WO2007028133A2 - Traitement et procede de prevention de la demence vasculaire - Google Patents

Traitement et procede de prevention de la demence vasculaire Download PDF

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WO2007028133A2
WO2007028133A2 PCT/US2006/034432 US2006034432W WO2007028133A2 WO 2007028133 A2 WO2007028133 A2 WO 2007028133A2 US 2006034432 W US2006034432 W US 2006034432W WO 2007028133 A2 WO2007028133 A2 WO 2007028133A2
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selectin
administration
pbs
rats
administrations
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PCT/US2006/034432
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WO2007028133A3 (fr
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John M. Hallenbeck
Hideaki Wakita
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Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
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Publication of WO2007028133A2 publication Critical patent/WO2007028133A2/fr
Publication of WO2007028133A3 publication Critical patent/WO2007028133A3/fr
Priority to US12/072,914 priority Critical patent/US7897575B2/en
Priority to US12/859,048 priority patent/US8940700B2/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention relates to compositions and methods for treatment of vascular dementia in a mammalian subject that involve inducing tolerance to E- selectin in the subject.
  • Vascular dementia is defined as the loss of cognitive function resulting from ischemic, ischemic-hypoxic, or hemorrhagic brain lesions as a result of cardiovascular diseases and cardiovascular pathologic changes. See, e.g., G. C. Roman, Med. Clin. North. Am., 86, pp. 477-99 (2002).
  • Vascular dementia is a chronic disorder. The symptoms of vascular dementia include cognitive loss, headaches, insomnia and memory loss.
  • Vascular dementia may be caused by multiple strokes (MID or post-stroke dementia) but also by single strategic strokes, multiple lacunes, and hypoperfusion lesions such as border zone infarcts and ischemic periventricular leukoencephalopathy (Binswanger's disease). See, G. C. Roman, supra. In Asian countries such as China, Japan and Korea, vascular dementia is observed in over 60% of patients with dementia. Primary and secondary prevention of stroke and cardiovascular disease decreases the burden of vascular dementia.
  • vascular dementia typically involves control of risk factors (i.e., hypertension, diabetes, smoking, hyperfibrinogenemia, hyperhomocystinemia, orthostatic hypotension, cardiac arrhythmias).
  • risk factors i.e., hypertension, diabetes, smoking, hyperfibrinogenemia, hyperhomocystinemia, orthostatic hypotension, cardiac arrhythmias.
  • hormone replacement therapy and estrogen replacement therapy could delay the onset of dementia in women.
  • hormone replacement therapy has negative side effects.
  • aspirin is widely prescribed for vascular dementia, there is very limited evidence that aspirin is actually effective in treating vascular dementia patients. See, P. S. Williams et al., Cochrane Database Syst.
  • Nimodipine has been implicated as a drug demonstrating short-term benefits in vascular dementia patients, but has not been justified as a long-term anti-dementia drug. See, J. M. Lopez- Arrieta and J. Birks, Cochrane Database Syst. Rev., 3, CD000147 (2002). In addition, clinical efficacy data of piracetam does not support the use of this drug in the treatment of dementia or cognitive impairment. L. Flicker and G. Grimley Evans, Cochrane Database Syst. Rev., 2, CDOOlOI l (2001).
  • the invention involves methods and compositions for preventing and treating vascular dementia.
  • vascular dementia can be treated by inducing immunological tolerance to E- selectin, a cell adhesion molecule that mediates the adhesion of various leukocytes, including neutrophils, monocytes, eosinophils, natural killer (NK) cells, and a subset of T cells, to activated endothelium.
  • E- selectin a cell adhesion molecule that mediates the adhesion of various leukocytes, including neutrophils, monocytes, eosinophils, natural killer (NK) cells, and a subset of T cells.
  • NK natural killer
  • one aspect of the invention is a pharmaceutical formulation comprising a pharmaceutically acceptable carrier and an effective amount of E- selectin, wherein the formulation is formulated for mucosal administration of E- selectin.
  • the mucosal administration can be intranasal, oral, enteral, vaginal, rectal, or respiratory administration.
  • the formulation is formulated for intra nasal administration, for example, as an aerosol.
  • the aerosol can be a dry aerosol.
  • the aerosol can be an atomized aqueous solution.
  • the E-selectin is an E-selectin polypeptide.
  • Such an E-selectin polypeptide can be a mammalian E-selectin polypeptide, for example, a human E-selectin, a bovine E-selectin, a murine E-selectin, a rat E-selectin or any other E-selectin polypeptide from a mammalian source.
  • the pharmaceutical formulation of the invention is typically _ administered in an effective amount (i ⁇ e., a therapeutically effective amount).
  • an effective amount of E-selectin is generally sufficient to induce tolerance to E-selectin in a mammal, hi some embodiments, an effective amount of E- selectin is sufficient to promote bystander-effect tolerance to E-selectin in a mammal.
  • effective amounts of E-selectin include ranges of E- selectin of about 0.005 mg to about 500 mg.
  • Another example of an effective amount of E-selectin is a range of E-selectin of about 5 ⁇ g to about 50 mg.
  • Another aspect of the invention is a method for treating or preventing vascular dementia in a mammal comprising mucosal administration of an amount of E-selectin polypeptide sufficient to induce bystander immune tolerance in the mammal.
  • vascular dementia can involve reduced blood flow to the brain.
  • the E-selectin is administered to mucosal surfaces of the mammal.
  • mucosal administration of E-selectin can include nasal, oral, enteral, vaginal, rectal, or respiratory administration, hi some embodiments, the administration is nasal or intranasal.
  • the inventive methods can involve a series of separate E-selectin administrations, hi some embodiments, the method involves a first series of administrations of E-selectin over a period of about two weeks. Such a first series of administrations can include about three to about seven administrations of E-selectin over the period of about two weeks.
  • the method can further comprise at least one booster series of administrations of E-selectin after at least two weeks from the first series of administrations. In some embodiments, each booster series of administrations comprise about three to about seven administrations of E-selectin over the period of two weeks.
  • Another aspect of the invention is a method for treating or preventing vascular dementia in a mammal comprising mucosal administration of an amount of E-selectin polypeptide sufficient to induce bystander immune tolerance in the mammal.
  • FIG. IA-B is the mucosal tolerance induction schedules employed for illustrative experiments described herein. For induction of mucosal tolerance, intranasal application of E-selectin was carried out.
  • FIG. IA shows the schedule for rats that received just a single series of E-selectin or PBS (control) administrations; rats receiving the single series of administrations are sometimes referred to herein as the "non-booster group.”
  • FIG. IB shows the schedule for rats that received a series of booster administrations of E-selectin or PBS (control) every three weeks; animals receiving such a series of administrations are sometimes referred to herein as the "booster group.”
  • FIG. 2 graphically illustrates the delayed type hypersensitivity (DTH) reaction in E-selectin-toIerized rats compared with PBS-tolerized rats.
  • DTH delayed type hypersensitivity
  • FIG. 3 graphically illustrates the discrimination indices of E-selectin tolerized and non-tolerized rats for the object recognition test.
  • the discrimination indices of the E-selectin and sham groups were significantly increased as compared with the PBS group. *: ⁇ 0.05, ⁇ *: ⁇ 0.01, ***: pO.OOl by Fisher's protected least significant difference procedure, as compared to PBS- treated animals.
  • FIG. 4 graphically illustrates of the percent of alternation by rats on the T-maze spontaneous alternation test. Rats have an instinctive behavioral tendency to alternate their choices between the arms of the T-maze more often than they repeat their initial choice. In the E-selectin-treated animals, the percent of rats that alternated was significantly increased at 90 days, as compared with the PBS-treated animals. *: p ⁇ 0.05 by ⁇ 2 test.
  • FIG. 5A-C graphically illustrates the percentages of correct arm entrance on the T maze left/right discrimination memory retention test.
  • FIG. 5A shows the percentage of correct choices made by E-selectin tolerized, PBS control and sham operated animals two weeks after carotid artery ligation.
  • FIG. 5B shows the percentage of correct choices made by E-selectin tolerized, PBS control and sham operated animals six weeks after carotid artery ligation.
  • FIG. 5C shows the percentage of correct choices made by E-selectin tolerized, PBS control and sham operated animals ten weeks after carotid artery ligation.
  • FIG. 6A-I shows photomicrographs of luxol fast blue stained sections of the corpus callosum (A, B, C), caudoputamen (D 5 E, F) and optic nerve (G, H, I) from rats that were subjected to a sham operation (C, F, I) or to bilateral ligation of the carotid arteries in animals that also received intranasal PBS (A, D, G) or E-selectin (B, E, H) on a booster tolerization schedule. Note that the extent of the white matter rarefaction was less severe in the E-selectin treated and sham- operated rats as compared with PBS group.
  • FIG. 7A-F shows photomicrographs of sections immunohistochemical stained using a cocktail of monoclonal antibodies directed against non- phosphorylated neurofilaments (SMI 311) that were obtained from the corpus callosum (A, B, C) and caudoputamen (D, E, F) from rats.
  • the rats were subjected to a sham operation (C, F) or to bilateral ligation of the carotid arteries in animals that also received intranasal PBS (A, D) or E-selectin (B, E) on a booster tolerization schedule. Note that the extent of the white matter rarefaction was less severe in the E-selectin treated and sham-operated rats as compared with PBS group.
  • FIG. 8 graphically illustrates the fiber densities of the corpus callosum, caudoputamen and optic nerve in rats subjected to a sham operation or to bilateral ligation of the carotid arteries and intranasal administration of either PBS or E-selectin on a booster tolerization schedule.
  • the fiber densities in E- selectin-treated animals were significantly higher than those in PBS-treated animals.
  • FIG. 9A-F shows photomicrographs of sections immunohistochemically stained for MHC class II antigens from the corpus callosum of rats that were subjected to a sham operation (C, F) or of rats that were subjected to bilateral ligation of the carotid arteries and intranasal PBS (A, D) or E-selectin (B, E) on a booster tolerization schedule.
  • C, F sham operation
  • A, D intranasal PBS
  • B, E E-selectin
  • FIG. 10 shows histograms of the numerical densities of MHC class II immunopositive microglia/macrophages in the corpus callosum of rats subjected to a sham operation, or to bilateral ligation of the carotid arteries in animals that also received intranasal PBS or E-selectin on a booster tolerization schedule.
  • FIG. 1 IA-B illustrates that CD4 positive T cells infiltrate brain tissues after carotid artery occlusion.
  • FIG. 11 A-B show rat corpus callosum sections immunohistochemically stained for CD4 (a marker for T cells). Sham-treated rats exhibited little or no CD4 positive T cell infiltration (FIG. 1 IA). In contrast, after carotid artery occlusion, increased numbers of CD4 positive T cells were observed in the corpus callosum of rats (FIG. 1 IB).
  • FIG. 12A-F shows photomicrographs of immunohistochemically stained sections for detection of TNF- ⁇ in the corpus callosum.
  • the rats were subjected to a sham operation (C, F) or to bilateral ligation of the carotid arteries in animals that also received intranasal PBS (A, D) or E-selectin (B, E) on a booster tolerization schedule.
  • C, F sham operation
  • A, D intranasal PBS
  • B, E E-selectin
  • FIG. 13 shows histograms of the numerical density of TNF- ⁇ - immunopositive vessels in the corpus callosum of rats subjected to a sham operation, or to bilateral ligation of the carotid arteries in animals that also received intranasal PBS or E-selectin on a booster tolerization schedule.
  • the number of TNF- ⁇ - immunopositive vessels was significantly reduced as compared with the PBS- treated animals.
  • FIG. 14A-F shows photomicrographs of sections immunohistochemically stained for detection of E-selectin in the corpus callosum.
  • the rats were subjected to a sham operation (C, F) or to bilateral ligation of the carotid arteries in animals that also received intranasal PBS (A, D) or E-selectin (B, E) on a booster tolerization schedule.
  • the sections were taken 90 days after carotid ligation.
  • E-selectin-treated and sham-operated animals E-selectin- immunopositive vessels were less prominent as compared to the PBS-treated animals.
  • FIG. 15 shows histograms of the numerical density of E-selectin- immunopositive vessels in the corpus callosum of rats subjected to a sham operation, or to bilateral ligation of the carotid arteries and either intranasal PBS or intranasal E-selectin on a booster tolerization schedule.
  • the number of E-selectin-immunopositive vessels was significantly reduced as compared with the PBS-treated animals.
  • FIG. 16 illustrates that occlusion of blood vessels feeding brain tissues leads to a number of problems, including disturbances in axonal transport, demyelination, induction of metalloproteinases (MMPs), blood brain barrier problems, activation of glial cells, infiltration of lymphocytes, edema, inflammation and immunological reactions that all lead to heightened tissue damage and further vascular injury.
  • MMPs metalloproteinases
  • the invention provides compositions and methods for treating and preventing negative consequences of vascular dementia.
  • tolerance refers to an antigen-induced immune unresponsiveness upon re-exposure to the antigen.
  • the antigen has previously been administered to induce such immune unresponsiveness.
  • the induced immune unresponsiveness may be specific for the administered antigen or may be antigen-non-specific as a result of production of an antigen-non-specific suppressor substance such as transforming growth factor beta (TGF ⁇ ), interleuldn-4 (IL-4) or interleukin-10 (IL-IO).
  • TGF ⁇ transforming growth factor beta
  • IL-4 interleuldn-4
  • IL-IO interleukin-10
  • suppressor tolerance means that T-cells, which are primed to recognize a specific antigen (E-selectin), release immune system suppressive cytokines after subsequent stimulation by that antigen (E-selectin).
  • E-selectin a specific antigen
  • Such suppressor T cells arise in the mucosal immune system and migrate to systemic sites where, upon antigen-specific reactivation, the suppressor T cells release TGF ⁇ , IL-4, IL-10 and other suppressive cytokines
  • a delayed type hypersensitivity reaction as used herein is a measure of whether the immune system actively reacts to an antigen or whether the immune system exhibits tolerance towards the antigen.
  • An antigen is introduced intradermally, and after about 48-72 hours post-injection the site of intradermal administration is observed. If the immune system does not exhibit tolerance, the injection site will appear red, inflamed, thickened, and tender. The swelling and thickening of the skin are a result of an immune response. The lack of a delayed type hypersensitivity response to the antigen indicates that the immune system is tolerant of the antigen.
  • a "subject" is a mammal or bird to which the E-selectin compositions of the invention are administered.
  • the subject can be bovine, rat, mouse, dog, pig, horse, goat, monkey, ape, human or other domestic or zoo mammal.
  • the subject can be chicken, turkey, parrot or other domestic or zoo bird.
  • E-selectin (also known as ELAM-I, CD62, and CD62E) is a cytokine- inducible cell surface glycoprotein that is found on endothelial cells.
  • E-selectin is a cell adhesion molecule that mediates the adhesion of various leukocytes, including neutrophils, monocytes, eosinophils, natural killer (NK) cells, and a subset of T cells, to activated endothelium.
  • E-selectin is expressed in vascular endothelial tissue. Pober, J. S., et al., J. Immunol. 136: 1680 (1986); Bevilacqua M. P., et al., Proc. Natl. Acad. Sci. 84: 9238 (1987). Expression of E-selectin is induced in response to the cytokines IL-I and TNF, as well as bacterial lipopolysaccharide (LPS), through transcriptional up-regulation. Pobor et al., supra; see also, Montgomery, et al., "Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-I) gene transcription" Proc. Natl. Acad.
  • ELAM-I endothelial-leukocyte adhesion molecule 1
  • E-selectin belongs to a family of adhesion molecules termed "selectins.” This family also includes P-selectin and L-selectin. Review articles relating to these selectins are provide in Lasky, "Selectins: interpreters of cell- specific carbohydrate information during inflammation” Science 258: 964 (1992) and Bevilacqua and Nelson, "Selectins” J. Clin. Invest. 91: 379 (1993). These molecules are characterized by common structural features such as an amino- terminal lectin-like domain, an epidermal growth factor (EGF) domain, and a discrete number of complement repeat modules (approximately 60 amino acids each) similar to those found in certain complement binding proteins.
  • EGF epidermal growth factor
  • nucleic acid and amino acid sequences for different types and species of E-selectin can be found in the art, for example, in the NCBI database. See website at ncbi.nlm.nih.gov. Thus, for example, the NCBI database provides a human E-selectin precursor amino acid sequence as accession number P16581 (gi: 126180). This sequence is provided below for easy reference as SEQ ID NOl .
  • the mature sequence for this human E-selectin extends from about amino acid 22 to amino acid 610.
  • the sequence for this mature E-selectin polypeptide is therefore as follows (SEQ ID NO:2).
  • An extracellular E-selectin domain may be used for tolerization of a subject.
  • the extracellular domain of the human E-selectin provided above includes a sequence of about amino acid 22 to about amino acid 556 and therefore has the following sequence (SEQ ID NO:3).
  • human E-selectin may be administered to a subject.
  • human E-selectin amino acid sequences can be found in the NCBI database, for example, as accession numbers AANO1237 (gi: 22536178), CAA17434 (gi: 3115964), AAA52376 (gi; 537524), CAI19357 (gi: 56417699), among others.
  • any such human E- selectin polypeptides can be used for tolerization of a subject to E-selectin.
  • non-human E-selectin may optimally induce tolerization to E-selectin in some human subjects. Therefore, the invention is directed to administering non-human E- selectin to subjects.
  • many sources and examples of non-human E-selectin are available. For example, nucleic acid and amino acid sequences for different types of non-human E-selectin can be found in the art, for example, in the NCBI database. See website at ncbi.nlm.nih.gov.
  • bovine, rat, mouse, dog, pig, horse, goat, monkey, ape or other mammalian E-selectin polypeptides can be administered to tolerize a subject to E-selectin.
  • Sequences for such mammalian E-selectins are available, for example, in the NCBI database.
  • bovine E-selectin polypeptide sequence that can be found in the NCBI database is the bovine E-selectin sequence with accession number P98107 (gi: 1346435). This bovine E-selectin sequence is the precursor sequence and is provided below for easy reference (SEQ ID NO:4). 1 MIVSQYLSAL TFVLLLFKES RTWSYHASTE MMTFEEARDY
  • the mature sequence for this bovine E-selectin extends from about amino acid 23 to amino acid 485.
  • the sequence for this mature bovine E-selectin polypeptide is therefore as follows (SEQ ID NO:5).
  • An extracellular E-selectin domain may be used for tolerization of a subject.
  • the extracellular domain of the bovine E-selectin provided above includes a sequence of about amino acid 23 to about amino acid 430 and therefore has the following sequence (SEQ ID NO:6).
  • bovine E-selectins As is known to the skilled artisan, some sequence variation exists among bovine E-selectins. Thus, other bovine E-selectin amino acid sequences can be found in the NCBI database, for example, as accession numbers S36772 (gi: 480377) and NP 776606 (gi: 27806407), among others. According to the invention, any such bovine E-selectin polypeptides can be used for tolerization of a subj ect to E-selectin.
  • rat E-selectin polypeptide sequence that can be found in the NCBI database is the rat E-selectin sequence with accession number P98105 (gi: 1346437). This rat E-selectin sequence is the precursor sequence and is provided below for easy reference (SEQ ID NO:7).
  • SEQ ID NO:7 MNASCFLSAL TFVLLIGKSI AWYYNASSEL MTYDEASAYC
  • the mature sequence for this rat E-selectin extends from about amino acid 22 to amino acid 549.
  • the sequence for this mature rat E-selectin polypeptide is therefore as follows (SEQ ID NO: 8).
  • An extracellular E-selectin domain may be used for tolerization of a subject.
  • the extracellular domain of the rat E-selectin provided above includes a sequence of about amino acid 22 to about amino acid 494 and therefore has the following sequence (SEQ ID NO:9).
  • mouse E-selectin polypeptide sequence that can be found in the NCBI database is the mouse E-selectin sequence with accession number B42755 (gi: 25295806). This mouse E-selectin sequence is the precursor sequence and is provided below for easy reference (SEQ ID NO: 10).
  • the mature sequence for this mouse E-selectin extends from about amino acid 22 to amino acid 612.
  • the sequence for this mature mouse E-selectin polypeptide is therefore as follows (SEQ ID NO:11).
  • An extracellular E-selectin domain may be used for tolerization of a subject.
  • the extracellular domain of the mouse E-selectin provided above includes a sequence of about amino acid 22 to about amino acid 557 and therefore has the following sequence (SEQ ID NO: 12).
  • Sources of E-selectin that can be used with the current invention include E-selectin that has been substantially purified from natural sources, recombinant E-selectin produced in prokaryotic or eukaryotic host cells by methods available in the art, and fragments of E-selectin. Furthermore, small organic molecules or peptides with structures that mimic an immunoreactive portion of E-selectin can also be used.
  • the immune system has the remarkable ability to mount a highly specific response against invading pathogens while ignoring self molecules.
  • This specificity is determined in part by the T lymphocyte, which expresses a randomly generated and unique T-cell receptor (TCR) that recognizes a peptide antigen bound to a major histocompatibility complex (MHC) molecule.
  • TCR T-cell receptor
  • MHC molecules can bind both to self peptides as well as to foreign peptides, where the self peptides are from the same organism as the MHC molecules (i.e., the host) and the foreign peptides are from a different, foreign organism.
  • the specificities of the peripheral TCR repertoire and/or the function of self-reactive T cells must be regulated such that the immune system ignores the self peptides or responds in a way that does not injure the host.
  • the physical elimination of autoreactive T cells during thymocyte development is the primary mechanism used by the immune system to establish such self- tolerance.
  • the immune system must either ignore a tissue-specific self peptide, or develop an active self-tolerance that relies on the suppression, physical elimination, or functional inactivation of mature autoreactive T cells.
  • tolerance refers to a selective inability of the immune system to respond to antigens and, for purposes of this invention, is a "learned" phenomenon; (2) both foreign and self-antigens can be targets of tolerogenic processes; (3) although tolerance can be mediated by suppressor cells, tolerance is not the same as immune suppression, either mechanistically or clinically; (4) tolerance can be maintained by active or passive processes and can result from cell inactivation, altered cellular function, or cell death; and (5) tolerance can be induced centrally (in the thymus) or peripherally.
  • immune tolerance is generated by exposure of mucosal surfaces to a tolerizing antigen (here, E-selectin).
  • a tolerizing antigen here, E-selectin.
  • Immune responses in mucosal tissues are self-limited, and repeated challenge with selected antigens results in a diminished response.
  • Mucosal administration of both high- and low- dose antigen results in immune tolerance, in which the immune response to subsequent systemic administration of antigen is blocked.
  • at least two mechanisms of immune tolerance may exist. Tolerance to high-doses of an antigen appears to occur by inactivation or clonal deletion of ThI and Th2 cells.
  • IL-4 interleukin-4
  • IL-IO interleukin-10
  • TGF- ⁇ TGF- ⁇
  • clonal anergy Inactivation of T cells by the clonal deletion tolerance mechanism is called clonal anergy and was originally described using a tissue culture system of cloned T cells.
  • Clonal anergy has since been defined as a reversible, induced tolerance state in which the T lymphocyte cannot produce its autocrine growth factor IL-2 or proliferate in response to the antigen it recognizes. In vitro, this unresponsive state is induced by stimulation of the T cell through its TCR in the absence of costimulatory signals, such as those occurring as a result of the interaction of B7 molecules on the antigen presenting cell (APC) with CD28 receptors on the T cell. In the absence of such costimulatory signals, T cells fail to proliferate, and TCR occupancy unaccompanied by proliferation down- regulates the T cell's responsiveness.
  • costimulatory signals such as those occurring as a result of the interaction of B7 molecules on the antigen presenting cell (APC) with CD28 receptors on the T
  • GALT gut-associated lymphoid tissue
  • BALT bronchial associated lymphoid tissue
  • MALT mucosa associated lymphoid tissue
  • these regulatory cells can be activated by the presence of the antigen, and will secrete immunosuppressive cytokines (IL-4, IL-10, and TGF- ⁇ ), thereby leading to suppression of ongoing immune responses to the antigen against which tolerance was induced and to unrelated self antigens.
  • immunosuppressive cytokines IL-4, IL-10, and TGF- ⁇
  • clonal deletion or clonal anergy of antigen-reactive lymphocytes generally occurs at high dosages.
  • IL-4, IL-10 and TGF- ⁇ are antigen-nonspecific immunosuppressive factors that suppress immune attack regardless of the antigen that triggers the attack.
  • TGF- ⁇ is thought to be one of the most important cytokines for bystander tolerance.
  • IL-4 enhances Th2 response (i.e., acts on T-cell precursors and causes them to differentiate preferentially into Th2 cells at the expense of ThI responses).
  • IL-4 also indirectly inhibits ThI exacerbation.
  • IL-IO is a direct inhibitor of ThI responses.
  • inducing E-selectin tolerance has many utilities. For example, it can be used in preventing and treating vascular dementia, strokes and other forms of vascular disease. Additionally, it can be used in treating disorders in which E-selectin has been determined, or may be determined, to play a role, such as, for example, lung injury, psoriasis, contact dermatitis, inflammatory bowel disease, arthritis, and the like.
  • lung injury psoriasis
  • contact dermatitis e.g., contact dermatitis, inflammatory bowel disease, arthritis, and the like.
  • Vascular Dementia is comprised of a number of heterogeneous pathological conditions, which results from ischemic or hemorrhagic brain lesions as well as from lesions that develop during protracted hypoperfusion.
  • the subcortical ischemic form of vascular dementia is a common type of vascular cognitive impairment and dementia, and one of the major causes of cognitive decline in elderly people.
  • Subcortical ischemic vascular dementia mainly results from small-vessel disease, which causes lacunes and extensive white matter lesions, and can be compared to large vessel dementia or cortical vascular dementia (Roman GC, Neurology. 1993 ;43 :250-260, Roman GC
  • ischemic lesions in subcortical ischemic vascular dementia particularly affect the frontal-subcortical circuits an observation that explains the major cognitive and clinical neurological effects of vascular dementia (Ishii N, Neurologyl986; 36: 340-45, Cummings JL, Arch Neuroll993; 50:873-80).
  • Subcortical ischemic vascular dementia is also caused by persistent hypertension (de Leeuw FE, Brain. 2002; 125 -.765-772) and hypoperfusion due to congestive heart failure (Roman GC. Neurol Res. 2004;26:454-458), atrial fibrillation (de Leeuw FE, Neurology. 2000;54:1795- 1801), and obstructive sleep apnea (Kamba M, J. Neurol. Neurosurg. Psychiatry. 2001; 71; 334-339).
  • Ischemic white matter lesions a common finding in elderly people, are the characteristic pathological changes in subcortical ischemic vascular dementia and cognitive impairment, and cognitive dysfunctions are related to lesion severity (Hachinski VC, Arch Neurol. 1987;4:21-23, Pantoni L, Alzheimer Dis. Assoc. Disord. 1999;13(su ⁇ l 3):S49-S54, de Groot JC, Neurology2001; 56:1539-1545).
  • Cerebrovascular white matter lesions constitute the core pathology in several types of vascular dementia, such as Binswanger's disease, cerebral amyloid angiopathy, and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).
  • cerebrovascular white matter lesions can be experimentally induced in the rat brains as a result of protracted hypoperfusion induced by the permanent occlusion of both common carotid arteries (Waldta H, Acta Neuropathol. (Berl) 1994;87: 484-492).
  • cerebral blood flow decreases to about 40 to about 82 % of normal over extended periods of time (Tsuchiya M, Exp. Brain Res. 89:87-92 (1992): Otori T, Cerebrovasc. Dis. 6(su ⁇ pl): 71 (1996); Tomimoto H, Brain Nerve 49:639-644 (1997); Ouchi Y, J Nucl Med. 39:198-202 (1998)).
  • E-selectin a glycoprotein
  • endothelial cells Bevilacqua MP, Science 243(4895):1160-1165 (1989)). It mediates the interaction between leukocytes, platelets, and the endothelium (Bevilacqua (1989)).
  • Normal resting endothelial cells do not express E-selectin (Pigott R, BBRC 187:584-9 (1992)).
  • the expression of E-selectin is induced in response to proinflammatory cytokines, such as IL-I and TNF, and its increased surface expression is a reflection of endothelial activation (Bevilcqua MP, Annu. Rev.
  • vessel activation and E-selectin expression play a pivotal role in the inflammatory process and subsequent tissue injury after cerebral ischemia through leukocyte- endothelial attachment and infiltration of leukocytes.
  • a novel method to induce generation of regulatory T cells targeted to activating blood vessels has been developed involving administration of E- selectin to induce mucosal tolerance to that antigen.
  • E-selectin participates in inflammation and immunological responses during and after an ischemic insult and serves to target immunomodulatory regulatory T cells to blood vessel segments that are undergoing endothelial cell activation. As illustrated in a previous application by the inventors U.S. Ser. No. 10/296,423 (filed June 11, 2003, and incorporated herein in its entirety), these regulatory T cells may prevent stroke and protect against ischemic brain damage through "bystander suppression.”
  • One aspect of the current invention is a method for inducing E-selectin tolerance in a subject.
  • This method involved administering E-selectin to mucosal tissues of a subject.
  • any E-selectin that can induce immune tolerance in the subject to E-selectin can be used.
  • an E-selectin with any of SEQ ID NO: 1-12 can be used in the invention.
  • Tolerance to an antigen such as E-selectin can be induced by administration to many types of mucosal tissues including oral, nasal, enteral, vaginal, rectal and respiratory mucosa. By reducing enzymatic degradation in the gastrointestinal tract, lower doses of antigen may sometimes be used for nonoral routes of administration. In some embodiments, tolerance is induced by intranasal administration of E-selectin.
  • Tolerance can be induced by a single series of E-selectin administrations.
  • E-selectin tolerance or E-selectin bystander tolerance can be induced by an administration protocol involving a single series of five administrations of E-selectin over a period of two weeks. In other embodiments, this regimen of five administrations over two weeks is repeated at least once. Repeating a series of E-selectin administration is referred to herein a "booster" series of administrations.
  • a single series of E-selectin dosages is administered within about one to two weeks.
  • the "booster" administrations repeat this series of E-selectin administrations after a period of several weeks without any E-selectin administrations, hi some embodiments, this booster regimen is repeated every three weeks for the remainder of the life of the subject.
  • E-selectin sources can vary.
  • minimum and maximum effective dosages vary depending on the method of administration.
  • Suppression of the clinical and histological changes associated with vascular dementia can occur within a specific dosage range, which, however, varies depending on the organism receiving the dosage, the route of administration, whether E-selectin is administered in conjunction with other co-stimulatory molecules, and the specific regimen of E-selectin administration.
  • nasal administration requires a smaller dosage than oral, enteral, rectal, or vaginal administration.
  • dosages are used that range from about 0.005 to about 500 mg/day, or from about 0.05 to about 50 mg/day.
  • mucosal dosages are from about 0.5 ⁇ g to about 50 mg per administration, or from about 0.5 ⁇ g to about 5 mg per administration.
  • optimization of the dosage necessary for immune suppression involves no more than routine experimentation.
  • E-selectin formulations of the present invention may comprise inert constituents including pharmaceutically-acceptable carriers, diluents, solubilizing agents, emulsifying agents, salts, and the like, as are available in the art.
  • Preferred E-selectin formulations are intranasal formulations including normal saline solutions, such as, for example, isotonic and physiologically buffered saline solutions and phosphate-buffered saline (PBS) solutions.
  • the total volume of the intranasal formulations is typically less than 1 milliliter, preferably less than 100 ⁇ l.
  • tablets may be formulated in accordance with conventional procedures employing solid carriers well-known in the art.
  • Capsules employed for oral formulations to be used with the methods of the present invention may be made from any pharmaceutically acceptable material, such as gelatin or cellulose derivatives.
  • Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are also contemplated, such as those described in U.S. Pat. No. 4,704,295, "Enteric Film-Coating Compositions," issued Nov. 3, 1987; U.S. Pat. No. 4,556,552, "Enteric Film- Coating Compositions," issued Dec. 3, 1985; U.S. Pat. No.
  • solid carriers include starch, sugar, bentonite, silica, and other commonly used carriers.
  • carriers and diluents which may be used in the formulations of the present invention include saline, syrup, dextrose, and water. E-selectin can also be administered in an aerosol or inhaled form.
  • formulations for tolerizing agents administered by inhalation are provided in Weiner, H. et al, "Improved treatment of autoimmune diseases by aerosol administration of auto antigens," WO9108760 (1991).
  • the antigens can be administered as dry powder particles or as an atomized aqueous solution suspended in a carrier gas (e.g., air, N.sub.2, and the like).
  • E-selectin formulations may be in the form of dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 microns, preferably between 2 and 3 micrqns : Finely divided particles may be prepared by pulverization and screen filtration using techniques available in the art. The particles may be administered by inhaling a predetermined quantity of the finely divided or powdered material.
  • the E-selectin formulations of the present invention may also be administered in soluble form as an aerosol spray using, for example, a nebulizer such as those described in U.S. Pat. No. 4,624,251 issued Nov. 25, 1986; U.S. Pat. No. 3,703,173 issued Nov. 21, 1972; U.S. Pat. No. 3,561,444 issued Feb. 9, 1971; and U.S. Pat. No. 4,635,627 issued Jan. 13, 1971.
  • Other systems of aerosol delivery such as the pressurized metered dose inhaler (MDI) and the dry powder inhaler (see, e.g., Newman, S. P. in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds. pp. 197-224, Butterworths, London, England, 1984) can be used when practicing the present invention.
  • MDI pressurized metered dose inhaler
  • dry powder inhaler see, e.g., Newman, S.
  • SHR-SP rats One useful animal model for the analysis of E-selectin formulations and their effectiveness in treating or preventing stroke is the stroke-prone and spontaneously hypertensive SHR-SP rat (Okamoto, K. et al., "Establishment of the stroke-prone spontaneously hypertensive rat (SHR)," Circ. Res. (Suppl.) 34, 35: 1 (1974)).
  • SHR-SP rats maybe obtained from professor Yukio Yamori, graduate School of Human and Environmental Studies, Kyoto University, Yoshida Nihonmatsu-cho, Sakyo-ku, Kyoto, 606-8316, Japan.
  • SHR-SP rats typically die of early-onset cardiovascular disease, sometimes as early as 14 weeks of age, although some SHR-SP rats live to more than 56 weeks of age. Frequently, the cardiovascular disease manifests as a stroke in these rats.
  • the occurrence of a stroke in these rats is diagnosed by measuring behavioral status that could be divided into 4 patterns: no abnormalities (grade 1), irritable (grade 2), lethargic (grade 3), akinetic (grade 4) (Yamori, U. et al., Japanese Circulation Journal 46: 274 (1982)).
  • the brains of SHR-SP rats at the time of death typically contain numerous infarcts and intraparenchymal hemorrhage areas that can be counted and measured through microscopic analysis of brain sections.
  • the effectiveness of an E-selectin formulation can be determined by comparing infarct and intraparenchymal hemorrhage numbers and areas in SHR-SP rats that have been treated with control or test E-selectin formulations.
  • the administration regimen can be evaluated in a similar manner.
  • the control formulations can consist of only carrier components or non-specific antigens (e.g., ovalbumin).
  • the efficacy of a regimen of booster administrations versus a single series of E-selectin administration can be compared. Examples of these procedures and comparisons are disclosed in the Examples section of this specification.
  • Another useful animal model for the analysis of E-selectin formulations and their effectiveness in treating or preventing vascular dementia involves occlusion of carotid arteries in rats. See, e.g., Sarti et al., Persistent impairment of gait performances and wor ⁇ ng memory after bilateral common carotid artery occlusion in the adult Wistar rat, BEHAVIOURAL BRAIN RESEARCH 136: 13-20 (2002).
  • cerebrovascular white matter lesions can be experimentally induced in the rat brain as a result of chronic cerebral hypoperfusion. This model is created by permanent occlusion of both common carotid arteries.
  • Wistar rats can be anesthetized, the bilateral common carotid arteries are exposed through a midline cervical incision and the common carotid arteries are double-ligated with silk sutures bilaterally.
  • the cerebral blood flow (CBF) then initially decreases by about 30 to 50% of the control after ligation.
  • the CBF values later range from 40 to 80 % of control after about 1 week to about 1 month.
  • white matter rarefaction is detected in rats with occluded carotid arteries after 3 days or more of ligation
  • Microglia and astroglia were activated after arterial occlusion in a manner that predicts the extent and severity of the subsequent white matter damages.
  • a few lymphocytes, labeled with CD4 or CD8 antibodies, can be detected as scattered in the white matter after arterial occlusion.
  • Other pathological changes include axonal damage and demyelination in white matter lesions. Blood-brain barrier disruptions have also been observed as well as increased matrix metalloproteinase activity in white matter lesions. These changes appear very similar to those in human cerebrovascular white matter lesions.
  • This model is a useful tool to assess the effectiveness of E-selectin tolerization on the pathophysiology of chronic cerebral hypoperfusion, and to provide data for determining optimal dosages and dosage regimens for preventing the cognitive impairment and white matter lesions in patients with cerebrovascular disease.
  • an E-selectin formulation for treating or preventing vascular dementia can therefore be determined by observing the gait performance, memory, learning abilities and the incidence and severity of white matter lesions in rats with carotid artery occlusions. Similarly, the E-selectin dosage and administration schedule can be adjusted pursuant to the memory and learning abilities of human patients being treated for vascular dementia. Assessment of the effect of E-selectin formulations on an immune response to E-selectin can also be made, for example, by determining diminution in certain inflammation markers, such as the number of activated T-cell clones directed against activated vascular tissue. Immunological tolerance can be measured by a number of methods that are well-known in the art.
  • delayed type hypersensitivity (DTH) response can measured in animals by injecting E-selectin, for example, into the footpad or ear flap of an organism to be analyzed and then administering a challenge injection, for example into a footpad or an ear, at a later time, typically more than 1 week later, most preferably 2 weeks later.
  • DTH reactions can be measured after the elicitation injection as the increase in swelling at the site of the antigen rechallenge.
  • Footpad or ear swelling can be measured at, for example, 0, 24 and 48 hr after challenge.
  • the optimum dosage of E-selectin is one that induces E-selectin tolerance, for example, bystander tolerance. In other embodiments, the optimum dosage of E-selectin is one that generates the maximum protective effect in preventing vascular dementia, stroke and the like. In other embodiments, the optimum dosage of E-selectin is one generating the maximum beneficial effect on damaged tissue caused by arterial occlusion. An effective dosage causes at least a statistically or clinically significant attenuation of at least one marker, symptom, or histological evidence characteristic of vascular dementia.
  • Markers, symptoms and histological evidence characteristic of vascular dementia include memory loss, confusion, disturbances in axonal transport, demyelination, induction of metalloproteinases (MMPs), activation of glial cells, infiltration of lymphocytes, edema, inflammation and immunological reactions that lead to tissue damage and further vascular injury. Stabilization of symptoms or diminution of tissue damage, under conditions wherein control patients or animals experience a worsening of symptoms or tissue damage, is one indicator of efficacy of a suppressive treatment. Ascertaining the effective dosage range as well as the optimum amount of E-selectin is accomplished using the teachings of the present application as well as any available teachings in the art.
  • an optimum regimen for administering E-selectin is determined in light of the information disclosed herein and well known information concerning administration of bystander antigens and autoantigens. Routine variation of dosages, combinations, and duration of treatment is performed under circumstances wherein the effects of such variations on the organism can be measured.
  • dosages for mammals and humans can be determined by beginning with a relatively low dose (e.g., 1 microgram) and progressively increasing the dosage while measuring appropriate responses (e.g., number of TGF- ⁇ , IL-4, and/or IL-IO secreting cells; number and activation of immune attack T-cells in the blood (e.g., by limiting dilution analysis and ability to proliferate); and/or disease severity).
  • the optimum dosage provides maximal prevention from vascular dementia or the maximum protection from tissue damage caused by vascular occlusion while minimizing undesirable side effects.
  • Potential side effects include the generation of pathogenic autoantibodies (Hu, W. et al., "Experimental mucosal induction of uveitis with the 60-kDa heat shock protein-derived peptide 336-351,” Eur. J. Immunol. 28: 2444 (1998); Genain C.
  • An effective dosage causes at least a statistically or clinically significant attenuation of at least one symptom of vascular dementia, or at least a statistically or clinically significant attenuation of the occurrence rate or time to onset of vascular occlusion.
  • the maximum effective dosage of a bystander antigen in humans can be ascertained by testing progressively higher dosages in clinical trials starting with a relatively low dosage, for example 0.5 ⁇ g per administration.
  • Preferred dosages for intranasal instillations are from about 0.5 to about 50 mg per administration, preferably for humans approximately from about 0.5 ⁇ g to 5 mg per administration. For rats, one preferred dosage is 5 ⁇ g per administration.
  • Preferred aerosol pharmaceutical formulations may comprise, for example, a physiologically-acceptable buffered saline solution containing between about 0.1 mg and about 300 mg, or about 1 mg and about 300 mg of E- selectin.
  • E-selectin is administered in a series of administrations. Typically these administrations are spaced apart over a period of 1 to 2 weeks. For example and as further detailed in the Examples, E-selectin can be administered in five intranasal administrations over a two week period.
  • This protocol can involve administering E-selectin every other day for ten days.
  • the administration regimen is repeated in booster administrations that are generally administered several weeks apart.
  • booster administrations are given after every three weeks.
  • Booster administrations may include a series of administrations, as described above for initial administrations.
  • Cytokine and non-cytokine synergists can be used in conjunction with E- selectin in the present invention to enhance the effectiveness of E-selectin tolerization.
  • Administration "in conjunction with” encompasses simultaneous and sequential administration, as well as administration in combined form or separately. Oral and parenteral use of cytokine synergists (Type I interferons) has been described in Hafler, D. A.
  • Th2 enhancing cytokines is described in Weiner H. L., et al., "Treatment of autoimmune disease using oral tolerization and/or Th2-enhancing cytokines," WO95275000(1995).
  • IL-4 and IL-IO can be administered in the manner described in Weiner et al. Id.
  • Non-limiting examples of non-cytokine synergists for use in the present invention include bacterial lipopolysaccharides from a wide variety of gram negative bacteria such as various subtypes of E. coli and Salmonella (LPS, Sigma Chemical Co., St. Louis, Mo.; Difco, Detroit, Mich.; BIOMOL Res. Labs., Madison, Pa.), Lipid A (Sigma Chemical Co., St. Louis, Mo.; ICN Biochemicals, Cleveland, Ohio; Polysciences, Inc., Warrington, Pa.); immunoregulatory lipoproteins, such as peptides covalently linked to tripalmitoyl-S-glycarylcysteinyl-seryl-serine (P. sub.3 C55) which can be obtained as disclosed in Deres, K. et al. (Nature, 342: 561-564, "In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine,"
  • the effective dosage range for noncytokine synergists for mammals is from about 15 ng to about 15 mg per kg weight and preferably 300 ng-12 mg per kg weight.
  • the effective dosage range for oral Type I interferon for mammals is from 1,000-150,000 units with no maximum effective dosage having been discerned.
  • Another active compound that may be useful in combination with E- selectin is methotrexate which is known to cause a marked Th2 immune deviation with greatly increased IL-4 secretion when given on a pulse regimen (Weiner et al., "Treatment of Autoimmune Disease Using Tolerization in Combination with Methotrexate," U.S. Pat. No. 5,935,577 (1999).
  • the co-stimulatory agent is preferably administered within 24 hours of administration of E-selectin. More preferably, it is administered at the same time as E-selectin. Most preferably, both are administered in a combined oral formulation.
  • EXAMPLE 1 Reduction of brain infarcts by administration of E-selectin. This Example illustrates the effects of administering E-selectin on reducing the incidence and size of infarcts in the brains of stroke-prone rats. Further information on stroke treatment by E-selectin tolerization can be obtained in a related application, PCT Application Ser. No. PCT/USOl/16583, which is incorporated by reference herein in its entirety. Materials and Methods
  • E-selectin and control preparations were administered in the following manner: SHR-SP rats were divided into three groups: (1) a saline (PBS) control group, (2) an E-selectin administration group (ES group), and (3) an ovalbumin (OVA) administration group (OVA group).
  • ES and OVA groups were divided into single (non-booster) and repetitive (booster) administration groups.
  • PBS phosphate-buffered saline
  • E-selectin in 20 ⁇ l PBS was administered into each nostril every other day for 10 days for a total of 5 administrations.
  • an initial 2.5 ⁇ g of E-selectin in 20 ⁇ l PBS was administered as above for the non-booster group; additionally, 2.5 ⁇ g of E-selectin in 20 ⁇ l of PBS was administered intranasally into each nostril every other day for 10 days (3 weeks after the first E-selectin course) and repeated every 3 weeks until the animals were sacrificed.
  • OVA non- booster group 2.5 ⁇ g ovalbumin in 20 ⁇ l PBS was administered into each nostril every other day for 10 days for a total of 5 administrations.
  • OVA booster group an initial 2.5 ⁇ g of ovalbumin in 20 ⁇ l PBS was administered into each nostril as above for the non-booster group; additionally, 2.5 ⁇ g of ovalbumin in 20 ⁇ l of PBS was administered intranasally into each nostril every other day for 10 days (3 weeks after the first ovalbumin course) and repeated every 3 weeks until the animals were sacrificed.
  • the rats were evaluated for physical and neurological signs of stroke. These evaluations included an assessment of excitement (i.e., piloerection, hyperkinesis), hyperirratibility (i.e., jumping, trying to escape), behavioral and psychological depression (i.e., hypokinesis, hyposthenia, hyporesponsiveness), motion disturbance (i.e., transient episode of repetitive lifting of paws, ataxia, paresis, paralysis), and late symptoms observed near the time of death (i.e., apathy, coma, urinary incontinence). The rats were also monitored by measuring arterial blood pressure, body weight, heart weight, and arterial blood gas using methods available in the art.
  • excitement i.e., piloerection, hyperkinesis
  • hyperirratibility i.e., jumping, trying to escape
  • behavioral and psychological depression i.e., hypokinesis, hyposthenia, hyporesponsiveness
  • motion disturbance i.e., transient episode of repetitive lifting of paws, ataxia
  • Infarcts were evaluated in the following manner. When animals showed signs of cardiac failure, kidney failure, or stroke, they were perfused and their brains were removed for histology and image processing. Sections from 8 predetermined stereotactic levels were cut from each brain (total of 240 sections). The number and area of infarcts or hemorrhages were determined for each section from each animal. Statistical significance of E-selectin administrations was determined by comparing E-selectin groups to control groups by a Cox Proportional Hazards Model.
  • the animals lived for variable periods from 14 weeks to the termination of the experiment at 56 weeks. Deaths were caused by heart failure and kidney failure secondary to severe hypertension (mean systolic blood pressure 215 mm Hg), as well as by strokes. Average age at time of death and average systolic blood pressure did not differ among the experimental groups.
  • Intraparenchyrnal hemorrhages were absent from the E-selectin booster group, but were present at an average number of from about 3.2 to about 2.3 per brain section analyzed in control and single E-selectin administration groups (see Tables I-IV).
  • This Example provides data showing that tolerance to E-selectin was induced by the intranasal administration protocol of E-selectin described above, which resulted in decreased stroke-related tissue damage.
  • E-selectin or control PBS preparations were administered to rats as described in Example 1 for the non-booster groups.
  • 2.5 ⁇ g E-selectin in 20 ⁇ l PBS was administered into each nostril every other day for 10 days for a total of 5 administrations.
  • DTH delayed-type hypersensitivity
  • results of the delayed-type hypersensitivity assay demonstrated that intranasal instillation of human E-selectin induced tolerance.
  • Administration of E-selectin intranasally before footpad and ear injection resulted in a significant suppression of ear swelling compared to control groups, as measured with Mitsutoyo microcalipers.
  • rats "tolerized” with PBS exhibited a an approximate 55% change in ear thickness (about 0.36 mm swelling), while the E-selectin tolerized rats exhibited only about a 20% change in ear thickness
  • EXAMPLE 3 Vascular Dementia Animal Model This Example provides information about the animal model used for evaluation of vascular dementia and the effects of E-selectin tolerization on vascular dementia.
  • the experimental model used for vascular dementia was hypoperfusion of Wistar rat brains.
  • previous work has shown that cerebrovascular white matter lesions can be experimentally induced in the rat brain as a result of chronic cerebral hypoperfusion and that such hypoperfusion leads to impaired memory.
  • This model is created by permanent occlusion of both common carotid arteries as described below.
  • the animals were anesthetized with 5 % isoflurane for induction and 1.5 % isoflurane for maintenance in 30 % O 2 /70 %N 2 O by facemask.
  • the core body temperature was monitored and maintained at 37.0 ⁇ 0.5 0 C using a heating pad and a heating lamp.
  • both common carotid arteries were exposed and double-ligated with 5-0 silk sutures as previously described by Wakita H., Acta Neuropathol. (Berl) 1994;87: 484-492.
  • the rats were kept in cages with food and water ad libitum.
  • four animals were subjected to the same surgical procedures without bilateral carotid ligation.
  • Cerebral blood flow (CBF) after carotid artery occlusion was 30 to 50 % of the control several days after ligation.
  • the CBF decreased to values ranging from 40 to 80 % of control over a prolonged period (1 week - 1 month).
  • FIGs. 6-13 The effects of carotid artery occlusion upon brain tissues are illustrated in FIGs. 6-13 by comparing results for the PBS treated animals, who received bilateral carotid ligation after administration of PBS during the tolerization schedule (shown in FIG. 1), with the sham-operated animals that did not receive bilateral carotid ligation.
  • FIGs. 6-8 white matter becomes rarefied after carotid artery occlusion (compare PBS vs. Sham-operated tissue sections in FIG. 6-7 and graphic summary in FIG. 8).
  • FIG. 9 shows that glial cells become activated in white matter after carotid artery occlusion.
  • FIG. 9 shows that glial cells become activated in white matter after carotid artery occlusion.
  • microglia and astroglia were activated briefly after vascular occlusion. Moreover, such activation was predictive of the extent and severity of the subsequent white matter damage.
  • CD4 or CD8 antibodies FIG. 4
  • FIG. 11 greater numbers of CD4 positive T cells infiltrated the corpus callosum of rats who received bilateral carotid ligation (“Ligated” rats) than was observed in rats that did not receive bilateral carotid ligation (“Sham” operated rats).
  • CD4 or CD8 positive T cells were scattered in the white matter after occlusion.
  • FIGs. 12 show that the numbers of TNF- ⁇ immunopositive blood vessels increase after carotid artery occlusion.
  • This Example illustrates that mucosal tolerization to E-selectin protects against several forms of memory dysfunction and white matter damage in the rat model of vascular cognitive impairment.
  • the tolerization schedule involved a single series of administrations or a single series of administrations plus a booster series of administrations as follows (see FIG. 1):
  • Ear thickness increase over baseline was measured with microcalipers (Mitsutoyo Co, Ltd, Kawasaki, Kanagawa, Japan) 2 days later.
  • the animals were anesthetized with 5 % isoflurane for induction and 1.5 % isoflurane for maintenance in 30 % O 2 /70 %N 2 O by facemask.
  • the core body temperature was monitored and maintained at 37.0 ⁇ 0.5 0 C using a heating pad and a heating lamp.
  • both common carotid arteries were exposed and double-ligated with 5-0 silk sutures as previously described by Wakita H., Acta Neuropathol. (Berl) 1994;87: 484-492.
  • the rats were kept in cages with food and water ad libitum.
  • four animals were subjected to the same surgical procedures without bilateral carotid ligation.
  • Behavioral assessment consisted of object recognition, T-maze spontaneous alternation, and T-maze left/right discrimination memory retention tests. An observer who was blind to the treatments performed behavioral assessment.
  • Object Recognition test This test evaluates non-spatial working memory related to the frontal subcortical circuits (Ennaceur A. Behav Brain Res 1988;31:47-59, Sarti C, Behav Brain Res. 2002;136:13-20).
  • the apparatus was formed by a glass box (30 X 60 X 30 cm). The apparatus was illuminated by a 100 W lamp suspended 70 cm above the box in a darkened room. The day before testing, rats were habituated to the test environment by exploring the box for 6 min without objects. On the day of the test, a session of two trials was given. The inter-trial interval was 60 min.
  • T-ma ⁇ e spontaneous alternation This test evaluates spatial working memory related to the frontal subcortical circuits (Bartolini L., Pharmacol Biochem Behav. 1992;43:1161-1164, Sarti C, Behav Brain Res. 2002;136:13- 20).
  • Spontaneous alternation was investigated in an acrylic T shaped runway. It consisted of a start box (20 X 18 cm) and start arm (60 cm long), and two identical goal arms (both 50 cm long). All arms were 10 cm wide and 10 cm high. Spontaneous alternation refers to the instinctive behavioral tendency by which rats typically alternate their choices between the arms of the T-maze more often than they repeat their initial choice. Rats were placed in the start box of the T-maze and a maximum time of 5 min was allowed for them to explore the maze. Spontaneous alternation was defined as following: the rat entered with all four feet into one goal arm, came back, and then entered with all four feet into the opposite goal arm. The number of rats who alternated was recorded.
  • T-maze left/right discrimination memory retention This test evaluates spacial reference memory related to the hippocampus and caudoputamen (Oliveira MG, Neurobiol Learn Mem 1997;68:32-41). This test was repeated at 2, 6 andlO weeks after surgery. The dimensions of the T-maze apparatus were described above. The exit of the start box and the entrances of the goal arms could be blocked by guillotine doors. Careful consideration was given to avoid providing the animals with any spatial cues. To minimize olfactory cues, the maze was wiped carefully after each run with 70 % alcohol.
  • Training sessions for left/right discrimination memory retention The day before training, after the spontaneous alternation test, rats were habituated for 15 min to the presence of food pellets (Bacon Softies; Bio-Serv, Frenchtown, NJ, USA) placed at the end of each arm in the T-maze. On days 1 to 3, the rats were food-deprived for 8 to 12 hours each day before the T-maze left/right discrimination training.
  • This training consisted of 3 stages. In the performance of the training, half of the rats from each group were randomly selected and reinforcement (food reward) placed on the right arm; for the other half of the rats from each group, the reinforcement was placed on the left arm. The reinforced arm then remained consistent throughout the training period. The first stage consisted of 5 trials.
  • a guillotine door was placed to close off one arm, and the animal was forced to enter the open arm, which was baited with a food reward that the animal was allowed to eat .
  • the second stage consisted of 5 trials, hi this stage, a guillotine door was placed to close off the same arm as that in the first stage, and the animal was forced to enter the open arm, which was not baited with a food reward.
  • a food reward was given and the animal was allowed to eat the food.
  • the animals remained on the maze for 2 min and were then placed in the start box for 2 min.
  • a guillotine door was removed, and the animal could enter into either arm (correct side and incorrect side). If the animal chose the arm on the correct side the animal received a food reward and was allowed to eat for 2 min after which it was placed in the start box for 2 min. If the animal chose the incorrect side-arm, the animal was picked up immediately and placed in the start box for 2 min.
  • the third stage was continued until the animals made 4 consecutive correct choices or until they had had 20 training sessions (the training ceiling). This procedure was performed daily on three successive days (on days 1 to 3).
  • Left/right discrimination memory retention test session The retention of left/right discrimination memory was evaluated at 1, 2, 3, 5, 7, 10 and 14 days after the training session. The animals were given 10 trials on each testing day. An entry was defined as all four paws entering the arm. The total number of correct entries was recorded.
  • Histopathology At 90 days after surgery, the animals were deeply anesthetized with sodium pentobarbital (100 mg/kg, intraperitoneally), perfused transcardially with 0.01 M PBS, and then perfused with a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Coronal brain blocks including the caudoputamen or optic nerve were embedded in paraffin for histological examination. Two micrometer-thick paraffin sections were then cut on a microtome. The luxol fast blue stain was used to evaluate the myelin damage.
  • the sections were subsequently incubated with a biotinylated anti-mouse IgG raised in donkey (Jackson Immuno Research Labs, West Grove, PA, USA, 1 :2000) for 1 hr, and then incubated with an avidin-biotin peroxidase complex solution (Vector Laboratories, Burlingame, CA, USA, 1:100) for 1 hr. After each incubation, the sections were rinsed for 30 min with 0.1 M PBS containing 0.3% Triton X-100. The immunoreaction products were visualized with diaminobenzidine (DAB kit, Vector Laboratories, Burlingame, CA, USA).
  • DAB kit Vector Laboratories, Burlingame, CA, USA
  • the severity of the white matter lesions was evaluated by the fiber density of luxol fast blue-stained sections.
  • Monochromatic photo images of both sides of the corpus callosum, the traversing fiber bundles of the caudoputamen bilaterally and both optic nerves were taken by means of a microscope with a x40 objective connected to a digital camera (MetaMorph Image Processing System, Universal Imaging Corp, Downingtown, PA, USA). These images were converted into PICT files by Photoshop (Adobe Systems Incorporated, San Jose, CA, USA) and the fiber density of each PICT file was analyzed with the NIH image computer program. To account for the variation of the fiber density between right and left sides of the corpus callosum and caudoputamen, the average of the fiber densities of both sides was calculated.
  • the sections were incubated overnight with the following antibodies (mouse or goat anti-rat) (dilutions in parentheses): against the major histocompatibility complex (MHC) class II (Ia) antigen (OX 6, Serotec, Raleigh, NC, USA, 1:100), against TNF (YC032, Yanaihara Institute, Fujinomiya, Shizuoka, Japan 1 :800) and against E-selectin (R and D systems, Minneapolis, MN, USA 50 ⁇ g/ml).
  • MHC major histocompatibility complex
  • Ia antigen
  • TNF YC032, Yanaihara Institute, Fujinomiya, Shizuoka, Japan 1 :800
  • E-selectin R and D systems, Minneapolis, MN, USA 50 ⁇ g/ml.
  • the sections were subsequently incubated with a biotinylated anti-mouse IgG or a biotinylated anti-goat IgG (Vector
  • the level of plasma TNF concentration was measured by a Rat TNF US ELISA kit (BioSource International, Camarillo, CA, USA) following the manufacturer's instructions.
  • the O.D. values (450 nm) were measured by SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA) and the concentration of the plasma TNF was calculated.
  • Cerebral blood flow (CBF) without E-Selectin tolerization Cerebral blood flow (CBF) was 30 to 50 % of the control several days after ligation. The CBF decreased to values ranging from 40 to 80 % of control over a prolonged period (1 week- 1 month).
  • FIG. 2 shows that rats treated with E-selectin had an ear thickness of slightly less than 0.05 mm whereas control rats that received only PBS had an ear thickness of almost 0.07 mm.
  • tolerization with E-selectin significantly improved the learning and memory impairment in the object recognition test (FIG. 3), T-maze memory retention (FIG. 5) and the ability to handle changes in the T-maze (FIG. 4), compared with the control group of rats that received only PBS.
  • the Pearson correlation coefficient between the fiber density of the optic nerve and the discrimination index in the E-selectin treated animals was minus 0.470. This correlation was not significantly different from O (p 0.2537). A few dark neurons were detected in the unilateral hippocampus of the three E-selectin- treated (27.3%), three PBS-treated animals (27.3%) and one sham-operated animal (25%). There were no obvious differences in the number of the dark neurons among three groups (data not shown). In the white matter of the sham-operated animals, there was positive immunostaining for the MHC class II (Ia) antigen in only a few glial cells.
  • Ia MHC class II
  • TNF- ⁇ was prominently expressed in endothelial cells in blood vessels of the white matter, such TNF- ⁇ expression was markedly attenuated in E-selectin-tolerized and sham-operated animals (FIG. 12).
  • E-selectin was expressed in endothelial cells of vessels in the brains of the PBS-treated animals.
  • the E-selectin immunoreactive vessels were decreased in number in the E-selectin-treated group as compared to the PBS-treated group (FIG. 14).
  • the mucosal tolerance to E-selectin had a suppressive effect against the activation of vessels in the brain induced by protracted hypoperfusion.
  • Immunoassay The sham group had a statistically significantly lower level of plasma TNF than the E-selectin and PBS groups by one-factor ANOVA (p ⁇ 0.05). However, there were no significant differences on the level of plasma TNF between the E-selectin and PBS groups.
  • E-selectin-treated group as compared to the PBS-treated group, the potential effect of differential visual acuity on behavioral tests such as the object recognition test should be considered.
  • the discrimination ability preserved by E-selectin treatment was not correlated with the degree of protection from fiber loss in the optic nerve.
  • One explanation for this discrepancy is that humans primarily base their choices on a memory of visual the properties of the sample object. In contrast, when rats explore an object, they sniff it, palpate it with vibrissae, and look at it. In the rodents, differential exploration of familiar objects and novel objects reflects to some extent their memory for olfactory and tactile properties of the sample object, although visual properties may also be remembered and contribute to discrimination.
  • Clonal anergy/deletion can be induced by a single feeding of very high-dose antigen (Chen Y., Nature. 376(6536):177-180 (1995)) and the production of regulatory T cells occurs after repetitive administration of low- dose antigen (Groux H., Nature 389:737-742 (1997); Chen Y., Science
  • Lymphocytes that are tolerized to an antigen and have become antigen-specific regulatory T-cells tend to migrate to the locale of the protein molecule to which they have been primed. In that location, they release immunomodulatory cytokines, such as TGF- ⁇ and IL-10 that counteract the effect of pro-inflammatory cytokines including TNp andjsuppress inflammation and immune responses after ischemia (Pang L., Stroke. 2001;32:544-552 (2001), Hallenbeck J.M., Trends in Immunology 26:550-556 (2005)).
  • immunomodulatory cytokines such as TGF- ⁇ and IL-10 that counteract the effect of pro-inflammatory cytokines including TNp andjsuppress inflammation and immune responses after ischemia
  • TNF expressed by endothelium has proinflammatory and procoagulant effects on endothelium (Pober JS, Physiol Rev. 70:427-451 (1990), Hallenbeck JM. Nat Med. 8:1363-1368 (2002)).
  • E-selectin appears to function by suppressing local vessel activation and the surrounding immunological and inflammatory processes rather than by systemic immunosuppression. Other mechanisms may also contribute to the protective effect in the present study.
  • White matter injury involves glial cells, which are abundant in white matter (Goldberg M, Stroke 34:330-332 (2003)). In this model, microglial activation with expression of MHC class II antigens was detected preferentially in the white matter (Wakita H, Acta Neuropathol.
  • TGF- ⁇ and IL-10 inhibit the activation of microglia (Suzumura A, J. Immunol. 151:2150-2158 (1993), Frei K, J Immunol. 152:2720-2728 (1994)), mucosal tolerization to E-selectiii suppresses the activated microglia through local production of these cytokines by the regulatory T cells.
  • the number of MHC class II positive activated microglia/macrophages in the white matter showed a trend toward suppression in the E-selectin group as compared to the PBS group.
  • Activated microglia may enhance a variety of inflammatory responses (Morioka T, J. Cereb. Blood Flow Metab.
  • Microglia are the major source of pro-inflammatory cytokines including IL-I and TNF, which may induce the expression of E-selectin in the ischemic cerebral vasculature.
  • the suppression of the microglia may inhibit both local vessel activation and the expression of E-selectin.
  • Activated microglia also release an array of cytotoxic substances that include other pro-inflammatory cytokines, prostanoids, proteases, reactive oxygen radicals and nitrogen intermediates.
  • the protective effect may be mediated by suppressing the release of these cytotoxic substances as well. The net effect decreases inflammation and preserves vessel integrity.
  • the present study demonstrates the protective effect of mucosal tolerance to E-selectin against ischemic cerebrovascular white matter damage and memory impairment during protracted cerebral hypoperfusion.

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Abstract

L'invention porte sur des compositions et sur des méthodes de traitement ou de prévention de la démence vasculaire chez un mammifère, ces méthodes consistant à administrer par les muqueuses une quantité suffisante du polypeptide E-sélectine pour induire une tolérance immune de voisinage chez le mammifère. L'invention porte également sur des compositions utiles dans le traitement ou la prévention de la démence vasculaire.
PCT/US2006/034432 2000-05-24 2006-08-30 Traitement et procede de prevention de la demence vasculaire WO2007028133A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1871802A2 (fr) * 2005-03-10 2008-01-02 Novavax, Inc. E-selectine de recombinaison produite dans des cellules d'insectes
WO2008045488A2 (fr) * 2006-10-09 2008-04-17 Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services National Institutes Of Health Traitement de l'inflammation, de la démyélinisation et de la perte neuronale/axonale
WO2008140449A1 (fr) 2007-05-11 2008-11-20 Thomas Jefferson University Méthodes de traitement et de prévention de maladies et de troubles neurodégénératifs
US7897575B2 (en) 2000-05-24 2011-03-01 The United States Of America As Represented By The Department Of Health And Human Services Treatment and prevention of vascular dementia
US10391068B2 (en) 2012-08-06 2019-08-27 Trustees Of Boston University Prion protein ligands as therapeutic agents for neurodegenerative disorders

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001089557A2 (fr) * 2000-05-24 2001-11-29 The United States Of America, As Represented By Secretary Of The Department Of Health & Human Services Methodes de prevention des accidents vasculaires cerebraux moyennant l'induction d'une tolerance a e-selectin

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001089557A2 (fr) * 2000-05-24 2001-11-29 The United States Of America, As Represented By Secretary Of The Department Of Health & Human Services Methodes de prevention des accidents vasculaires cerebraux moyennant l'induction d'une tolerance a e-selectin

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WANG X ET AL: "DEMONSTRATION OF INCREASED ENDOTHELIAL-LEUKOCYTE ADHESION MOLECULE-1 MRNA EXPRESSION IN RAT ISCHEMIC CORTEX" STROKE, AMERICAN HEART ASSOCIATION, DALLAS TX, US, vol. 26, no. 9, 1995, pages 1665-1669, XP001064744 ISSN: 0039-2499 *
ZOPPO DEL G ET AL: "INFLAMMATION AND STROKE: PUTATIVE ROLE FOR CYTOKINES, ADHESION MOLECULES AND iNOS IN BRAIN RESPONSE TO ISCHEMIA" BRAIN PATHOLOGY, ZUERICH, CH, vol. 10, no. 1, January 2000 (2000-01), pages 95-112, XP001008248 ISSN: 1015-6305 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7897575B2 (en) 2000-05-24 2011-03-01 The United States Of America As Represented By The Department Of Health And Human Services Treatment and prevention of vascular dementia
US8940700B2 (en) 2000-05-24 2015-01-27 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, National Institutes Of Health E-selectin compositions and use thereof for inducing E-selectin tolerance
EP1871802A2 (fr) * 2005-03-10 2008-01-02 Novavax, Inc. E-selectine de recombinaison produite dans des cellules d'insectes
EP1871802A4 (fr) * 2005-03-10 2008-12-24 Novavax Inc E-selectine de recombinaison produite dans des cellules d'insectes
WO2008045488A2 (fr) * 2006-10-09 2008-04-17 Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services National Institutes Of Health Traitement de l'inflammation, de la démyélinisation et de la perte neuronale/axonale
WO2008045488A3 (fr) * 2006-10-09 2008-10-23 Us Gov Health & Human Serv Traitement de l'inflammation, de la démyélinisation et de la perte neuronale/axonale
WO2008140449A1 (fr) 2007-05-11 2008-11-20 Thomas Jefferson University Méthodes de traitement et de prévention de maladies et de troubles neurodégénératifs
EP2977452A2 (fr) 2007-05-11 2016-01-27 Thomas Jefferson University Procédés de traitement et de prévention de maladies et de troubles neurodégénératifs
US10391068B2 (en) 2012-08-06 2019-08-27 Trustees Of Boston University Prion protein ligands as therapeutic agents for neurodegenerative disorders

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