# Patent

## Title
Methods for treating reperfusion injuries using flagellin

## Abstract

The present invention relates to treating a tissue in a mammal from the effects of reperfusion using flagellin.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is the national stage of International Application No. PCT/US2009/052493, filed on Jul. 31, 2009, which claims the benefit of U.S. Provisional Application No. 61/085,766, filed on Aug. 1, 2008, the contents of which are incorporated herein by reference.


FIELD OF THE INVENTION
This invention relates to the use of flagellin related polypeptides to treat tissues from the effects of reperfusion.
BRIEF DESCRIPTION OF SEQUENCE LISTING
Reference is made to the Sequence Listing, which is contained in the file named “Sequence.txt” (134 Kb, created Jan. 25, 2012), the contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Tissues deprived of blood and oxygen undergo ischemic necrosis or infarction with possible irreversible organ damage. Once the flow of blood and oxygen is restored to the organ or tissue (reperfusion), the organ does not immediately return to the normal preischemic state. Reperfusion of coronary flow is necessary to resuscitate the ischemic or hypoxic tissue or organ. Timely reperfusion facilitates salvage of cells and decreases morbidity and mortality. Reperfusion of an ischemic area may result in a paradoxical dysfunction including marked endothelial cell dysfunction, which results in vasoconstriction, platelet and leukocyte activation, increased oxidant production, and increased fluid and protein extravasation.
Over the past two decades has witnessed several pharmacological interventions designed to limit reperfusion injury. Unfortunately, the success of some agents has been limited to experimental model of ischemia and reperfusion. The lack of consistent clinical benefit may be related to a variety of factors, including poor clinical trial design, inadequate pharmacokinentic/pharmacodynamic studies and the complexity of the human in vivo model.
There is a need in the art to distinguish therapeutic strategies for ischemia vs. reperfusion, and it is possible that a combination of agents is required to elicit the maximum clinical benefit.
SUMMARY OF THE INVENTION
Provided herein is a method of treating a tissue of a mammal from the effects of reperfusion, which may comprise administering to a mammal in need thereof a composition comprising flagellin. The composition may be administered in combination with an antioxidant, which may be selected from the group consisting of amifostine and vitamin E.
The reperfusion may be caused by an injury, which may be ischemia or hypoxia. The ischemia may be selected from the group consisting of tachycardia, infarction, hypotension, embolism, thromboemoblism (blood clot), sickle cell disease, localized pressure to extremities to the body, and tumors. The hypoxia may be selected from the group consisting of hypoxemic hypoxia (carbon monoxide poisoning; sleep apnea, chronic obstructive pulmonary disease, respiratory arrest; shunts), anemic hypoxia (O2 content low), hypoxemic hypoxia, and histotoxic hypoxia. The localized pressure may be due to a tourniquet.
The composition may be administered prior to, together with, or after the influx of oxygen. The tissue may be selected from the group consisting of GI tract, lung, kidney, liver, cardiovascular system, blood vessel endothelium, central nervous system, peripheral nervous system, muscle, bone, and hair follicle.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 demonstrates the level of creatinine in the serum of mice over 5 days post intravenous administration of flagellin at concentrations of either 0.01 μg, 0.5 μg, 1.0 μg, or 5.0 μg/body.
FIG. 2 demonstrates the affect of flagellin administered to mice before imposition of renal ischemia and measuring survival and creatinine following reperfusion of the ischemic kidneys. Panel A shows the percent survival of mice who were pretreated with flagellin at concentrations of either 0.01 μg, 0.5 μg, 1.0 μg, or 5.0 μg/body or PBS as a control. Panel B shows the level of creatinine in the same group of pretreated and control mice.
FIG. 3 demonstrates the histopathology of ischemic kidney cells 24 hours after reperfusion that were pretreated with PBS or flagellin at concentrations of either 0.01 μg, 0.5 μg, 1.0 μg, or 5.0 μg/body. The Sham column indicates kidney cells isolated from mice that were not imposed with renal ischemia.
FIG. 4 demonstrates histopathology of kidney cells 7 days after reperfusion. In the first panel, the histopathology slide shows kidney cells isolated from mouse pretreated with PBS before renal ischemia and followed by reperfusion of the ischemic kidneys. In the first panel, the histopathology slide shows kidney cells isolated from mouse pretreated with PBS before renal ischemia and followed by reperfusion of the ischemic kidneys. In the second panel, the histopathology slide shows kidney cells isolated from mouse pretreated with flagellin at a concentration of 0.5 μg/body but not imposed with renal ischemia. The third panel demonstrates a histopathology slide showing kidney cells isolated from a mouse pretreated with flagellin at a concentration of 0.5 μg/body and imposed with renal ischemia and followed by reperfusion of the ischemic kidneys.
FIG. 5 demonstrates assessing leukocyte infiltration 9 hours and 24 hours after reperfusion into ischemic kidney cells isolated from mice pretreated with PBS or flagellin at 0.5 μg /body. FIG. 5a is kidney tissue cells immunohistochemically stained for levels of neutrophil infiltration 9 hours and 24 hours after reperfusion in ischemic and non-ischemic treated kidneys cells from mice pretreated with PBS or flagellin at 0.5 μg /body. FIG. 5b is number of neutrophils, macrophages, CD4+T cells, and CD8+T cells infiltrating into kidney tissue cells isolated from mice pretreated with PBS or flagellin a concentration of 0.5 μg /body. FIG. 5c shows the effects of flagellin on myeloperoxidase levels, as a measure of the activation of neutrophils, during reperfusion of ischemic kidneys as compared to controls.
FIG. 6a demonstrates critical role of flagellin preventing chemokines CXCL1/KC and CXCL2/KC in directing leukocyte infiltration into ischemic kidney tissues. FIG. 6b demonstrates mRNA levels of the acute phase proteins IL-1b and IL-6 but not TNFa were also decreased in ischemic kidneys at 9 hours post-reperfusion in flagellin preconditioned animals.
FIG. 7 demonstrates survival and creatinine levels in groups of C57BL/6 mice that were subjected to 45 minutes of bilateral renal pedicle occlusion and were administered 0.5 μg of flagellin at various times following the removal of the renal clamps. FIG. 7a shows the effects of administering flagellin 30 minutes before or within 30 minutes after declamping on the viability of mice subjected to ischemic injury. FIG. 7b shows the effects of administering flagellin 30 minutes before declamping or within 30 minutes following declamping on levels of serum creatinine after reperfusion of the ischemic kidneys. FIG. 7c shows the effects of flagellin on CXCL1 and CXCL2 levels at 9 and 24 hours after reperfusion.
FIG. 8 demonstrates administration of 0.5 μg flagellin within 30 minutes of reperfusion of ischemic kidneys of wild-type C57BL/6 mice reconstituted with wild-type bone marrow decreased CXCL1 and CXCL2 mRNA levels. In MyD88−/− recipients reconstituted with either MyD88−/− or wild-type bone marrow, little CXCL1 and CXCL2 mRNA was induced during reperfusion of ischemic kidneys and administration of flagellin during reperfusion of these kidneys did not decrease the mRNA levels of these chemokines. In contrast, wild-type recipients of bone marrow from MyD88−/− donors expressed high levels of CXCL1 and CXCL2 mRNA and these levels were decreased by administration of flagellin during reperfusion of the ischemic kidneys.
FIG. 9 demonstrates renal sections from wild-type C57BL/6 and BALB/c mice were stained with anti-TLR5 antibody. FIG. 9a shows renal sections from wild-type C57BL/6 and BALB/c mice stained with anti-TLR5 antibody. FIG. 9b demonstrates expression levels of TLR5 mRNA were low in kidneys prior to imposition of renal ischemia/reperfusion but increased quickly during reperfusion of ischemic kidneys.
FIG. 10 shows the domain structure of bacterial flagellin. The Ca backbone trace, hydrophobic core distribution and structural information of F41. Four distinct hydrophobic cores that define domains D1, D2a, D2b and D3. All the hydrophobic side-chain atoms are displayed with the Ca backbone. Side-chain atoms are color coded: Ala, yellow; Leu, Ile or Val, orange; Phe and Tyr, purple (carbon atoms) and red (oxygen atoms). c, Position and region of various structural features in the amino-acid sequence of flagellin. Shown are, from top to bottom: the F41 fragment in blue; three b-folium folds in brown; the secondary structure distribution with a-helix in yellow, b-structure in green, and b-turn in purple; tic mark at every 50th residue in blue; domains D0, D1, D2 and D3; the axial subunit contact region within the proto-element in cyan; the well-conserved amino-acid sequence in red and variable region in violet; point mutations in F41 that produce the elements of different supercoils. Letters at the bottom indicate the morphology of mutant elements: L (D107E, R124A, R124S, G426A), L-type straight; R (A449V), R-type straight; C (D313Y, A414V, A427V, N433D), curly33.
FIG. 11 shows a schematic of Salmonella flagellin domains, its fragments, and its interaction with TLR5. Dark bars denote regions of the flagellin gene used to construct fragments comprising A, B, C, A′ and B′.
FIG. 12 depicts flagellin derivatives. The domain structure and approximate boundaries (amino acid coordinates) of selected flagellin derivatives (listed on the right). FliC flagellin of Salmonella dublin is encoded within 505 amino acids (aa).
FIGS. 13A-K show the nucleotide and amino acid sequence for the following flagellin variants: AA′ (SEQ ID NO: 7-8), AB′ (SEQ ID NO: 9-10), BA′ (SEQ ID NO: 11-12), BB′ (SEQ ID NO: 13-14), CA′ (SEQ ID NO: 15-16), CB′ (SEQ ID NO: 17-18), A (SEQ ID NO: 19-20), B (SEQ ID NO: 21-22), C (SEQ ID NO: 23-24), GST-A′ (SEQ ID NO: 25-26), GST-B′ (SEQ ID NO: 27-28), AA′n1-170 (SEQ ID NO: 29-30), AA′n1-163 (SEQ ID NO: 33-34), AA′n54-170 (SEQ ID NO: 31-32), AA′n54-163 (SEQ ID NO: 335-36), AB′n1-170 (SEQ ID NO: 37-38), AB′n1-163 (SEQ ID NO: 39-40), AA′n1-129 (SEQ ID NO: 41-42), AA′n54-129 (SEQ ID NO: 43-44), AB′n1-129 (SEQ ID NO: 45-46), AB′n54-129 (SEQ ID NO: 47-48), AA′n1-100 (SEQ ID NO: 49-50), AB′n1-100(SEQ ID NO: 51-52), AA′n1-70 (SEQ ID NO: 53-54) and AB′n1-70 (SEQ ID NO: 55-56). The pRSETb leader sequence is shown in Italic (leader includes Met, which is also amino acid 1 of FliC). The N terminal  constant domain is underlined. The amino acid linker sequence is in Bold. The C terminal constant domain is underlined. GST, if present, is highlighted.
FIG. 14A shows histological of mice hind limb muscle 14 days after reperfusion following 3 hours of warm ischemia using a hematoxylin/eosin stain where the mouse had been given 0.5 μg of CBLB502 within 15 minutes of reperfusion. FIG. 14 B shows histological of mice hind limb muscle 14 days after reperfusion following 3 hours of warm ischemia using a hematoxylin/eosin stain where the mouse had been given vehicle (PBS) within 15 minutes of reperfusion. FIG. 14C shows the wet/dry ratio of tissue edema in the limb of mice administered either with CBLB502 or PBS within 15 minutes of reperfusion after 3 hours of ischemia. The ratio of edema was also measured in the limb of mice administered CBLB502 or PBS, but spared 3 hours of ischemia. FIG. 14D shows the wet/dry ration of vascular leaks using Blue Dye per gram weight limb of mice administered either CBLB502 or PBS within 15 minutes of reperfusion after 3 hours of ischemia. The ratio of vascular leaks was also measured in the limb of mice administered CBLB502 or PBS, but spared 3 hours of ischemia.
FIGS. 15A-C show a comparison of amino acid sequences of the conserved amino (FIG. 15A) and carboxy (FIG. 15B) terminus from 21 species of bacteria. The 13 conserved amino acids important for TLR5 activity are shown with shading. The amino acid sequences are identified by their accession numbers from TrEMBL (first letter =Q) or Swiss-Prot (first letter =P). The amino terminus sequences have SEQ ID NOs: 57-77, respectively, for each of the 21 bacterial species, and the carboxy terminus sequences have SEQ ID NOs: 78-98, respectively.



DETAILED DESCRIPTION
The inventors have made the surprising discovery that flagellin protects from the effects of reperfusion. The absence or reduction of oxygen and nutrients from blood creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than the restoration of normal function. The restored blood flow reintroduces oxygen within cells that damages cellular proteins, DNA and the plasma membrane. Damage to cell's membrane may in turn cause the release of more free radicals. Such reactive species also act in redox signaling to induce apoptosis of ischemic tissue cells. In addition, inflammatory response further damages the tissue. White blood cells carried to the area by the newly returning blood release a host of inflammatory factors such as interleukins as well as free radicals in response to tissue damage. Leukocytes may also build up in small capillaries, obstructing them and leading to more ischemia. While not being bound by theory, flagellin may provide protection from the effects of reperfusion by reducing the oxidative and inflammatory stresses to the tissue thereby preventing apoptosis and allowing faster recovery of the tissue to a normal state. This protective nature of flagellin can either by used at the onset of reperfusion or be used to prevent further damage due to reperfusion. The below-described invention relates in part to administration of flagellin to a treat tissue of a mammal from the effects of reperfusion.
1. Definitions
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
“Administer” may mean a dosage of an agent that induces NF-κB activity, means a single dose or multiple doses of the agent.
“Analog” may mean, in the context of a peptide or polypeptide, a peptide or polypeptide comprising one or more non-standard amino acids or other structural variations from the conventional set of amino acids.
“Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof, including Fab, F(ab′)2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody may be a monoclonal antibody, polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom. The antibody may also be a chimeric antibody. The antibody may be derivatized by the attachment of one or more chemical, peptide, or polypeptide moieties known in the art. The antibody may be conjugated with a chemical moiety.
“Apoptosis” may mean a form of cell death that includes progressive contraction of cell volume with the preservation of the integrity of cytoplasmic organelles; condensation of chromatin (i.e., nuclear condensation), as viewed by light or electron microscopy; and/or DNA cleavage into nucleosome-sized fragments, as determined by centrifuged sedimentation assays. Cell death occurs when the membrane integrity of the cell is lost (e.g., membrane blebbing) with engulfment of intact cell fragments (“apoptotic bodies”) by phagocytic cells.
A “peptide” or “polypeptide” may mean a linked sequence of amino acids and may be natural, synthetic, or a modification or combination of natural and synthetic.
“Treating,” “treatment,” or “to treat” each may mean to alleviate, suppress, repress, eliminate, prevent or slow the appearance of symptoms, clinical signs, or underlying pathology of a condition or disorder on a temporary or permanent basis. Preventing the disease involves administering a composition of the present invention to an animal prior to onset of the disease. Suppressing the disease involves administering a composition of the present invention to an animal after induction of the disease but before its clinical appearance. Repressing the disease involves administering a composition of the present invention to an animal after clinical appearance of the disease.
2. Treating the Effects of Reperfusion
Provided herein is a method of treating the effects of reperfusion by administering to a mammal in need thereof a composition comprising flagellin. Reperfusion may be caused by an injury.
Reperfusion may damage a body component when blood supply returns to the body component after the injury. The effects of reperfusion may be more damaging to the body component than the injury itself. There are several mechanism and mediators of reperfusion including oxygen free radicals, intracellular calcium overload, and endothelial dysfunction. Excessive quantities of reactive oxygen species, when reintroduced into a previously injured body component, undergo a sequential reduction leading to the formation of oxygen free radicals. Potent oxidant radicals, such as superoxide anion, hydroxyl radical, and peroxynitrite may be produced within the first few minutes of reflow to the body component and may play a crucial role in the development of reperfusion injury. Oxygen free radicals also can be generated from sources other than reduction of molecular oxygen. These sources include enzymes, such as xanthine oxidase, cytochrome oxidase, and cyclooxygenase, and the oxidation of catecholamines.
Reperfusion is also a potent stimulus for neutrophil activation and accumulation, which in turn serve as potent stimuli for reactive oxygen species production. Specifically, the main products of the neutrophil respiratory burst are strong oxidizing agents including hydrogen peroxide, free oxygen radicals and hypochlorite. Neutrophils are the most abundant type of phagocyte, normally representing 50 to 60% of the total circulating leukocytes, and are usually the first cells to arrive at the site of injured body component. Oxygen-derived free radicals produce damage by reacting with polyunsaturated fatty acids, resulting in the formation of lipid peroxides and hydroperoxides that damage the body component and impair the function of membrane-bound enzyme systems. Free radicals stimulate the endothelial release of platelet activating factor and chemokines such as neutrophil activator factor, chemokine (C-X-C motif) ligand 1, and chemokine (C-X-C motif) ligand 1 which attracts more neutrophils and amplifies the production of oxidant radicals and the degree of reperfusion injury. Reactive oxygen species also quench nitric oxide, exaggerating endothelial injury and tissue cell dysfunction. In addition to an increased production, there is also a relative deficiency in endogenous oxidant scavenging enzymes, which further exaggerates free radical-mediated cardiac dysfunction.
Reperfusion may further result in marked endothelial cell dysfunction. Endothelial dysfunction facilitates the expression of a prothrombotic phenotype characterized by platelet and neutrophil activation, important mediators of reperfusion. Once neutrophils make contact with the dysfunctional endothelium, they are activated, and in a series of well-defined steps (rolling, firm adherence, and transmigration) they migrate into areas of tissue injury through endothelial cell junctions as part of the innate immune response.
Changes in intracellular calcium homeostasis play an important role in the development of reperfusion. Reperfusion may be associated with an increase in intracellular calcium; this effect may be related to increased sarcolemmal calcium entry through L-type calcium channels or may be secondary to alterations in sarcoplasmic reticulum calcium cycling. In addition to intracellular calcium overload, alterations in myofilament sensitivity to calcium have been implicated in reperfusion. Activation of calcium-dependent proteases (calpain I) with resultant myofibril proteolysis has been suggested to underscore reperfusion injury, as has proteolysis of troponin.
Reperfusion of tissue cells subjected to an injury have an altered cellular metabolism, which in turn may contribute to delayed functional recovery. For example, an injury may induce anaerobic metabolism in the cell with a net production of lactate. Lactate release persists during reperfusion, suggesting a delayed recovery of normal aerobic metabolism. Likewise, the activity of mitochondrial pyruvate dehydrogenase (PDH) may be inhibited up to 40% after an injury and may remain depressed for up to 30 minutes after reperfusion.
Each of these events during reperfusion can lead to stress to the tissue cells and programmed cell death (apoptosis) and necrosis of the tissue cells. Apoptosis normally functions to “clean” tissues from wounded and genetically damaged cells, while cytokines serve to mobilize the defense system of the organism against the pathogen. However, under conditions of severe injury both stress response mechanisms can by themselves act as causes of death.
a. Flagellin
The flagellin may be a flagellin-related polypeptide. The flagellin may be from any source, including a variety of Gram-positive and Gram-negative bacterial species. Flagellin may have the amino acid sequence of one of 23 flagellins from bacterial species that are depicted in FIG. 7 of U.S. Patent Publication No. 2003/0044429, the contents of which are incorporated herein by reference. The nucleotide sequences encoding the flagellin polypeptides listed in FIG. 7 of U.S. 2003/0044429 are publicly available at sources including the NCBI Genbank database.
Flagellin may be the major component of bacterial flagellum. Flagellin may be composed of three domains (FIG. 10). Domain 1 (D1) and domain 2 (D2) may be discontinuous and may be formed when residues in the amino terminus and carboxy terminus are juxtaposed by the formation of a hairpin structure. The amino and carboxy terminus comprising the D1 and D2 domains may be most conserved, whereas the middle hypervariable domain (D3) may be highly variable. Studies with a recombinant protein containing the amino D1 and D2 and carboxyl D1 and D2 separated by an Escherichia coli hinge (ND1-2/ECH/CD2) indicate that D1 and D2 may be bioactive when coupled to an ECH element. This chimera, but not the hinge alone, may include IkBa degradation, NF-kB activation, and NO and IL-8 production in two intestinal epithelial cell lines. The non-conserved D3 domain may be on the surface of the flagellar filament and may contain the major antigenic epitopes. The potent proinflammatory activity of flagellin may reside in the highly conserved N and C D1 and D2 regions.
Flagellin may induce NF-kB activity by binding to Toll-like receptor 5 (TLR5). The TLR family may be composed of at least 10 members and is essential in innate immune defense against pathogens. The innate immune system may recognize pathogen-associated molecular patterns (PAMPs) that are conserved on microbial pathogens. TLR may recognize a conserved structure that is particular to bacterial flagellin. The conserved structure may be composed of a large group of residues that are somewhat permissive to variation in amino acid content. Smith et al., Nat. Immunol. 4:1247-53 (2003) have identified 13 conserved amino acids in flagellin that are part of the conserved structure recognized by TLR5. The 13 conserved amino acids of flagellin that may be important for TLR5 activity are shown in FIG. 11.
The flagellin may be from a species of Salmonella, a representative example of which is S. dublin (encoded by GenBank Accession Number M84972) (SEQ ID NO: 1). The flagellin related-polypeptide may be a fragment, variant, analog, homolog, or derivative of SEQ ID NO: 1, or combination thereof, that binds to TLR5 and induces TLR5-mediated activity, such as activation of NF-kB activity. A fragment, variant, analog, homolog, or derivative of flagellin may be obtained by rational-based design based on the domain structure of Flagellin and the conserved structure recognized by TLR5.
The flagellin may comprise at least 10, 11, 12, or 13 of the 13 conserved amino acids shown in FIG. 11 (positions 89, 90, 91, 95, 98, 101, 115, 422, 423, 426, 431, 436 and 452). The flagellin may be at least 30-99% identical to amino acids 1 174 and 418 505 of SEQ ID NO: 1.
The flagellin may be a flagellin polypeptide from any Gram-positive or Gram-negative bacterial species including, but not limited to, the flagellin polypeptides disclosed in U.S. Pat. Pub. 2003/000044429, the contents of which are incorporated herein, and the flagellin peptides corresponding to the Accession numbers listed in the BLAST results shown in FIG. 25 of U.S. Patent Pub. 2003/000044429, or variants thereof.
The flagellin may stimulate TLR5 activity. Numerous deletional mutants of flagellin have been made that retain at least some TLR5 stimulating activity. The flagellin may be a deletional mutant disclosed in the Examples herein, and may comprise a sequence translated from GenBank Accession number D13689 missing amino acids 185-306 or 444-492, or from GenBank Accession number M84973 missing amino acids 179-415, or a variant thereof.
The flagellin may comprise transposon insertions and changes to the variable D3 domain. The D3 domain may be substituted in part, or in whole, with a hinge or linker polypeptide that allows the D1 and D2 domains to properly fold such that the variant stimulates TLR5 activity.
The variant hinge elements may be found in the E. coli MukB protein and may have a sequence as set forth in SEQ ID NOS: 3 and 4, or a variant thereof.
Other agents may be used to target TLR5 receptors. These agents may be agonists of TLR5 and stimulate TLR5 activity. The agonist may be an anti-TLR5 antibody or other small molecule.
b. Injury
The effects of reperfusion may be caused by an injury to the body component. The injury may be due to ischemia, hypoxia, an infarction, or an embolism. Treatment of the injury may lead to reperfusion and further damage to the body component.
(1) Ischemia
Ischemia may be an absolute or relative shortage of blood supply to a body component. Relative shortage may be a mismatch, however small, of blood supplied (oxygen delivery) to a body component vs. blood required to a body component for the adequate oxygenation. Ischemia may also be an inadequate flow of blood to a part of the body due to a constriction or blockage of blood vessels supplying it and may affect any body component in the body. Insufficient blood supply causes body components to become hypoxic, or, if no oxygen is supplied at all, anoxic. This may cause necrosis. The mechanisms of ischemia may vary greatly. For example, ischemia to any body component may be due to tachycardia (abnormally rapid beating of the heart), atherosclerosis (lipid-laden plaque obstructing the lumen of arteries), hypotension (low blood pressure in septic shock, heart failure), thromboembolisms (blood clots), outside compression of blood vessels (tumor), embolisms (foreign bodies in the circulation, e.g., amniontic fluid embolism), sickle cell disease (abnormally shaped hemoglobin), infarctions, induced g-forces which restrict the blood flow and force the blood to extremities of the body, localized extreme cold due to frostbite, ice, improper cold compression therapy, and any other force that restricts blood flow to the extremities such as a tourniquet. Force to restrict blood flow to extremities may be required due to severe lacerations, incisions, puncture such as a knifing, crushing injuries due to blunt force trauma, and ballistic trauma due to gunshot or shrapnel wounds. Ischemia may be a feature of heart diseases, ischemic colitis, transient ischemia attacks, cerebrovascular accidents, acute renal injury, ruptured arteriovenous malformations, and peripheral artery occlusive disease.
(2) Hypoxia
Hypoxia may be a deprivation of adequate supply of oxygen. Hypoxia may be pathological condition in which the body as a whole (generalized hypoxia) or region of the body (tissue hypoxia) is deprived of adequate oxygen supply. A variation in levels of arterial oxygen may be due to a mismatch between supply and demand of oxygen by body components. A complete deprivation of oxygen supply is anoxia. Hypoxia may be hypoxemic hypoxia, anemic hypoxia, hypoxemic hypoxia, histotoxic hypoxia, histotoxic hypoxia, and ischemic hypoxia.
Hypoxemic hypoxia may be an inadequate supply of oxygen to the body as a whole caused by low partial pressure of oxygen in arterial blood. Hypoxemic hypoxia may be due to low partial pressure of atmospheric oxygen such as at high altitudes, replacement of oxygen in breathing mix of a modified atmosphere such as a sewer, replacement of oxygen intentionally as in recreational use of nitrous oxide, a decrease in oxygen saturation of the blood due to sleep apnea, or hypopnea, inadequate pulmonary ventilation such as chronic obstructive pulmonary disease or respiratory arrest, anatomical or mechanical shunts in the pulmonary circulation or a right to left shunt in the heart and lung. Shunts may cause collapsed alveoli that are still perfused or a block in ventilation to an area of the lung. Shunts may present blood meant for the pulmonary system to not be ventilated and prevent gas exchange because Thebesia vessels empty into the left ventricle and the bronchial circulation, which supplies the bronchi with oxygen.
Anemia hypoxia may be the total oxygen content is reduced but the arterial oxygen pressure is normal. Hypoxemic hypoxia may be when blood fails to deliver oxygen to target body components. Hypoxemic hypoxia may be caused by carbon monoxide poisoning which inhibits the ability of haemoglobin to release the oxygen bound to it, or methaemoglobinaemia, an abnormal haemoglobin that accumulates in the blood.
Histotoxic hypoxia may be due to being unable to effectively use oxygen due to disabled oxidative phosphorylation enzymes.
(3) Infarction
Infarction is a is a type of pathological condition that can cause ischemia. Infarction may be a macroscopic area of necrotic tissue caused the loss of an adequate blood supply due to an occlusion. The infarction may be a white infarction composed of platelets and causes necrosis in organ tissues such as heart, spleen, and kidneys. The infarction may be a red infarction composed of red blood cells and fibrin strands in organ tissues of the lung. Disease associated with infarction may include myocardial infarction, pulmonary embolism, cerebrovascular accident (stroke), acute renal failure, peripheral artery occlusive disease (example being gangrene), antiphospholipid syndrome, sepsis, giant cell arthritis, hernia, and volvulus.
(4) Embolism
Embolism is a type of pathological condition that can cause ischemia. Embolism may be an object that migrates from one part of the body and causes an occlusion or blockage of a blood vessel in another part of the body. An embolism may be thromboembolism, fat embolism, air embolism, septic embolism, tissue embolism, foreign body embolism, amniotic fluid embolism. Thromboembolism may be a blood clot that is completely or partially detached from the site of thrombosis. Fat embolism may be endogenous fat tissues that escape into the blood circulation. The fracture of bones is one example of a leakage of fat tissue into the ruptured vessels and arteries. Air embolism may be a rupture of alveoli and inhaled air that leaks into the blood vessels. The puncture of the subclavian vein or intravenous therapy are examples of leakage of air into the blood vessels. A gas embolism may be gasses such as nitrogen and helium because insoluble and forming small bubbles in the blood.
c. Body Component
This invention relates to treatment of a body component in a mammal. The body component may be an organ, a tissue, or a cell. The body component may be from an abdomen, acetabulum, adipose, adrenal cortex, adrenal gland, adrenal medulla, alveolar macrophage, amnion, aorta, artery, ascites, ascitic fluid, axilla lymph node, bladder, blood, bone, bone marrow, bowel, brain, breast, bronchus, cartilage, caudal trunk, cerebellum, cervix, chorionic villi, colon, conjunctiva, connective tissue, cornea, dermis, dorsal root ganglion, duodenum, dysplastic tongue mucosa, egg, embryo, endocrine, endometrium, endothelium, epidermis, epithelium, erythropoietic, eye, fibroblast, fin, foetus, foot, foreskin, Gasser's node, gingival stroma, gonad, groin lymph node, heart, humerus, ileum, intestine, ileocecal, ileum, islets of Langerhanm, kidney, larvae, larval, larynx, liver, lung, lung (bronchioalveolar), lymph, lymph node, lymphatic tissue, lymphoid, lymphoid organs, mammary, mammary alveolar nodules, mammary gland, mesonephros, mesothelium, moulting nymph, mouth, muscle, nasal, nasal septum, nervous system, neural, oesophageal gastric junction, oesophagus, oral, ovary, palatal mesenchyme, pancreas, papillary ovarian, penis, peripheral blood, peritoneum, pharynx, pituitary, placenta, pleural effusion, pleural fluid, prostate, pupal ovary, rectum, retina, right axial lymph node, salivary duct, sialaden, skeletal muscle, skin, small bowel, small intestine, soft tissue, spleen, sternum, stomach, tail, testicle, testis, thigh, thymus, thyroid, thyroid glands, tongue, tonsil, trachea, trunk, turbinate, umbilical cord, umbilicus, uterus, vagina, viscera, vulva, GI tract, lungs, kidneys, liver, cardiovascular system, blood vessel endothelium, central and peripheral nervous system, muscle, bone, hair follicles, and yolk sac.
3. Composition
This invention also relates to a composition comprising a therapeutically effective amount of flagellin. The composition may be a pharmaceutical composition, which may be produced using methods well known in the art. The composition may also comprise a coagent. As described above, the composition may be administered to a mammal for treating the effects of reperfusion.
a. Administration
Administration of the compositions using the method described herein may be orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, or combinations thereof. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, and intraarticular. For veterinary use, the composition may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian can readily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The compositions may be administered to a human patient, cat, dog, large animal, or an avian.
The composition may be administered simultaneously or metronomically with other treatments. The term “simultaneous” or “simultaneously” as used herein, means that the composition and other treatment be administered within 48 hours, preferably 24 hours, more preferably 12 hours, yet more preferably 6 hours, and most preferably 3 hours or less, of each other. The term “metronomically” as used herein means the administration of the composition at times different from the other treatment and at a certain frequency relative to repeat administration.
The composition may be administered at any point prior to reperfusion including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40 mins., 35 mins., 30 mins., 25 mins., 20 mins., 15 mins, 10 mins, 9 mins, 8 mins, 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins. prior to reperfusion. The composition may be administered at any point prior to the injury including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40 mins., 35 mins., 30 mins., 25 mins., 20 mins., 15 mins, 10 mins, 9 mins, 8 mins, 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins. prior to injury.
The composition may be administered at any point after reperfusion including about 1 min, 2 mins., 3 mins., 4 mins., 5 mins., 6 mins., 7 mins., 8 mins., 9 mins., 10 mins., 15 mins., 20 mins., 25 mins., 30 mins., 35 mins., 40 mins., 45 mins., 50 mins., 55 mins., 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40 hr, 42 hr, 44 hr, 46 hr, 48 hr, 50 hr, 52 hr, 54 hr, 56 hr, 58 hr, 60 hr, 62 hr, 64 hr, 66 hr, 68 hr, 70 hr, 72 hr, 74 hr, 76 hr, 78 hr, 80 hr, 82 hr, 84 hr, 86 hr, 88 hr, 90 hr, 92 hr, 94 hr, 96 hr, 98 hr, 100 hr, 102 hr, 104 hr, 106 hr, 108 hr, 110 hr, 112 hr, 114 hr, 116 hr, 118 hr, and 120 hr after reperfusion.
b. Formulation
The method may comprise administering a composition to treat for the effects of reperfusion. Compositions provided herein may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycollate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.
Compositions provided herein may also be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The compositions may also be formulated as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, nonaqueous vehicles and preservatives. Suspending agent include, but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Nonaqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid.
Compositions provided herein may also be formulated as suppositories, which may contain suppository bases including, but not limited to, cocoa butter or glycerides. Compositions provided herein may also be formulated for inhalation, which may be in a form including, but not limited to, a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane. Compositions provided herein may also be formulated as transdermal formulations comprising aqueous or nonaqueous vehicles including, but not limited to, creams, ointments, lotions, pastes, medicated plaster, patch, or membrane.
Compositions provided herein may also be formulated for parenteral administration including, but not limited to, by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents. The composition may also be provided in a powder form for reconstitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water.
Compositions provided herein may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection. The compositions may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).
c. Dosage
The method may comprise administering a therapeutically effective amount of the composition to a patient in need thereof. The therapeutically effective amount required for use in therapy varies with the nature of the condition being treated, the length of time desired to increase hematopoietic stem cells into the bloodstream, and the age/condition of the patient. In general, however, doses employed for adult human treatment typically are in the range of 0.001 mg/kg to about 200 mg/kg per day. The dose may be about 1 μg/kg to about 100 μg/kg per day. The desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day. Multiple doses may be desired, or required.
The dosage may be at any dosage including, but not limited to, about 0.1 μg/kg, 0.2 μg/kg, 0.3 μg/kg, 0.4 μg/kg, 0.5 μg/kg, 0.6 μg/kg, 0.7 μg/kg, 0.8 μg/kg, 0.9 μg/kg, 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg, 950 μg/kg, 975 μg/kg or 1 mg/kg.
4. Coagent
Flagellin or the composition may be coadministered with a coagent. The coagent may be any compound that slows or prevents the effects of reperfusion. The coagent may be an antioxidant. The antioxidant may be able to slow and prevent the oxidation of other molecules, cells, tissues or organs. The antioxidant may be vitamin E, ascorbic acid, glutathione, lipoic acid, uric acid, carotenes such as β-carotene and retinol, vitamin E, and coenzyme Q, thiols such as cysteine, cysteamine, glutathione, and bilrubin, amifostine, and flavanoids.
The coagent may be a sodium-hydrogen antiport inhibitor. Injury and reperfusion may result in marked intracellular acidosis. A sodium-hydrogen antiport inhibitor may be used to reduce proton extrusion and prevent increases in Ca2+. A sodium-hydrogen inhibitor may be cariporide.
The coagent may be insulin. Insulin may be used to stimulate PDH activity and prevent inhibition of PDH activity after reperfusion.
The coagent may be adenosine. Adenosine may be used to open mitochondrial KATP channels.
5. Combination Treatment
The method may be used in combination with other methods to treat the injury. The other methods may be treatments of myocardial infarction (heart attack), pulmonary embolism, cerebrovascular accident (stroke), peripheral artery occlusive disease (example being gangrene), antiphospholipid syndrome, sepsis, giant cell arteritis, hernia, volvulus, solid tumor cancers, decompression sickness, sickle cell anemia, puncture of the subclavian vein, bone fractures, high altitude sickness, recreational use of nitrous oxide, sleep apnea, hypopnea, shunts, anemia, carbon monoxide poisoning, methaemoglobinaemia, thromboembolism, fat embolism, air embolism, septic embolism, tissue embolism, foreign body embolism, amniotic fluid embolism, induced g-forces, and external pressure to prevent blood flow due to severe cuts, castration, or mangling. The method may also be used in combination with methods of treating reperfusion injuries such as administering low doses of hydrogen sulfate (H2S), glisoden, or wheat glialin, or performing therapeutic hypothermia or aortic cross-clamping.
The present invention has multiple aspects, illustrated by the following non-limiting examples.
Example 1
Dose-Dependent Protection of Flagellin on Renal Function [Flagellin May be a TLR5 Agonist]
At particular dosages, flagellin does not affect renal function. This effect was demonstrated by measuring the levels of creatinine in the serum of mice after systemic administration of different dosages of flagellin. C57BL/6 mice were injected with either 0.01 μg, 0.5 μg, 1.0 μg, or 5.0 μg of flagellin and levels of serum creatinine (mg/dl) were monitored daily as shown in FIG. 1. Administration of 5 μg of flagellin resulted in increased serum concentrations that was evident within 24 hours after administration. After 24 additional hours (48 hours total), the levels of creatinine peaked and then fell back to background levels by 72 hours after administration and then began to slowly rise to low levels again (FIG. 1). In contrast, administration of 1 μg of flagellin also induced a rise in serum creatinine levels, but this was only detected as a single peak after 48 hours and then fell to background levels by 72 hours after administration. Administration of 0.5 μg and 0.1 μg did not induce any measurable increases in serum creatinine levels throughout the study period.
Example 2
Dose-Dependent Effect of Flagellin on Renal Function
At particular dosages, flagellin is capable of protecting renal tissue of a mammal from the effects of acute renal ischemia. This effect was demonstrated by administering flagellin to mice before imposition of renal ischemia and measuring survival following reperfusion of the ischemic kidneys. Specifically, 30 minutes before being subjected to 45 minutes of bilateral renal pedicle occlusion, groups of C57BL/6 mice were given either various doses of flagellin (0.01 μg, 0.5 μg, 1.0 μg, or 5.0 μg per body) in 40 μg of PBS or PBS alone (400 μl) via intravenous administration. Survival of the mice, levels of serum creatinine, and histopathology data were then collected.
a. Survival
Bilateral renal pedicle occlusion was performed in the mice as previously detailed (REFERENCES). Mice were given 20 U (units/ml) sodium heparin via intraperitoneal administration 20 minutes before surgery. The mice were anesthetized with phenobarbital and kept warm under a 60-W light bulb until surgery. Under aseptic conditions, the abdominal cavity was opened with a midline incision and the bilateral renal pedicle was occluded non-traumatically with a microvascular clamp (World Precision Instruments, Sarasota, Fla.) and the wound was temporarily closed with 4-0 silk suture. Mice were placed on a heat pad under a 60 Watt light bulb and a sensor tip of the TraceableTM Certificate Memory Monitoring Thermometer (Fisher Scientific) was placed into the abdominal cavity to ensure temperature maintenance at 32° C. during the imposition of renal ischemia. Kidneys were subjected to ischemia for 45 minutes. After removal of the clamp, immediate and complete renal reperfusion was confirmed visually and the peritoneal cavity was closed. Sham-operated mice were treated in an identical manner except for the bilateral clamp of the renal pedicle.
In the control group given PBS without flagellin, 80% of the animals expired within 5 days following reperfusion of the ischemic kidneys (see FIG. 2a). All animals given 5 μg of flagellin before imposition of renal ischemia expired within 5 days following reperfusion. In contrast, all animals given either 1 or 0.5 μg of flagellin before renal ischemia survived more than 45 days after reperfusion. The protective effect of flagellin in acute renal ischemia was also observed to be dose-dependent in that animals given 0.1 or 0.01 μg were not protected against the injury.
b. Renal Function Measurement
Serum creatinine levels were also measured to determine the protective effect of flagellin on renal function. Sham operated mice and mice subjected to bilateral renal I/R injury were anesthetized with isofluorane and bled from the postorbital plexus using a heparin-coated microcapillary tube at 24-hour intervals. The serum was stored at −80° C. until measurement. Serum creatinine levels were measured using the Creatinine Kit (Sigma Diagnostics, Inc., St. Louis, Mo.). The protective effect of flagellin was reflected by the low levels of serum creatinine determined at 24 hours post-reperfusion in animals given 1.25 or 0.5 μg flagellin 30 minutes before imposition of ischemia (see FIG. 2a). Animals given the non-protective low doses of flagellin (0.1 and 0.01 μg) had higher levels of creatinine levels that fell just below those observed in the control group that received PBS 30 minutes before bilateral pedicle occlusion.
c. Histology Studies of Renal Tissue
High serum creatinine levels, an indication of renal dysfunction induced by imposition of ischemia-reperfusion injury, were supported by the histopathology of the ischemic kidneys 24 hours after reperfusion (see FIG. 3). For immunohistochemistry, retrieved kidneys were halved, embedded in OCT compound (Sakura Finite U.S.A., Torrance, Calif.), and immediately frozen in liquid nitrogen. Coronal sections were cut (7 mm), mounted onto slides, dried for 1 hr, and then fixed in acetone for 10 minutes. Slides were immersed in PBS for 10 min and in 3% hydrogen peroxide/methanol for 5 minutes at room temperature to eliminate endogenous peroxidase activity. Endogenous biotin activity was blocked with the Biotin Blocking System (DAKO, Carpentaria, Calif.). After treating with normal rat serum (1:100), anti-mouse Gd-1 mAb (RB6.8C5) diluted at 1:100 in PBS with 1% bovine serum albumin (BSA) to detect neutrophils, or 1:50 dilutions of rat anti-mouse CD4 mAb (GK1.5) to detect CD4+ T cells, rat anti-mouse CD8a mAb (53-6.7) to detect CD8+ T cells, or rat anti-mouse macrophage (F4/80) mAb (SEROTEC, Raleigh, N.C.) was added to the sections. Control slides were incubated with rat IgG. After 1 hr, slides were washed 3× with PBS and incubated for 20 min with biotinylated rabbit anti-rat IgG antiserum (Sigma Aldrich) diluted 1:100 in PBS/1% BSA. After 3 washes in PBS, slides were incubated with streptavidin-horseradish peroxidase (DAKO) for 20 min. The DAB (3,3′-diaminobenzidine) substrate-chromagen solution (Vector Laboratories, Inc., Burlingame, Calif.) was applied to the slides for 0.5-3 min. After rinsing in dH2O, slides were counterstained with hematoxylin, washed with dH2O, cover-slipped, and viewed by light microscopy. Images were captured using Image Pro Plus (Media Cybernetics, Silver Spring, Md.).
To stain TLR5, 1 mg of anti-TLR5 mAb (ABR-Affinity Bioregions, Inc., Golden, Colo.) was applied to slides and incubated for 1 hr at room temperature and after washing biotinylated goat anti-mouse IgG antibody diluted 1:100 for 30 min at room temperature. After applying the DAB, the slides were washed with tap water, dipped for 3 sec in hematoxylin and then washed. The slides were dehydrated with increasing concentrations of ethanol to 50% and then immersed in Citra solve twice for 10 min each. The slides were washed with tap water, cover-slipped, and viewed by light microscopy.
The use of serum creatinine levels as an indication of renal dysfunction induced by imposition of ischemia-reperfusion injury was supported by the histopathology of the ischemic kidneys 24 hours after reperfusion (see FIG. 3). Control group animals given PBS 30 min before imposition of renal ischemia had severe tubular necrosis with caste formation evident 24 hours after reperfusion. Consistent with the induction of renal dysfunction by administration of 5 μg of flagellin, there was evidence of renal pathology 30 min after administration of 5 μg of flagellin without imposing ischemia and this increased in severity following imposition of renal ischemia and reperfusion with obvious hemorrhage, thrombosis, and caste formation. In contrast, animals given 0.5 μg flagellin 30 min before imposition of ischemia had low levels of leukocyte infiltration 24 hours after reperfusion but the renal architecture appeared relatively normal. The low, non-protective dose, 0.1 μg flagellin, did not rescue the renal pathology induced by ischemia/reperfusion injury. When kidneys of surviving animals were examined at day 7 post-reperfusion, marked decreases in tubular necrosis and leukocyte infiltration as well as the absence of thrombosis and case formation were observed in animals given 0.5 μg flagellin prior to imposition of renal ischemia. (see FIG. 4).
d. Neutrophil Infiltration to Damaged Renal Tissue
Since neutrophil infiltration and activation is a major contributor to the tissue injury following renal ischemia-reperfusion, ischemic kidneys were retrieved 9 and 24 hours after reperfusion from animals treated with PBS alone or with 0.5 μg flagellin before imposition of ischemia and the levels of neutrophil infiltration was assessed by immunohistochemically staining of prepared tissue sections.
To directly determine the number of neutrophils, macrophages, CD4+ T cells and CD8+ T cells in ischemic kidneys during reperfusion, one quarter pieces of the retrieved kidney were cut and weighed. The kidneys were incubated in RPMI 1640 culture medium with 2% fetal calf serum for 1 hr and then were pushed through a 70 mm cell strainer using a syringe plunger. The cells were collected and the erythrocytes lyses using ACK Lying Buffer (GIBCO, Grand island, NY). After 2 washes, viable cells were counted using Trepan blue exclusion. Aliquots of the cells were preincubated with anti-CD16/CD32 Cf receptor antibody (BD Harlingen, San Diego, Calif.) for 5 min to block nonspecific antibody binding and then samples were incubated with FITC-conjugated anti-CD45 mAb as well as PE-conjugated antibody to detect macrophages (F4/80) or CD8+ T cells (53-6.7) and APC-conjugated antibody to detect neutrophils (RB6.8C5) or CD4+ T cells (GK1.5) (all antibodies from BD Harlingen) for 30 min at 4° C. Cells were analyzed using two-color flow cytometry on a FACSCalibur (BD Biosciences, San Jose, Calif.). The forward scatter and FL1 (CD45+) channels were used to gate the leukocytes in the kidney tissue followed by analysis of the specific leukocyte populations. For each sample, 200,000 events were accumulated. The data were analyzed using CellQuest software (BD Biosciences). Total numbers of each leukocyte population were calculated by: (the total number of leukocytes counted)×(% of the leukocyte population counted in the CD45+ cells)/100. The data are reported as number of each leukocyte population/g kidney tissue from sham and I/R animals.
Marked decreases in neutrophil infiltration were observed 9 and 24 hours after reperfusion when animals were given 0.5 μg flagellin (see FIG. 5a). Direct quantitation of leukocytic infiltration into the ischemic kidneys indicated that 0.5 μg flagellin reduced neutrophil infiltration almost to the levels observed in the sham-operated control animals (see FIG. 5b). Decreases in the number of CD4 and CD8 T cells and macrophages were observed in ischemic kidneys 24 hours after reperfusion and administration of 0.5 μg of flagellin 30 minutes before ischemia decreased the number of both CD4 and CD8 T cells further.
Example 3
Flagellin Condition Decreases Pro-inflammatory Cytokine Expression During Reperfusion of Ischemic Kidneys
This example demonstrates the critical role of flagellin preventing chemokines CXCL1/KC and CXCL2/KC in directing leukocyte infiltration into ischemic kidney tissues. Previous studies have indicated that peak levels of the neutrophil chemoattractants CXCL1/KC and CXCL2/KC in ischemic kidneys occurs at 9 hours post-reperfusion.
To begin to investigate mechanisms underlying the decreased leukocytic infiltration into ischemic kidneys when animals were conditioned with 0.5 ug flagellin, kidneys were removed 9 and 24 hours after reperfusion and the mRNA and protein levels of neutrophil and macrophage chemoattractants were determined (FIG. 6). One-quarter pieces were cut from harvested kidneys and frozen in liquid nitrogen. Total tissue RNA was extracted using RNeasyTM Mini Kit (QIAGEN, Valencia, Calif.) and reverse transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, Calif.). Real time PCR was performed on a Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.) with test KC/CXCL1, MIP-2/CXCL2 and MCP-1/CCL2 primers and Mrp132 used as the control (Applied Biosystems, Foster City, Calif.).
Kidneys samples stored in liquid nitrogen were dissolved in 500 ml of PBS with 0.01 M EDTA and a proteinase inhibitor cocktail (10 mg/ml phenylmethyl solfonyl fluoride, 2 mg/ml aprotinin, 2 mg/ml leupeptin, 100 mg/ml Pefabloc SC, and 100 mg/ml chymostatin), and then 1 ml of 1.5% Triton X-100 in PBS was added. After incubation with agitation for 1 hr at 4° C., samples were centrifuged, the supernatant was collected, and the total protein concentration was determined using the BCATM Protein Assay Kit (Pierce, Rockford, Ill.). KC/CXCL1, MIP-2/CXCL2 and MCP-1/CCL2 concentrations were measured by sandwich ELISA using Quantikine M Kits (R&D Systems, Minneapolis, Minn.). To determine the activation of neutrophils during reperfusion of ischemic kidneys, the concentration of myeloperoxidase (MPO) was measured using the Mouse MPO ELISA test kit (Cell Sciences, Canton, Mass.). Results are reported as concentration of test protein per mg of total tissue protein ±SD.
Preconditioning with protective doses of flagellin (1.25 or 0.5 ug) resulted in significant decreases in mRNA expression and protein levels of the neutrophil chemoattractants CXCL1 and CXCL2 at 9 hours post-reperfusion. Expression of CCL2 mRNA or protein levels were low at both 9 and 24 hours after reperfusion and were not further influenced by preconditioning with flagellin. In addition, mRNA levels of the acute phase proteins IL-1b and IL-6 but not TNFa were also decreased in ischemic kidneys at 9 hours post-reperfusion in flagellin preconditioned animals (FIG. 6b).
Example 4
Protective Effect of Flagellin When Administered During Reperfusion of Ischemic Kidneys
This example demonstrates that flagellin provides a protective effect to acute ischemic treated kidneys when given after the initiation of reperfusion. As described above, bilateral renal pedicle occlusion was performed in the mice and serum creatinine levels were measured to determine the protective effect of flagellin on renal function after initiation of reperfusion.
Specifically, groups of C57BL/6 mice were subjected to 45 minutes of bilateral renal pedicle occlusion and were administered 0.514 of flagellin at various times following the removal of the renal clamps (See FIG. 7). Administration of flagellin 30 minutes before or within 30 min after declamping rescued the viability of all mice subjected to the ischemic injury. Flagellin administration 1 hour as well as at later times after initiation of reperfusion failed to rescue any of the mice from the injury. The protective effect of administering the flagellin 30 minutes before declamping or within 30 minutes following declamping was reflected by the low levels of serum creatinine monitored 24 hours after reperfusion of the ischemic kidneys (FIG. 7b).
Example 5
Protective Effect of Flagellin Requires TLR5 Signaling on Renal Parenchymal Cells
This example demonstrates the target source of protective effect of flagellin treatment during reperfusion of tissue. As discussed in Examples 1-4, reperfusion studies were performed on ischemic kidneys.
Radiation-induced bone marrow reconstituted chimeras were generated between wild-type C57BL/6 and B6.MyD88−/− mice. Radition-induced bone marrow reconstituted chimeras were generated by cutting the tips of femurs and tibias from wild-type C57BL/6 and B6.MyD88−/− mice and flushing with RPMI 1640 to collect the bone marrow cells. Bone marrow recipient mice first received 1100 Rad g-irradiation and then 3 hours later received 20×106 bone marrow cells intravenously. Irradiated CD90.1 recipients received bone marrow from congenic CD90.1 donors or vice versa. The reconstituted recipients received antibiotics (0.2 mg/ml sulfamethoxazole and 0.4 mg/ml trimethoprim) in the drinking water from day 1 to 7 as prophylaxis. The recipients were allowed to recover for 8-12 weeks and complete chimerism was confirmed by staining peripheral blood cells with FITC-conjugated 90.2 and PE-conjugated 90.1.
FIG. 8 shows that administration of 0.5 μg flagellin within 30 minutes of reperfusion of ischemic kidneys of wild-type C57BL/6 mice reconstituted with wild-type bone marrow decreased CXCL1 and CXCL2 mRNA levels. In MyD88−/− recipients reconstituted with either MyD88−/− or wild-type bone marrow, little CXCL1 and CXCL2 mRNA was induced during reperfusion of ischemic kidneys and administration of flagellin during reperfusion of these kidneys did not decrease the mRNA levels of these chemokines. In contrast, wild-type recipients of bone marrow from MyD88−/− donors expressed high levels of CXCL1 and CXCL2 mRNA and these levels were decreased by administration of flagellin during reperfusion of the ischemic kidneys. This demonstrates that the target of the flagellin was a parenchymal kidney cell rather than a leukocyte.
To investigate this further, renal sections from wild-type C57BL/6 and BALB/c mice were stained with anti-TLR5 antibody (FIG. 9a). The cells staining positively were primarily cells in the vasculature and staining was not apparent on renal tubular cells or glomeruli. Kidney sections from Moth Eaten mice that have a genetic defect in the expression of TLR 5 did not stain with the anti-TLR5 antibody. Expression levels of TLR5 mRNA were low in kidneys prior to imposition of renal ischemia/reperfusion but increased quickly during reperfusion of ischemic kidneys (FIG. 9b).
Example 6
Protective Effect of Flagellin in Hind Limb Ischemia Model
The potential protective effects of CBLB502 in a mouse hind limb ischemia model were investigated in a simulation of a tourniquet-induced ischemic injury. These studies originated from studies indicating that CBLB502 given to mice subjected to bilateral renal pedicle occlusion attenuated ischemic injury and renal dysfunction including decreased neutrophil chemoattractant production in response to reperfusion, decreased neutrophil infiltration into the ischemic kidney, and attenuation of rises in serum creatinine levels and loss of viability. The protectant could be given either before imposition of renal pedicle occlusion, or more importantly for clinical use, up to 30 min after reperfusion of the ischemic kidney.
The tourniquet-induced injury was modeled by tightening a wide rubber band on the left hind limb of mice for 2-4 hours. After the ischemic time, the rubber band was loosened and removed. The animals recovered from anesthesia but exhibited an inability to use the ischemic limb, which was dragged behind them for periods of up to 9 days. The ischemic injury also included edema of the limb that was clearly visible and that was quantified by wet-dry weight measures and comparison with the contralateral, non-ischemic hind limb, and induction of high levels of proinflammatory cytokines including neutrophil chemoattractants and intense neutrophil infiltration into the ischemic limb. In addition, injection of Evan's blue dye indicated considerable amounts of vascular leak in the ischemic limb (data not shown).
Studying the protective effects of CBLB502, mice were again subjected to a tourniquet-induced injury by tightening a wide rubber band on the left hind limb for 3 hours. After the ischemic time, the rubber bank was loosened and removed. Fifteen minutes upon removal of the rubber band and initiation of reperfusion, 0.5 μg of CBLB502 or vehicle (PBS) was administered intramuscularly into the left ischemic limb. Mice administered the CBLB502 has a more rapid recovery of limb usage and, by day 14 posted reperfusion, had a measurable grip strength for the ischemic limb of 10GF. In contrast, mice administered only PBS 15 minutes post reperfusion did not achieve this strength until day 21. Limbs given CBLB502 also had almost no evidence of edema 25 hours after reperfusion as evidence by a wet/dry weight ratio of 2.5 vs. 3.4 for the ischemic limb from mice given only vehicle at reperfusion (See FIG. 14C). With regard to vascular leak, the CBLB502 administered mice had a 7.4 μg Evan's Blue Dye per gram web weight limb tissue vs. 13.1 μg for vehicle administered mice, P<0.001) (See FIG. 14D). Finally, limbs treated with CBLB502 at reperfusion had significant decreases in tissue neutrophil and macrophage chemoattractant sCXCL2, CCL2, and myeloperoxidase (P<0.05 for each assay). A hematoxylin/eosin stain was performed on the hind limb muscle on day 14 after reperfusion following 3 hours of ischemia on mice treated with CBLB502 (FIG. 14A) or vehicle (FIG. 14B).
Injection of CBLB502 within 30 min of reperfusion also resulted in decreases in neutrophil chemoattractant production and neutrophil infiltration into the ischemic limb, visible decreases in edema, and accelerated recovery (day 4-6) of the use of the ischemic limbs. Histological examination also indicated greater muscle fiber bundle thickness in the ischemic limbs of animals treated with the protectant (data not shown).
These results will be further investigated through quantitative measurement of inflammation and limb dysfunction in animals subjected to hind limb ischemia with vs. without administration of the CBLB502 protectant. This will include quantification of other proinflammatory cytokines, direct quantification of neutrophil infiltration, quantitation of muscle fiber bundle thickness and apoptosis of muscle fibers, and magnitude and duration of edema.

## Claims

1. A method of treating a tissue of a mammal for the effects of reperfusion comprising administering to a mammal in need thereof a composition comprising flagellin.

2. The method of claim 1, wherein the reperfusion is caused by an injury.

3. The method of claim 2, wherein the injury is ischemia or hypoxia.

4. The method of claim 3, wherein the ischemia results from a condition selected from the group consisting of tachycardia, infarction, acute renal failure, stroke, hypotension, embolism, thromboembolism (blood clot), sickle cell disease, localized pressure to extremities to the body, and tumors.

5. The method of claim 3, wherein the hypoxia is selected from the group consisting of hypoxemic hypoxia resulting from carbon monoxide poisoning, sleep apnea, chronic obstructive pulmonary disease, respiratory arrest, or shunts; anemic hypoxia (O2 content low); and histotoxic hypoxia.

6. The method of claim 2, wherein the injury is selected from the group consisting of myocardial infarction, stroke, and acute renal injury.

7. The method of claim 4, wherein the localized pressure is due to a tourniquet.

8. The method of claim 1, wherein the composition is administered prior to, together with, or after the influx of oxygen resulting from reperfusion.

9. The method of claim 1, wherein the tissue is selected from the group consisting of: GI tract, lung, kidney, liver, cardiovascular system, blood vessel endothelium, central nervous system, peripheral nervous system, muscle, bone, and hair follicle.

10. The method of claim 1, wherein the composition is administered in combination with an antioxidant.

11. The method of claim 10, wherein the antioxidant is selected from the group consisting of amifostine and vitamin E.