previous studies have demonstrated that HIF-l activation protects against renal IR injury (Schodel et al. (2009)).
  • IL-11 also has a protective effect in the liver which has been injured by ischemia/ reperfusion (Example 8) and decreases necrosis in intestinal epithelial cells (Example 9).
  • IL-11 whether native, recombinant or a variant, whether or not an IL-11 analog, or whether or not modified by PEG, protects organs from IR injury.
  • IR injury most often is a result of surgery involving major organs, such as the kidney, liver, lungs, heart, aorta or intestines.
  • major organs such as the kidney, liver, lungs, heart, aorta or intestines.
  • the present invention encompasses the administration of IL-11 and IL-11 variants, and IL-11 analogs, including PEGylated IL-11 variants and analogs.
  • Preferred methods of administration include oral; mucosal, such as nasal, sublingual, vaginal, buccal, or rectal; parenteral, such as subcutaneous, intravenous, intraperitoneal, intradermal, bolus injection, intramuscular, or intraarterial; or continuous infusion; or transdermal administration to a subject.
  • the IL-1 lmust be in the appropriate form for administration of choice.
  • compositions for administration may comprise a therapeutically effective amount of the IL-11, IL-11 variants or IL-11 analogs, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human, and approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • Carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • a saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, bulking agents such as trehalose, mannitol, sorbitol, and sucrose, amino acids such as glycine, histidine, methionine, and arginine, and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • Detergents commonly used in pharmaceutical compositions include polysorbate-20, polysorbate-80, pluronic acid F68, sodium dodecyl sulfate, and sodium lauryl sulfate. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, cachets, troches, lozenges, dispersions, suppositories, ointments, cataplasms (poultices), pastes, powders, dressings, creams, plasters, patches, aerosols, gels, liquid dosage forms suitable for parenteral administration to a patient, and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.
  • Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable form of carrier so as to provide the form for proper administration to the patient.
  • compositions adapted for oral administration may be capsules, tablets, powders, granules, solutions, syrups, suspensions (in non-aqueous or aqueous liquids), or emulsions.
  • Tablets or hard gelatin capsules may comprise lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof.
  • Soft gelatin capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid polyols. Solutions and syrups may comprise water, polyols, and sugars.
  • An active agent intended for oral administration may be coated with or admixed with a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract. Thus, the sustained release may be achieved over many hours and if necessary, the active agent can be protected from degradation within the stomach.
  • Pharmaceutical compositions for oral administration may be formulated to facilitate release of an active agent at a particular gastrointestinal location due to specific pH or
  • compositions adapted for transdermal administration may be provided as discrete patches intended to remain in intimate contact with the epidermis of the recipient over a prolonged period of time.
  • compositions adapted for nasal and pulmonary administration may comprise solid carriers, such as powders, which can be administered by rapid inhalation through the nose.
  • Compositions for nasal administration may comprise liquid carriers, such as sprays or drops.
  • inhalation directly through into the lungs may be accomplished by inhalation deeply or installation through a mouthpiece.
  • These compositions may comprise aqueous or oil solutions of the active ingredient.
  • Compositions for inhalation may be supplied in specially adapted devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which can be constructed so as to provide predetermined dosages of the active ingredient.
  • Pharmaceutical compositions adapted for rectal administration may be provided as suppositories or enemas.
  • Pharmaceutical compositions adapted for vaginal administration may be provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the compositions substantially isotonic with the blood of the subject.
  • Other components which may be present in such compositions include water, alcohols, polyols, glycerine, and vegetable oils.
  • Compositions adapted for parental administration may be presented in unit-dose or multi-dose containers, such as sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile carrier, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include: Water for Injection USP; aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • a therapeutically effective dose will be determined by the skilled artisan considering several factors which will be known to one of ordinary skill in the art. Such factors include the particular form of the IL-11 protein, and its pharmacokinetic parameters such as bioavailability, metabolism, and half-life, which will have been established during the usual development procedures typically employed in obtaining regulatory approval for a pharmaceutical compound. Further factors in considering the dose include the condition or disease to be treated or the benefit to be achieved in a normal individual, the body mass of the patient, the route of administration, whether the administration is acute or chronic, concomitant medications, and other factors well known to affect the efficacy of administered pharmaceutical agents. Thus, the precise dose should be decided according to the judgment of the person of skill in the art, and each patient's circumstances, and according to standard clinical techniques.
  • IL-11 Human recombinant IL-11 (HR IL-11) and cysteine-residue specific PEG-conjugated (PEGylated IL-11) were synthesized at Bolder BioTechnology, Inc., Boulder, CO.
  • IL-11 and IL-11 (* 179C) proteins were expressed as fusion proteins in E. coli strain ER2566 using the pTYB 11 expression plasmid (New England Biolabs, Beverly, MA) essentially as described in U.S. Patent Nos. 7,495,087 and 8,133,480.
  • IL-1 1 (* 179C) is an IL-1 1 analog containing a cysteine residue added following the last amino acid of the native protein.
  • the expressed fusion protein comprises an N-terminal chitin binding domain joined to a yeast intein sequence followed by desProIL-11 or desProIL-11 (* 179C).
  • E. coli expression of the fusion protein was induced by addition of isopropyl-p-D-thiogalactopyranoside to the cultures. Following induction, the induced cells were lysed and the fusion protein captured on a chitin affinity column (New England Biolabs). The chitin column was washed with buffer containing 50 mM dithiothreitol to activate the intein domain, which cleaves IL-11 from the fusion protein.
  • the cleaved IL-11 proteins were eluted from the column and purified by S- Sepharose column chromatography.
  • the purified IL-11 (* 179C) protein was modified with a branched 40 kDa maleimide-PEG obtained from Nippon Oil and Fat Corporation (Irvine, CA) and the PEGylated protein purified from unreacted protein and unreacted PEG by S- Sepharose column chromatography.
  • RP-HPLC and SDS-PAGE analyses both proteins were approximately 95% pure.
  • HK-2 cells Cell culture Human kidney proximal tubule cells (HK-2 cells) (ATCC, Manassas, VA) were used for assessments at the cellular level.
  • Rat intestinal epithelial cells (IEC-2 cells) (ATCC, Manassas, VA) were also used for assessments at the cellular level.
  • the data were analyzed with Student's /-test when comparing means between two groups or one-way ANOVA plus Tukey's post hoc multiple comparison test when comparing multiple groups.
  • Two-way ANOVA plus Bonferroni posttest was used to test the effects of sham operation or renal IR injury on different mouse strains or treatment groups.
  • the ordinal values of the renal injury scores were analyzed by the Mann-Whitney nonparametric test. In all cases, a probability statistic ⁇ 0.05 was taken to indicate significance. All data are expressed throughout the text as means ⁇ SEM.
  • mice with renal IR were administered with the IL-11 prior to, and after renal injury, and tests for renal function performed.
  • Example IB Mice as described in Example IB were pretreated with saline (vehicle for HR IL-11), PEG (vehicle for PEGylated IL-11), HR IL-11 (0.1-1 mg/kg, i.p.) or long acting PEGylated IL-11 (0.1-1 mg/kg, i.p.) (both as described in Example 1A), 10 minutes prior to renal ischemia or sham-operation.
  • saline vehicle for HR IL-11
  • PEG vehicle for PEGylated IL-11
  • HR IL-11 0.1-1 mg/kg, i.p.
  • long acting PEGylated IL-11 0.1-1 mg/kg, i.p.
  • mice as described in Example IB were treated with saline, PEG, HR IL-11 (1 mg/kg, i.p.) or PEGylated LL-11 (1 mg/kg, i.p) (both as described in Example 1A) 30 minutes or 60 minutes after reperfusion of the ischemic kidney.
  • Plasma samples were collected 24 hours after IR injury to examine the severity of renal dysfunction by measurements of plasma creatinine, measured with an enzymatic creatinine reagent kit according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA) (Slot (1965)). Unlike the Jaffe method, this method of creatinine measurement largely eliminates the interferences from mouse plasma chromagens.
  • Elevated plasma creatinine values are indicative of renal dysfunction.
  • the kidney is the primary organ for filtering creatinine out of the blood and into the urine (via kidney glomerular filtration and proximal tubular secretion).
  • Levels of creatinine in blood and urine can be used to calculate creatinine clearance.
  • Plasma creatinine levels are directly related to the kidney glomerular filtration rate and are a commonly used indicator of kidney function. An increase in plasma creatinine levels indicates that kidney glomerular filtration is impaired, i.e., lower than normal glomerular filtration rate, and that kidney damage has occurred. When the glomerular filtration rate becomes too low, patients require dialysis.
  • Pretreatment with HR IL-11 or PEGylated IL-l l (10 minutes before renal ischemia, 0.1-lmg/kg, N 5-6 per group) partially but significantly attenuated the increases in plasma creatinine in mice after renal IR (Table 1).
  • IL-11 treatment after renal reperfusion also protected against renal IR injury.
  • Pre-or post-ischemic treatment with either HR IL-11 or PEGylated IL-11 significantly attenuated the increases in plasma creatinine after renal IR, indicating that the proteins protect the kidney from IR injury.
  • Lower plasma creatinine levels in the IL-11 and PEGylated IL-11 treated mice indicate that their kidney glomerular filtration rates are higher than those of vehicle-treated mice, and closer to the glomerular filtration rates in normal (untreated) mice.
  • Example 3- Further Evidence of the Renal Protective Effect of IL-11 Administration In order to further test whether HR IL-11 or PEGylated IL-1 1 administration before and after renal IR injury protects against injury, mice with renal IR were administered the IL- 1 1 prior to and after renal injury, and histological analysis was performed.
  • Kidney cortex and cortico-medullary junction samples were collected after IR injury to examine the severity of renal dysfunction (renal tubular necrosis, apoptosis and neutrophil infiltration).
  • H&E hematoxylin and eosin
  • Apoptosis after renal IR with TU EL staining was detected as described in Park et al. (2010) using a commercially available in situ cell death detection kit (Roche, Indianapolis, IN) according to the instructions provided by the manufacturer.
  • Kidney inflammation after renal IR was assessed by detection of neutrophil infiltration using immunohistochemistry 24 hours after IR as described in Park et al. (2010).
  • Neutrophils infiltrating the kidney were quantified in 5-7 randomly chosen 400X microscope images fields in the corticomedullary junction and results were expressed as neutrophils counted per 400X field.
  • Apoptotic TUNEL positive cells were quantified in 5-7 randomly chosen 200X microscope images fields in the corticomedullary junction and results were expressed as neutrophils counted per 200X field.
  • Figure 1A shows the severe necrotic renal injury in saline- or PEG-treated mice subjected to IR, 24 hours after injury.
  • the kidneys of vehicle-treated (saline or PEG) mice subjected to renal IR showed significant tubular necrosis, and proteinaceous casts with increased congestion (Figure 1A).
  • Table 3 - IL-11 and PEG IL-11 reduce Jablonski scale renal injury scores for subjected to renal IR
  • Renal ischemia and 24 hour of reperfusion resulted in severe apoptosis in the kidneys of saline- or PEG-treated mice.
  • the TU EL staining detected apoptotic renal cells in kidney of mice subjected to renal IR with predominant proximal tubule cell apoptosis (Figure IB, magnification 200X).
  • HR IL-11 or PEG IL-11 given before or after renal ischemia significantly reduced the number of apoptotic TUNEL-positive cells in the kidney ( Figure 1C).
  • Figure ID shows representative images of neutrophil immunohistochemistry of kidneys (magnification 400X) from mice subjected to 30 minutes of renal ischemia and 24 hour reperfusion. As shown by the dark brown color of the cells, there was significant neutrophil infiltration in the kidneys of mice treated with saline or PEG and subjected to 24 hour renal IR. In sham-operated mice, no neutrophils were found in the kidney (data not shown). Mice treated with HR IL-11 or PEG IL-11, before or after renal ischemia, had significantly reduced neutrophil infiltration in the kidney after LR ( Figure IE).
  • HR IL-11 or PEGylated DL-l l administration before or after renal IR injury protects against renal injuiy, as measured by a variety of histological criteria, including reduction in renal necrosis and renal tubular injury, reduction in numbers of apoptotic renal cells, and reduction in numbers of infiltrating neutrophils.
  • Example 4- IL-11 Reduces Necrosis and Apoptosis in Human Kidney Proximal Tubule Cells
  • HK-2 cells as described in Example 1C, were induced with necrotic injury by exposure to 5 mM H 2 0 2 for 3 hours and lactate dehydrogenase (LDH) released into cell culture media was measured as described in Lee and Emala (2002) using a commercial LDH assay kit (Promega, Madison, WI).
  • LDH lactate dehydrogenase
  • HK-2 cells were exposed to rumor necrosis factor (TNF)- (20 ng/mL) plus cycloheximide (10 ⁇ ⁇ ) for 16 hours as described previously (Lee et al. (2007)). Cycloheximide was added in addition to T F-cc to facilitate apoptosis. Cycloheximide has been shown to synergistically increase TNF-a cytotoxicity (Ruff and Gifford (1981); Wright et al. 1992)).
  • TNF rumor necrosis factor
  • HK-2 cell apoptosis was assessed by detecting poly-(adensosine diphosphate-ribose)- polymerase (PARP) and caspase 3 fragmentations on a Western Blot as previously described Lee et al. (2007).
  • PARP poly-(adensosine diphosphate-ribose)- polymerase
  • HK-2 cells were pretreated with 10-1000 ng/mL HR IL-11 or PEGylated IL-11 (as described in Example 1A) 30 minutes before induction of necrosis or apoptosis.
  • Lactate dehydrogenase (LDH) released after H 2 0 2 -induced necrosis (5 mM H 2 0 2 for 3 hours) in HK-2 cells (N 6 for each group, expressed as a percentage of total LDH released). Data are means ⁇ SEM. *P ⁇ 0.05 versus PEG-treated group.
  • Example 5- IL-11 Increases SKI Synthesis and Induces SK Activity in HK-2 Cells
  • SKI IL-11 induces SKI
  • SKI mR A and protein were measured in HK-2 cells after IL-1 1 treatment.
  • mRNA encoding human SKI or SK2 was measured 6 hours after HR IL-11 or PEGylated IL-11 treatment in HK-2 cells as described in Kim et al. (2010)(1). Table 5 lists the primer sequences utilized. HK-2 cell lysates were also collected for immunoblotting analyses of SKI, SK2 and ⁇ -actin (internal protein loading control) 16 hours after HR IL-11 or PEGylated IL-11 treatment as described previously (Kim at al. (2010)(1)).
  • SK activity was measured as described previously by Kim et al. (2007) using a modified protocol according to Vessey et al. (2005).
  • the assay buffer was supplemented with 250 mM KCI plus 0.5% Triton X-100 (Klawitter et al. (2007); Pitman et al. (2012)).
  • Table 5 Human RT-PCR primers bp, base pairs; GAPDH, glyceraldehyde 3 -phosphate dehydrogenase; SK, sphingosine kinase.
  • Respective anticipated RT-PCR product size, PCR cycle number for linear amplification and annealing temperatures used for each primer are also provided.
  • Example IIB SKI 7" and SK2 7" mice described in Example IB were treated with either 1 mg/kg HR IL-11, 1 mg/kg PEGylated IL-1 1, saline or PEG, and subjected to renal IR as described in Example 2.
  • Plasma samples were collected 24 hours after IR injury to examine the severity, of renal dysfunction by measurements of plasma creatinine, as described in Example 2.
  • SKI 7" mice were not protected against renal IR.
  • Example 7 Hypoxia-inducible factor (HIF)-la plays a critical role in IL-11 mediated SKI induction
  • HK-2 cells (Example 1C) were treated with HR IL-1 1 (100 ng/niL for 6 hours) or PEGylated IL-11 (100 ng/mL for 6 hours) (Example 1A).
  • Example 1C To inhibit HIF-la, other HK-2 cells (Example 1C) were pretreated with 10 ⁇ 2- methoxyestradiol (2ME, a posttranscriptional down-regulator of HIF-la) (Mabjeesh et al. (2003); Volpi et al. (2011)) or with 25 ⁇ 3-(5'-Hydroxymethyl-2 , -furyl)-l-benzylindazole (YC-1 , an inhibitor of HIF-la activity) (Hsu et al. (2003); Wu et al. (2003)) 30 min before HR IL-1 1 or PEGylated IL-11 treatment.
  • 2ME 2- methoxyestradiol
  • YC-1 an inhibitor of HIF-la activity
  • Nuclear extracts from HK-2 cells were prepared using the Transfactor Extraction Kit (Clontech, Mountain View, CA) according to the manufacturer's instructions.
  • HIF-l a DNA- binding activity in nuclear extracts was determined using a TransFactor Family Colorimetric kit specific for HIF-la (Clontech) according to the manufacturer's instructions.
  • Table 7 - IL-11 and PEG IL-11 induce nuclear translocation of HIF-la in HK-2 cells.
  • PEGylated IL-1 1 significantly and selectively increased SKI mRNA expression in HK-2 cells without affecting SK-2 mRNA expression.
  • HK-2 cells were pretreated with inhibitors of HIF-la signaling (2ME (10 ⁇ ) or YC-1 (25 ⁇ )) 30 minutes before exposure to HR IL-11 (data not shown) or PEGylated IL-11 (100 ng/mL for 6 hours)
  • induction of SKI mRNA was significantly attenuated without affecting SK2 mRNA expression (Table 8). Similar results were obtained by freating cells with HR IL-11.
  • Table 8- HIF-loc plays a critical role in IL-11 mediated SKI induction
  • mice with hepatic IR were administered the IL-11 prior to hepatic injury, and tests for hepatic function performed.
  • Example IB Mice as described in Example IB were pretreated with PEG (vehicle for PEGylated IL-11), HR IL-11 (1 mg/kg) or long acting PEGylated IL-11 (1 mg/kg) (both as described in Example 1A), 10 minutes prior to hepatic ischemia or sham-operation.
  • PEG vehicle for PEGylated IL-11
  • HR IL-11 1 mg/kg
  • long acting PEGylated IL-11 both as described in Example 1A
  • mice Male C57BL/6 mice (20-25 g, Harlan, Indianapolis, IN) were subjected to liver IR injury as described previously (Park et al. (2009)). This method of partial hepatic ischemia results in a segmental (approximately 70%) hepatic ischemia but spares the right lobe of the liver and prevents mesenteric venous congestion by allowing portal decompression through the right and caudate lobes of the liver. Sham operated mice were subjected to laparotomy and identical liver manipulations without the vascular occlusion.
  • Plasma samples were collected 5 and 24 hours after IR injury to examine the severity of hepatic dysfunction by measurements of plasma amino alanine transferase ("ALT") as a measure of liver injury using the InfinityTM ALT assay kit according to manufacturer's instructions (ThermoFisher Scientific, Waltham, MA.).
  • ALT plasma amino alanine transferase
  • ALT is an enzyme found primarily in the liver, but also to a lesser extent in other tissues, such as kidney, heart, muscles, and pancreas. Blood normally contains low levels of ALT, but when the liver is damaged the liver releases ALT into the bloodstream. Thus, elevated plasma ALT levels are indicative of liver dysfunction or injury.
  • Example 9- IL-11 reduces necrosis in human intestinal cells
  • IEC-6 cells with induced necrosis were administered with the IL-11 prior to injury induction.
  • IEC-2 cells as described in Example 1C, were induced with necrotic injury by exposure to 1 mM or 500 ⁇ H 2 0 2 for three hours and lactate dehydrogenase (LDH) released into cell culture media was measured as described in Lee and Emala (2002) using a commercial LDH assay kit (Promega, Madison, WI) at 2, 4, 6, and 8 hours.
  • LDH lactate dehydrogenase
  • IEC-6 cells were pretreated with 10-1000 ng/mL of HR IL-11 or PEGylated IL- 11 (as described in Example 1 A) for 30 minute prior to induction of necrosis.
  • G-protein-coupled receptor SlPl acts within endothelial cells to regulate vascular maturation. Blood 102: 3665-3667, 2003.
  • YC-1 inhibits proliferation of human vascular endothelial cells through a cyclic GMP- independent pathway. Biochem Pharmacol 66: 263-271, 2003.
  • Kidney-specific reconstitution of the Al adenosine receptor in Al adenosine receptor knockout mice reduces renal ischemia-reperfusion injury. Kidney Int 75: 809-823, 2009.
  • Kim M, Kim M, Park SW, Pitson SM and Lee HT Isoflurane protects human kidney proximal tubule cells against necrosis via sphingosine kinase and sphingosine- 1-phosphate generation. Am J Nephrol ?, !: 353-362, 2010 (1). Kim M, Park SW, Kim M, Chen SW, Gerthoffer WT, D'Agati VD and Lee HT.
  • Leclercq TM and Pitson SM Cellular signalling by sphingosine kinase and sphingosine 1-phosphate. IUBMB Life 58: 467-472, 2006.
  • I - phosphate protects kidney and liver after hepatic ischemia and reperfusion in mice through S1P(1) receptor activation. Lab Invest 90: 1209-1224, 2010.
  • I I - induced heat shock protein 25 confers intestinal epithelial-specific cytoprotection from oxidant stress. Gastroenterology 124: 1358-1368, 2003.
  • Sphingosine kinase- 1 is a hypoxia-regulated gene that stimulates migration of human endothelial cells. Biochem Biophys Res Commun 368: 1020-1025, 2008.
  • Interleukin-11 and interleukin-6 protect cultured human endothelial cells from H202-induced cell death. Am J Respir Cell Mol Biol 29: 513-522, 2003.
  • AMPK AMP-activated protein kinase

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Abstract

La présente invention concerne une méthode de prévention ou de traitement d'une lésion due à une ischémie suivie par une reperfusion par le biais de l'administration de l'IL-11, que ce soit sous une forme non modifiée ou substituée par un PEG. L'administration de l'IL-11 devrait avoir lieu avant l'événement provoquant la lésion, dans le cas par exemple d'une chirurgie connue, ou après l'événement provoquant la lésion, dans le cas par exemple d'un accident cérébro-vasculaire.
PCT/US2013/049039 2012-07-03 2013-07-02 Utilisation de l'interleukine-11 pour la protection contre l'ischémie et une lésion de reperfusion WO2014008242A1 (fr)

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WO2016140983A1 (fr) * 2015-03-03 2016-09-09 Avalon Biologics Limited Compositions et méthodes pour une il-11 pégylée
US10335492B2 (en) 2015-03-03 2019-07-02 Nansha Biologics (Hong Kong) Limited Increasing serum half-life of human IL-11 variants by N-terminal branched PEGylation
US10881739B2 (en) 2015-03-03 2021-01-05 Nansha Biologies (Hong Kong) Limited Interleukin-11 PEGylation reaction intermediate composition
US10946103B2 (en) 2015-03-03 2021-03-16 Nansha Biologics (Hong Kong) Limited Methods of treatment using a branched amino terminal pegylated Il-11 composition
US11931417B2 (en) 2015-03-03 2024-03-19 Nansha Biologics (Hong Kong) Limited Methods of preparing a pegylated human IL-11 composition

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