WO2014165043A1 - Fragments, mutants et protéines de fusion chimériques de la leptine pour le traitement de la maladie d'alzheimer - Google Patents

Fragments, mutants et protéines de fusion chimériques de la leptine pour le traitement de la maladie d'alzheimer Download PDF

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WO2014165043A1
WO2014165043A1 PCT/US2014/024217 US2014024217W WO2014165043A1 WO 2014165043 A1 WO2014165043 A1 WO 2014165043A1 US 2014024217 W US2014024217 W US 2014024217W WO 2014165043 A1 WO2014165043 A1 WO 2014165043A1
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leptin
cognitive
beta
fragment
disease
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PCT/US2014/024217
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Nikolaos Tezapsidis
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Neurotez, Inc.
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22Hormones
    • A61K38/2264Obesity-gene products, e.g. leptin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/28Drugs 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

Definitions

  • the present invention relates to methods for treating, preventing, or diagnosing the pathology of progressive cognitive disorders resulting from accumulation of an amyloid peptide or tau
  • lipid homeostasis meaning the multi-layered regulatory networks of lipid metabolism, transport, and signal transduction
  • lipid-lowermg agents e.g., statins
  • amyloid ⁇
  • APP amyloid precursor protein
  • NFTs Neurofibrillary tangles
  • tau is phosphoryiated abnormally at proline directed serine/threonine phosphorylation sites, which can be detected using specific antisera.
  • serine/threonine (Ser/Thr) phosphorylation sites include Ser--202/Thr-205 (ATS site), Ser-214 and/or Ser-214, Ser-181, and/or Ser-212 (AT 100 site), Thr-231 and/or Ser-235 (TG3 site), and Ser-396/Ser-404 (PHF-I site).
  • mice models expressing a repressihle human tau still developed NFTs, neuronal loss, and behavioral impairments; after tau suppression, the behavioral deficits stabilized, yet NFTs continued to accumulate.
  • NFTs axorsal pathology with accumulation of tau proceeded plaque deposition, NFTs (and presumable "intermediates") exist within the cytoplasm of viable neurons. Only in advanced disease are large numbers of extracellular NFTs identified.
  • NFTs are not specific for AD, particularly if a broader definition of NFTs includes different tau isoforms or if one expands the expectation of the morphological characteristics of NFTs.
  • the two classical lesions of AD, neuritic plaques and NFTs, can occur
  • Amyloid plaques are decorated with proteins of the complement system, eicosanoids and cytokines, integral components of ongoing inflammatory processes that augment the harmful effects of NI (Emmerling et al., Biochim Biophys Acta 1502: 158-71 (2000)).
  • Important regulators of the immune system include the cytokines and chemokines secreted by leukocytes (B or T cells, normally scarce in the brain) or antigen presenting cells (APC) (microglia, perivascular macrophages, astrocytes in the brain), in AD brain, both pro-inflammatory cytokines and anti-inflammatory cytokines are expressed (Benveniste et al., Neurochem intl, 39: 381-91 (2001)). In addition to immune function, cytokines may directly affect the processing of APP (Blasko et al., FASEB J. 13: 63-68 (1999)). Leptin has very similar structural and functional characteristics to the cytokines (Heshka, J. T., and P.
  • Leptin is a peptide hormone that controls adaptive metabolic mechanisms to energy availability leading to storage or mobilization of fat (Schwartz et al., Nature. 404: 661-71 (2000)).
  • Adipocyte-derived leptin primarily exerts its central action through the arcuate nucleus neurons (an aggregation of neurons in the mediobasal hypoth lamus); however, it can affect other populations, including hippocampal neurons and cells of the periphery (Shanley et al., Nat Neurosci. 5:299-300 (2002)).
  • Ablation of leptin or of leptin signaling is sufficient to cause obesity as exemplified by leptin-deficient obese, hyperinsulinemic mice having the genotype ob/ob; diabetic mice with a mutation in the leptin receptor gene having the genotype db/db, which produce but are non-responsive to leptin: rats of the genotype fa/fa, which have the "fatty" obesity gene, which is a mutated leptin receptor; and in a few rare genetic cases (Schwartz et al., Nature. 404: 661-71 (2000)).
  • Leptin has been investigated as a novel therapy for obesity. While the peptide was well tolerated by humans and exhibited an excellent safety profile in clinical trials, it failed to demonstrate efficacy for the targeted condition. [0010] The primary amino acid sequence of Leptin indicated that it adopts a 4-helix bundle structure similar to that of the cytokine IL-2, which has since been verified by x-ray
  • Leptin contains all three LR-interacting domains at both its N- and C-termini as well as a single disulfide bond at its C-terminus, resulting in formation of a C -terminal loop at positions 117-167 (FIG. 8).
  • domain II and III of Leptin are well described, however less is known about the role of domain I (Table 2).
  • Domain II is responsible for binding of the Leptin molecule to the LR signaling chain, with limited effect on receptor activation, while domain III is involved in signaling, with limited effect on receptor binding (Peelman, F, et al., J. Biol. Chem. 279 (2004) 41038-41046).
  • Domain I incurs a modest effect on receptor activation, but is hypothesized to function in binding to the non-signaling a-chain of the LR (Peelman, F. et al., J. Biol. Chem. 279 (2004) 41038-41046).
  • the leptin receptor (ObR), a member of the class I cytokine receptor superfamily (Lord,
  • isoform refers to a version of a protein that has the same function as another version of the protein but that has some small differences in its sequence. All isoforms of ObR share an identical extracellular ligand-binding domain (Couce et al., N euro endoc i ology. 66:145-50 (1997)).
  • Leptin's functional receptor (ObRb), the b isoform, is expressed not only in the hypothalamus, where it regulates energy homeostasis and neuroendocrine function, but also in other brain regions and in the periphery, including all cell types of innate and adaptive immunity (Lord, G. M., et al., Nature 394:897 (1998); Zhao, Y., R. et al, Biochem. Biophys. Res. Common. 300: 247 (2003)); Zarkesh-Esfahani, H., G. et al., J. Immunol 157: 4593 (2001) CaldefieChezet, F., A, et al., J, Leukocyte Biol. 69:414 (2001)).
  • Leptin binding to its functional receptor recruits Janus tyrosine kinases and activates the receptor, which then serves as a docking site for cytoplasmic adaptors such as STAT (Baumann,
  • STAT proteins initially are present in inactive forms in the cytoplasm Following ligand stimulation and receptor dimerization, the JA1C/STAT pathway is activated by activation of receptor-bound JAK kinases. These JAK kinases subsequently phosphorylate the receptor at tyrosine residues, which recruits STATs to the receptor. STATs then are
  • phosphoSTATs phosphorylated to form phosphoSTATs, dimerized, and translocated to the nucleus, where the phosphoSTAT dimers bind to specific sequences in the promoter regions of their target genes, and stimulate the transcription of these genes (Schindler et al., Ann. Rev. Biochem. 64: 621-51 (1995)), including negative regulators, such as the suppressor of cytokine signaling 3 (Bjorbaek, C, K. et al. ./. Biol. Chem. 274:30059 (1999)) and the protein tyrosine phosphatase IB (Cheng, A. N. et al. Dev. Cell 2:497 (2002), Schwartz et al, Nature, 404:661-71 (2000), Louis A.
  • negative regulators such as the suppressor of cytokine signaling 3 (Bjorbaek, C, K. et al. ./. Biol. Chem. 274:30059
  • M phosphatidylinositol 3 '-kinase
  • Leptin may have a physiologic role as a liporegulatory hormone responsible for maintaining intracellular homeostasis in the face of wide variations in caloric intake (Linger R H. 2003. Annu Rev Physiol . 65:333-47). This is achieved by directly stimulating lipoiysis, (meaning fat breakdown), and inhibiting lipogenesis (meaning fat synthesis) (Lee Y, et al., J. Biol Chem. 276(8):5629-35 (2001)). Leptin can improve insulin resistance and hyperglycemia by a mechanism not completely understood (Toyoshima et al.. Endocrinology 146: 4024-35 (2005)), despite insulin's ability to stimulate lipogenesis (Kersten, EM BO Reports 2(4): 282-286 (2001). Both insulin and ⁇ are degraded by insulin degrading enzyme (IDE).
  • IDE insulin degrading enzyme
  • SREBPs Sterol Regulatory Element-Binding Proteins
  • SREBPs are transcription factors that regulate the expression of genes for both cholesterol and fatty acid synthesis.
  • the inactive precursor form of S EBPs resides in cytoplasmic membranes.
  • Intracellular lipid depletion triggers proteolytic cleavage of the SREBPs, allowing the amino terminus to enter the nucleus and activate the expression of enzymes, including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), major biosynthetic enzymes for fatty acid synthesis.
  • enzymes including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), major biosynthetic enzymes for fatty acid synthesis.
  • CNS central nervous system
  • metabolic pathways involving lipids serve mainly to provide the building blocks for membranes, vitamins, second messengers and to modify proteins by acylation, because there are no main mechanisms for utilizing triglyeerides/fatty acids as energ sources.
  • AD Amyloid ⁇
  • lipid rafts refers to membrane microdomains enriched in cholesterol, glycosphingolipids and glucosyiphosphatidyl-inositol-(GPI)-tagged proteins implicated in signal transduction, protein trafficking and proteolysis, Within the LRs it is believed that Ap's precursor, Amyloid Precursor Protein (APP), a type I membrane protein, is cleaved first by the protease ⁇ -secretase (BACE) to generate the C-terminal intermediate fragment of APP, CAPPp, which remains imbedded in the membrane.
  • APP Amyloid Precursor Protein
  • FIG. 1 a The amino acid sequence of ⁇ peptide showing its cleavage sites and membrane domain is shown in FIG. 1 a.
  • CAPPP is subsequently cleaved at a site residing within the lipid bilayer by y-secretase, a high molecular weight multi-protein comple containing presenilin, (PS 1/PS2), nicastrin, PEN-2, and APH-1 or fragments thereof (De Strooper, Neuron. 38: 9-12 (2003)).
  • finally is released outside the cell, where it can: a) start accumulating following oligomerization and exerting toxicity to neurons or b) be removed either by mechanisms of endocytosis (involving apolipoprotein-E (apoE) and LRP or Scavenger Receptors) or by degradation by extracellular proteases including insulin-degrading enzyme (IDE) and neprilysin CFarris et aL Proc Natl Acad Sci USA. 100:4162-4167 (2003)) (FIG. 1 b).
  • endocytosis involving apolipoprotein-E (apoE) and LRP or Scavenger Receptors
  • IDE insulin-degrading enzyme
  • Fatty acid and cholesterol availability and cellular composition modifies the transbiiayer distribution of cholesterol and, consequently, overall membrane fluidity, function and localization of lipid rafts, a process which changes with aging (Wood et al., Neurobiol Aging. 23:685-694 (2002)). Therefore, it was hypothesized that leptin's lipolytic/antilipogenic activity could affect the composition of the LRs, affecting ⁇ turnover.
  • U.S. Pat. No. 8,227,408 describes leptin's ability to modify the levels of ⁇ both in vitro and in vivo.
  • Leptin similarly to methyl- ⁇ -eyclodextrin, reduces ⁇ -secretase activity in neuronal cells, possibly, but without being limited by theory, by altering the lipid composition of membrane LRs. This contrasts the results of treatments with cholesterol and etomoxir (an inhibitor of carnitine-palmitoyi transferase- 1).
  • etomoxir an inhibitor of carnitine-palmitoyi transferase- 1
  • inhibitors of acetyl CoA carboxylase and fatty acid synthase mimicked leptin's action.
  • leptin was able to increase apoEdependent ⁇ uptake in vitro.
  • leptin can modulate indirectly bidirectional ⁇ kinesis, reducing its levels extracellulariv.
  • Chronic administration of leptin to AD-transgenic animals reduced the brain ⁇ load, illustrating its therapeutic potential.
  • the described invention discloses that fragments of leptins, derivatives of leptins, mutants of leptins, and chimeric fusion proteins of leptins share these properties.
  • the described invention provides a method for treating a progressive cognitive disease, cognitive disorder, or cognitive condition resulting from accumulation of an amyloid peptide, comprising: administering to a subject in need thereof a first composition comprising: (i) a therapeutic amount of a leptin, a leptin mimic, or a pharmaceutically acceptable salt thereof, and (ii) a pharmaceutically acceptable carrier, wherein the leptin or the leptin mimic is a recombinant human leptin, a pegylated recombinant human leptin (PEG-OB), a recombinant human methionyl leptin, a leptin peptidomimetic, a biologically active fragment of leptin, a fusion peptide of leptin with an Fc fragment of immunoglobulin, a fusion peptide of the biologically-acti ve fragment of leptin with the Fc fragment of immunoglobulin
  • the method further comprises monitoring circulating levels of the amyloid peptide.
  • the circulating levels of amyloid peptide are detected in a sample of cerebrospinal fluid or blood.
  • the method further comprises placing the subject on a low fat diet.
  • the progressive cognitive disease, cognitive disorder, or cognitive condition is a dementia, an amyloidosis, Down's syndrome, or cerebral amyloid angiopathy.
  • the progressive cognitive disease, cognitive disorder, or cognitive condition is Alzheimer's disease.
  • the progressive cognitive disease, cognitive disorder, or cognitive condition is senile systemic amyloidosis.
  • the progressive cognitive disease, cognitive disorder, or cognitive condition is cerebrovascular amyloidosis.
  • the amyloid peptide is an amyloid ⁇ ( ⁇ ) peptide.
  • the first composition further comprises (iii) a therapeutically effective amount of one or more lipoiytic/antilipogenic compounds wherein the one or more lipoiytic/antilipogenic compounds reduce amyloid ⁇ ( ⁇ ) production, increase apoE- ⁇ ( ⁇ ) uptake, or both.
  • the first composition modulates accumulation of the amyloid peptide in the cerebral nervous system.
  • the first composition is administered by at least one route selected from the group consisting of orally, buccally, parenterally, intranasally, rectally, and topically.
  • the method further comprises serially administering a second composition comprising a therapeutically effective amount of one or more lipolytic/antilipogenic compounds, wherein the one or more Sipolytic/antiSipogenic compounds reduce amyloid ⁇ ( ⁇ ) production, increase apoE- ⁇ ( ⁇ ) uptake, or both.
  • the method further comprises placing the subject on a low- fat diet.
  • the described invention provides a method of improving resilience of cognitive function in a subject in need thereof, the method comprising: (a) administering to the subject a composition comprising: i. a cognitive function- enhancing or cognitive function stabilizing amount of leptin, a leptin mimic, or a pharmaceutically acceptable salt thereof, wherein the leptin or the leptin mimic is a recombinant human leptin, a pegylated recombinant human leptin (PEG- OB), a recombinant human methionyl leptin, a leptin peptidomimetic, a biologically-active fragment of leptin, a fusion peptide of leptin with an Fc fragment of immunoglobulin, a fusion peptide of the biologically-active fragment of leptin with the Fc fragment of immunoglobulin, a leptin agonist, and a combination thereof, and i
  • the composition is administered orally, buccally, parenterally, intranasally, rectally, virally or topically.
  • the method further comprises (b) measuring the subject's ability to perform mental tasks.
  • the subject's ability to perform mental tasks is measured by at least one test for memory, computation, or attention.
  • the biologically active fragment of leptin comprises an amino acid sequence selected from the group consisting of SEQ ID Os: 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 43, 44 and 45.
  • the biologically active fragment of leptin comprises a first and a second fragment, wherein the first fragment has amino acid sequence SEQ ID NO: 41 , wherein the second fragment has amino acid sequence SEQ ID NO: 42, and wherein the first fragment is cova!entiy linked to the second fragment via a disulfide bond between cysteine at amino acid residue 96 of SEQ ID NO: 41 and cysteine at amino acid residue 8 of SEQ ID NO: 42.
  • the therapeutic amount is from about 0.01 mg per kg (of body weight) per day to about 0.5 mg per kg (of body weight) per day.
  • the subject in need thereof has a systemic leptin deficiency.
  • the composition restores, replenishes, or increases leptin levels.
  • the biologically active fragment of leptin has an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 43, 44 and 45.
  • the biologically active fragment of leptin comprises a first and a second fragment, wherein the first fragment has amino acid sequence SEQ ID NO: 41, wherein the second fragment has amino acid sequence SEQ ID NO: 42, and wherein the first fragment is covalently linked to the second fragment via a disulfide bond between cysteine at amino acid residue 96 of SEQ ID NO: 41 and cysteine at amino acid residue 8 of SEQ ID NO: 42.
  • the cognitive function-enhancing amount of the leptin, the leptin mimic, or the pharmaceutically acceptable salt thereof reduces amyloid ⁇ ( ⁇ ) production, increases apoE-Abeta ( ⁇ ) uptake, or both.
  • the amyloid peptide is an amyloid ⁇ ( ⁇ ) peptide.
  • the subject in need thereof has a systemic leptin deficiency.
  • the composition restores, replenishes, or increases leptin levels.
  • the cognitive function-enhancing amount is from about 0.01 mg per kg (of body weight) per day to about 0.5 mg per kg (of body weight) per day.
  • the composition further comprises (iii) a therapeutically effective amount of one or more lipolytic/antilipogenic compounds, wherein the one or more lipolytic/antilipogenic compounds reduce amyloid ⁇ ( ⁇ ) production, increase apoE- ⁇ ( ⁇ ) uptake, or both.
  • the method further comprises placing the subject on a low fat diet.
  • the method further comprises serially administering a second composition comprising a therapeutically effective amount of one or more lipoiyiic/antilipogenic compounds, wherein the one or more lipolytic/antilipogenic compounds reduce amyloid ⁇ ( ⁇ ) production, increase apoE- ⁇ ( ⁇ ) uptake, or both.
  • the described invention provides a method for treating a progressive cognitive disease, cognitive disorder, or cognitive condition resulting from an increase in tau phosphorylation, comprising: administering to a subject in need thereof a first composition comprising (i) a therapeutic amount of a leptin, a leptin mimic, or a
  • the leptin or the leptin mimic is a recombinant human leptin, a pegylated recombinant human leptin (PEG-OB), a recombinant human methionyl leptin, a leptin peptidoniimetic, a biologically active fragment of leptin, a fusion peptide of leptin with an Fc fragment of immunoglobulin, a fusion peptide of the biologically- active fragment of leptin with the Fc fragment of immunoglobulin, a leptin agonist, or a combination thereof, wherein the therapeutic amount of the leptin or the leptin mimic is effective to decrease tau phosphorylation in brain.
  • PEG-OB pegylated recombinant human leptin
  • a recombinant human methionyl leptin a leptin peptidoniimetic
  • the progressive cognitive disease, cognitive disorder, or cognitive condition is a dementia, an amyloidosis, Down's syndrome, or cerebral amyloid angiopathy.
  • the progressive cognitive disease, cognitive disorder, or cognitive condition is Alzheimer's disease.
  • the progressive cognitive disease, cognitive disorder, or cognitive condition is senile systemic amyloidosis.
  • the progressive cognitive disease, cognitive disorder, or cognitive condition is cerebrovascular amyloidosis.
  • the composition is administered orally, buccally, parenterally, intranasally, reclaUy, or topically.
  • the biologically active fragment of leptin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 43, 44 and 45.
  • the described invention provides a method for increasing neuronal cell viability in a subject suffering from a progressive cognitive disease, cognitive disorder, or cognitive condition comprising: administering to a subject in need thereof a first composition comprising (i) a therapeutic amount of a leptin, a leptin mimic, or a
  • the leptin or the leptin mimic is a recombinant human leptin, a pegylated recombinant human leptin (PEG-OB), a recombinant human methionyl leptin, a leptin peptidomimetic, a biologically active fragment of leptin, a fusion peptide of leptin with an Fc fragment of immunoglobulin, a fusion peptide of the biologically- active fragment of leptin with the Fc fragment of immunoglobulin, a leptin agonist, or a combination thereof, wherein the therapeutic amount of the leptin or the leptin mimic is effective to increase neuronal cell viability in the subject.
  • PEG-OB pegylated recombinant human leptin
  • a recombinant human methionyl leptin a leptin peptidomimetic
  • the progressi ve cognitive disease, cognitive disorder, or cognitive condition is a dementia, an amyloidosis, Down's syndrome, or cerebral amyloid angiopathy .
  • the progressive cognitive disease, cognitive disorder, or cognitive condition is Alzheimer's disease.
  • the progressive cognitive disease, cognitive disorder, or cognitive condition is cerebrovascular amyloidosis.
  • the composition is administered orally, bueeally, parenterally, intranasally, rectally, or topically.
  • the biologically acti ve fragment of leptin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 43, 44 and 45.
  • FIG. la shows the amino acid sequence, cleavage sites and membrane domain of ⁇ .
  • FIG. 1 b shows mechanisms of ⁇ production and clearance.
  • FIG. 2 shows pathways related to or affected by Ieptin, leading to inhibition of lipogenesis and stimulation of lipolysis, inhibiting ⁇ production.
  • FIG. 3 shows the amyioidogenic and anti-amyloidogenic pathways (from
  • FIG. 4 shows that Ieptin affects ⁇ production through BACE in rafts. Asterisks indicate that the value is significantly different from that of the corresponding control (set at P ⁇ 0.05).
  • Neuro2a ceils stably transfected with hyg-sal 34 were treated for about 2 h or about 5 h with about 100 ng/ml Ieptin, Ob (black); about 125 mg/ml cyclodextrin, CDX (gray stripe): about 5 mg/ml cholesterol, Ch (pale gray); Ieptin plus cholesterol, Ob- Ch (medium gray).
  • Neuro2a cells stably transfected with hyg-sal34 were treated for about 2 h or 5 h with about 400 ng/ml Ieptin, Ob (black); about 250 mg/ml cyclodextrin, CDX (gray stripe), 10 mg/ml cholesterol, Ch (pale gray ) and Ieptin plus cholesterol, Ob+Ch (medium gray).
  • sucrose gradient fractions in (d) were assayed for ⁇ -seeretase activity using a fluorescence-quenching assay (QTL Biosystems, NM).
  • FIG. 5 shows that leptin affects apoE -dependent ⁇ -uptake and the possible involvement of SREBPs.
  • FIG. 6 shows that leptin modulates free cholesterol-rich membrane domains and that surplus cholesterol may trigger leptin.
  • Neural cultures from El 5 rat cerebral cortex were processed for enrichment in neurons (a-d) or astrocytes (e-h) and, after about 7 days to 10 days in culture, treated for about 5 h with about 10 ⁇ ig/ml cholesterol (b, f) or about 400 ng/ml leptin plus cholesterol (c, g) or leptin alone (d, h).
  • Controls (a, e) were treated with media alone. Cells were stained for filipin.
  • FIG. 7 shows the deficiency of leptin in AD transgenic mice and the effect of leptin supplementation on amyloid load
  • HFD high fat diets
  • LFD low fat diets
  • Plasma total AP ( ⁇ 40 plus ⁇ 42/43) was measured in 10 month Tg2576 mice following a 2 month LFD or HFD with (+) or without (-) leptin infusion.
  • FIG. 8 shows a graphical representation of the leptin molecule's structure and functional domains.
  • FIG. 9 shows the effect of full length leptin and leptin fragments on RA-SY5Y ceil viability.
  • Panel A shows the effect of full length leptin and leptin fragments on RA-SY5Y cell viability in the absence of cholesterol.
  • Panel B shows the effect of full length leptin and leptin fragments on RA-SY5Y cell viability in the presence of cholesterol.
  • FIG. 10 shows the effect of full length leptin and leptin fragments on tau
  • Panel A shows the effect of full length leptin and leptin fragments on tau phosphorylation in RA-S Y5Y cells in the absence of linoleic acid.
  • Panel B shows the effect of full length leptin and leptin fragments on tau phosphorylation in RA- SY5Y cells in the presence of linoleic acid.
  • FIG. 11 shows the effect of full length leptin and leptin fragments on ⁇ production in RA-SY5Y cells.
  • Panel A shows the effect of full length leptin and leptin fragments on ⁇ production in RA-SY5Y cells in the absence of ceramide.
  • Panel B shows the effect of full length leptin and leptin fragments on ⁇ production in RA-SY5Y cells in the presence of ceramide.
  • AD Alzheimer's disease
  • Extracellular amyloid deposits are known as neuritic or senile plaques. Amyloid deposits can also be found within and around blood vessels.
  • APP amyloid precursor protein
  • can be detected in plasma and cerebrospinal fluid (CSF) in vivo, and in ceil culture media in vitro.
  • CSF cerebrospinal fluid
  • the terms "amyloid peptide” "amyloid 0 peptide” and " ⁇ " are used interchangeably herein to refer to the family of peptides generated through proteolytic processing of the amyloid precursor protein (APP).
  • APP exists as three different spliced isoiorms, one having 770 amino acids (isoform a) (SEQ ID NO: l), one having 751 amino acids (isoform b) (SEQ ID NO:2), and one having 695 amino acids (SEQ ID NO:3).
  • the term "APP” as used herein refers to all three isoforms.
  • the terms "amyloid peptide” "amyloid 0 peptide” and " ⁇ ” include, but are not limited to, ⁇ 40 (SEQ ID NO:4), ⁇ 42 (SEQ ID N():5) and ⁇ 43 (SEQ ID NO:6).
  • ⁇ 40 SEQ ID NC:4
  • ⁇ 42 SEQ ID N():5
  • ⁇ 43 SEQ ID NO: 6
  • administer refers to the giving or supplying of a medication, including in vivo administration, as well as
  • compositions may be administered systernically either orally, bucally, parenteraily, topically, by inhalation or insufflation (i.e., through the mouth or through the nose) or rectally in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants and vehicles as desired, or may be locally administered by means such as, but not limited to, injection, implantation, grafting, topical application or parenteraily.
  • agent and “therapeutic agent” are used interchangeably herein to refer to a drug, molecule, composition, or other substance that provides a therapeutic effect.
  • active agent refers to the ingredient, component or constituent of the compositions of the present invention responsible for the intended therapeutic effect.
  • amino acid residue or amino acid or “residue” are used
  • amino acids that are incorporated into a protein, a polypeptide, or a peptide, including, but not limited to, a naturally occurring amino acid and known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids, including amino acids with secondary modifications such as acetylation or other substitutions.
  • the amino acids may be L- or D-amino acids.
  • An amino acid may be replaced by a synthetic amino acid which is altered so as to increase the half-life of the peptide or to increase the potency of the peptide, or to increase the bioavailability of the peptide.
  • the following represent groups of amino acids that are conservative substitutions for one another:
  • Isoleucine (1), Leucine (L), Methionine (M), Valine (V); and
  • APP amyloid precursor protein
  • amyloidoses refers to a group of conditions of diverse etiologies characterized by the accumulation of insoluble fibrillar proteins (amyloid) in various organs and tissues of the body, wherein eventually organ function is compromised.
  • the associated disease states may be inflammatory, hereditary or neoplastic and the deposition of the amyloid peptide may be localized, generalized or systemic.
  • the described invention provides a method for treating or preventing the pathology of a disease, disorder or condition resulting from accumulation of an amyloid peptide in a subject.
  • the amyloid peptide is an amyloid ⁇ peptide.
  • Such a disease, disorder or condition may be any cognitive impairment, including, but not limited to, a dementia; amyloidoses, such as AD and senile systemic amyloidosis; Down ' s syndrome (patients with Down's syndrome, characterized by trisomy 21 , have an extra copy of APP and develop senile plaques from about 12 years of age); cerebral amyloid angiopathy (CAA), also known as congophilic angiopathy or cerebrovascular amyloidosis (a disease of small blood vessels in the brain in which deposits of amyloid protein in the vessel walls may lead to stroke, brain hemorrhage, or dementia); as well as diseases, disorders or conditions co- morbid with (meaning occurring in association with) AD or with any of the above diseases, disorders or conditions, such as Parkinson's disease and epilepsy.
  • CAA cerebral amyloid angiopathy
  • CAA also known as congophilic angiopathy or cerebrovascular amyloidosis
  • cognitive function refers to the intellectual processes resulting in an understanding, perception, or awareness of one's ideas as well as the ability to perform mental tasks, such as thinking, learning, judging, remembering, computing, controlling motor functions, and the like.
  • ementia refers to a decline or a progressive decline in cognitive function due to damage or disease in the brain beyond what might be expected from normal aging.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition, or disorder, substantially ameliorating clinical or aestiietical symptoms of a condition, substantially preventing the appearance of clinical or aesthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying stimuli.
  • disease or “disorder” as used herein refers to an impairment of health or a condition of abnormal functioning.
  • disorder refers to a pattern of symptoms indicative of some disease or condition.
  • injury refers to damage or harm to a structure or function of the body caused by an outside agent or force, which may be physical or chemical.
  • condition refers to a variety of health states and is meant to include disorders or diseases caused by any underlying mechanism or disorder, injury, and the promotion of healthy tissues and organs.
  • the term "subject” as used herein includes animal species of mammalian origin, including humans, it further includes cells and tissues derived from these species. [0066] According to one embodiment, accumulation of amyloid peptide in the disease, disorder or condition may occur ex trace Slularly, meaning located or occurring outside a cell or cells. In a further embodiment, the accumulation of amyloid peptide is in the central nervous system (CNS) of the subject, and may be either in the brain or on cerebral blood vessels walls.
  • CNS central nervous system
  • the method of the present invention comprises the step of administering to a subject susceptible to or having a disease, disorder or condition resulting from accumulation of an amyloid peptide a composition comprising (i) a therapeutically effective amount of leptin, a leptin mimic, a leptin derivative, or a leptin agonist, and (ii) a pharmaceutically acceptable carrier, and thereby modulating accumulation of the amyloid peptide.
  • the term “modulate” or “modulating” refers to adjusting, changing, or manipulating the function or status of amyloid peptide accumulation. Such modulation may be any change in the rate of accumulation, including an undetectable change.
  • the method comprises monitoring circulating levels of amyloid peptide. Such monitoring may be performed one or more times at any point, i.e., before, during, or after, administration of leptin to a subject. Methods for monitoring include measuring leptin levels detected in a sample of cerebrospinal fluid or blood collected from the subject.
  • peptidomimetic is a compound containing non-peptidic structural elements capable of mimicking or antagonizing (meaning neutralizing or counteracting) the biological action(s) of a natural parent peptide.
  • a leptin agonist is a compound capable of activating the ieptin receptor and/or downstream effectors (see FIG. 2) and modulating amyloid peptide levels in a subject.
  • an activator of AMP-dependent protein kinase may have anti- amyioidogenic activity, based on AMPK's ability to promote lipolysis and inhibit lipogenesis upon activation.
  • AMPK AMP-dependent protein kinase
  • AICAR 5--aniinoimidazole-4-carboxaniide riboside
  • the antidiabetic drugs metformin and rosigiitazone may also exert some of their pharmacological actions through AMPK.
  • blood brain barrier ' or "blood-CSF barrier '” are used to describe naturally- occurring systems for excluding substances from the brain and for transporting substances from blood to CSF or brain and vice versa to preserve homeostasis in the nervous system.
  • the baniers facilitate entry of necessary metabolites, but block entry or facilitate removal of unnecessary metabolites or toxic substances.
  • the efficacy of the exclusion or the transport is determined by morphological and functional characteristics of the brain and spinal cord capillaries and by the biochemical and biophysical characteristics of the solute.
  • the barrier systems include carrier-mediated transport systems. Since lipid solubility enhances the transport of substances, ionized polar compounds enter the brain slowly unless there is a specific transport system for them.
  • Also useful according to the present invention is a leptin blocker; mimic, mimetic or peptidomimetic of a leptin blocker, such as a leptin-binding protein; or a leptin antagonist, which increases amyloid peptide levels.
  • compounds capable of inhibiting AMPK e.g., compound C
  • such blockers or inhibitors are useful in providing an experimental approach to accelerate AD-like pathology in existing animal models of AD, and for in vitro experimental approaches.
  • derivative refers to an amino acid sequence produced from a ieptin-derived peptide, either directly or by modification or partial substitution of the leptin- derived peptide.
  • derivatives of leptin include truncated and fusion leptin products (see infra),
  • peptide refers to a molecule of two or more amino acid chemically linked together.
  • a peptide may refer to a polypeptide, protein or peptidomimetic.
  • the peptides of the invention may comprise D- amino acids (which are resistant to L-amino acid-specific proteases in vivo), a combination of D- and L-amino acids, and various "designer” amino acids (e.g., ⁇ -methyl amino acids, C-a-methyl amino acids, and N-a-methyl amino acids, etc.) to convey special properties.
  • Synthetic amino acids include ornithine for lysine, and norleucine for leucine or isoleucine.
  • the peptides can have peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties.
  • a peptide may be generated that incorporates a reduced peptide bond, i.e., R 5 —
  • CH 2 NH -R 2 where 3 ⁇ 4 and R 2 are amino acid residues or sequences.
  • a reduced peptide bond may be introduced as a dipeptide subunit.
  • Such a peptide would be resistant to protease activity, and would possess an extended half-live in vivo. Accordingly, these terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.
  • peptide is also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. In some embodiments, the peptide is of any length or size. Use herein of the terms “peptide”, “peptides”, or “peptidomimetic” should be takers to include reference to “derivatives" of such compounds, unless the context requires otherwise, and to include “prodrugs.”
  • sequence relationships between two or more nucleic acids or polynucleotides are used herein to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.
  • reference sequence refers to a sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • comparison window refers to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison windov.' may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be at least 30 contiguous nucleotides in length, at least 40 contiguous nucleotides in length, at least 50 contiguous nucleotides in length, at least 100 contiguous nucleotides in length, or longer.
  • a gap penalty typically is introduced and is subtracted from the number of matches.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981 ); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad.
  • the BLAST family of programs which can be used for database similarity searches, includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
  • sequence identity/similarity values refer to the value obtained using the BLAST 2.0 suite of programs using default parameters. Altschui et al,, Nucleic Acids Res. 25:3389-3402 (1997). Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information at ncbi.nlm.nih.gov. This algorithm involves first identifying high scoring sequence pairs ( ⁇ ISPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • ⁇ ISPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et a ., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits then are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST? program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Nail. Acad. Sci. USA, 1989, 89: 10915).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nail. Acad. Sci. USA, 1993, 90: 5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • BLAST searches assume that proteins may be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids.
  • Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar.
  • a number of low-complexity filter programs may be employed to reduce such low-complexity alignments.
  • the SEG Wang and Federhen, Comput. Chem., 1993, 17:149-163
  • XNU Choverie and States, Comput. Chem., 1993, 17: 191-201
  • low-complexity filters may be employed alone or in combination.
  • sequence identity in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence identity refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
  • Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity.” Means for malting this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1 , The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer AppHc. Biol ScL, 1988, 4: 11-17, e.g., as implemented in the program PC/GENE (Intelligeneti.es, Mountain View, ( Calif., USA).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) relative to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • polynucleotide comprises a sequence that has at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity and at least 95% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • these values may be adjusted appropriately to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.
  • Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, or at least 70%, at least 80%, at least 90%, or at least 95%. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions.
  • nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is that the polypeptide that the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70 percent sequence identity to a reference sequence, at least 80 percent, at least 85 percent, at least 90 percent or 95 percent sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, /. Mol. Biol. 48:443 (1970).
  • An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • Peptides which are "substantially similar” share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes.
  • the administered composition according to the described invention may further comprise a therapeutically effective amount of one or more lipolytic/antilipogenic compounds.
  • lipolytic compound refers to a compound whose activity pertains to, is characterized by, or causes lipolysis (meaning the disintegration or splitting of fats).
  • antilipogenic compound refers to a compound whose activity pertains to, is characterized by, or causes inhibition of lipid synthesis.
  • the lipolytic/antilipogenic compound may be an acetyl CoA carboxylase inhibitor (such as 5-(tetraecyloxy)-2-furoic acid (TOFA)), a fatty acid synthase inhibitor (such as cerulenin), an acetyl CoA carboxylase inhibitor and a fatty acid synthase inhibitor, or an AMPK activator.
  • TOFA 5-(tetraecyloxy)-2-furoic acid
  • a fatty acid synthase inhibitor such as cerulenin
  • an acetyl CoA carboxylase inhibitor and a fatty acid synthase inhibitor or an AMPK activator.
  • the administered composition may be used in conjunction with other pharmaceuticals.
  • the subject in need of treatment according to the method of the described invention has indications of other complications, such as cardiovascular disease, diabetes, or is a carrier of the apoE G4 allele
  • the subject also may be instructed to follow additional varied treatment regimens.
  • allele refers to an alternative DNA coding of the same gene occupying a given gene locus.
  • the G 4 allele of the apoE gene likely constitutes a major risk factor for amyloid ⁇ peptide accumulation and late-onset AD.
  • One such regimen may be to follow a low-fat diet in combination with treatments described herein.
  • the described invention provides a method of modulating amyloid peptide accumulation in a subject comprising interfering with (meaning affecting or disrupting) at least one step in at least one metabolic or signaling pathway associated with leptin.
  • the metabolic pathways or signaling pathways associated with leptin include, but are not limited to, the amyloidogenic pathways (which lead to generation of the A beta peptide), the LRP pathway (which leads to endocytosis/clearance of the A beta peptide), the insulin degrading pathway (which leads to degradation of the A beta peptide), and any other pathway(s) affected by, or associated with, leptin. (See Fig. 2 for signaling pathways associated with leptin).
  • amyloidogenic pathway refers to the cellular mechanisms by which APP is proteolytically processed to generate amyloid- beta, as shown in FIGS. 1 and 3. APP is proteolytically processed either through the amyloidogenic pathway or the antiamyloidogenic pathway.
  • fragment or "peptide fragment” as used herein refers to a small part derived, cut off, or broken from a larger peptide, polypeptide or protein, which retains the desired biological activity of the larger peptide, polypeptide or protein. In the amyloidogenic pathway, consecutive cleavage of APP by beta - and gamma-secretase generates A beta.
  • cleavage of APP by the protease beta - secretase occurs at the N-terminus of the A beta domain to yield the secreted sAPP beta (SEQ ID NO:7) as well as a C-terminal fragment of APP of 99 amino acids (C99) (SEQ ID NO:8).
  • C99 is further cleaved within its transmembrane domain by y-secretase, leading to the secretion of the A beta peptide and the generation of the APP intracellular domain (AICD).
  • the A beta peptide so generated is prone to aggregation.
  • a beta peptide oligomers are neurotoxic and lead to an impairment of long-term potentiation (LTP).
  • LTP long-term potentiation
  • large amounts of A beta peptide are deposited in amyloid plaques, which are the characteristic pathological hallmarks of AD.
  • a-secretase is a member of the ADAM (A Disintegrin And Metalloproteinase) family of metalloproteases.
  • ADAM Disintegrin And Metalloproteinase
  • a- Cleavage of APP can be induced upon overexpression of ADAM 10 or by the activation of second messenger cascades.
  • LRP lipoprotein receptor related protein pathway
  • LRP LDL receptor-related protein
  • a2M alpha 2-macroglobulin
  • insulin degrading pathway refers to the pathway by which insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, degrades A beta peptides.
  • IDE insulin-degrading enzyme
  • the described invention also provides a method for diagnosing a cognitive disorder, disease, condition or precondition comprising measuring circulating leptin levels.
  • the described invention also provides methods of improving cognitive function in a subject in need thereof, the method comprising the step of administering to the subject (i) a composition comprising (i) leptin, a leptin mimic, a leptin derivative, a leptin agonist, an AMP-dependent protein kinase (AMPK) activator, or a leptin blocker, a mimic of a leptin blocker, a leptin antagonist, or an AMPK inhibitor and (ii) a pharmaceutically acceptable carrier to the subject.
  • a composition comprising (i) leptin, a leptin mimic, a leptin derivative, a leptin agonist, an AMP-dependent protein kinase (AMPK) activator, or a leptin blocker, a mimic of a leptin blocker, a leptin antagonist, or an AMPK inhibitor and (ii) a pharmaceutically acceptable carrier to the subject.
  • cognitive function is as defined in above to refer to the intellectual processes resulting in an understanding, perception, or awareness of one's ideas as well as the ability to perform mental tasks, such as thinking, learning, judging, remembering, computing, controlling motor functions, and the like.
  • cognitive function enhancing amount refers to that amount of the compositions of the described invention that will noticeably impact the ability to perform mental tasks, as measured by tests for memory, computation, attention, or other mental or cognitive attribute, or as suggested by an individual's perception of his or her abilities in these realms.
  • compositions of the invention may be administered orally, buccally, parenterally, intranasally, rectally, or topically.
  • compositions of the described invention may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups or elixirs.
  • oral or “orally” refer to the introduction into the body by mouth whereby absorption occurs in one or more of the following areas of the body: the mouth, stomach, small intestine, lungs (also specifically referred to as inhalation), and the small blood vessels under the tongue (also specifically referred to as sublingually).
  • Compositions intended for oral use may be prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide pharmaceutically elegant and palatable
  • Tablets may contain the active ingredient(s) in admixture with non-toxic pharmaceutically-acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example, magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They also may be coated for controlled release.
  • compositions of the described invention also may be formulated for oral use as hard gelatin capsules, where the active ingredient(s) is(are) mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or soft gelatin capsules wherein the active ingredient(s) is (are) mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • an oil medium for example, peanut oil, liquid paraffin, or olive oil.
  • compositions of the described invention may be formulated as aqueous suspensions wherein the active ingredient(s) is (are) in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide such as lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyl- eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene
  • compositions of the described invention may be formulated as oily suspensions by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil, such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
  • compositions of the described invention may be formulated in the form of dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water.
  • the active ingredient in such powders and granules is provided in admixture with a dispersing or wetting agent, suspending agent, and one or more preservatives.
  • a dispersing or wetting agent, suspending agent, and one or more preservatives are exemplified by those already mentioned above. Additional excipients, or example, sweetening, flavoring and coloring agents also may be present.
  • compositions of the invention also may be in the form of an emulsion.
  • An emulsion is a two-phase system prepared by combining two immiscible liquid carriers, one of which is disbursed uniformly throughout the other and consists of globules that have diameters equal to or greater than those of the largest colloidal particles.
  • the globule size is critical and must be such that the system achieves maximum stability. Usually, separation of the two phases will not occur unless a third substance, an emulsifying agent, is incorporated.
  • a basic emulsion contains at least three components, the two immiscible liquid carriers and the emulsifying agent, as well as the active ingredient.
  • compositions of the invention may be in the form of an oil-in-water emulsion.
  • the oily phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example a liquid paraffin, or a mixture thereof.
  • Suitable emulsifying agents may be naturally-occurring gums, for example, gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.
  • the emulsions also may contain sweetening and flavoring agents.
  • compositions of the invention also may be formulated as syrups and elixirs.
  • Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations also may contain a demulcent, a preservative, and flavoring and coloring agents.
  • Demulcents are protective agents employed primarily to alleviate irritation, particularly mucous membranes or abraded (meaning torn or cut) tissues.
  • Others include acacia, agar, benzoin, carbomer, gelatin, glycerin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, propylene glycol, sodium alginate, tragacanth, hydrogels and the like.
  • compositions of the described invention may take the form of tablets or lozenges formulated in a conventional manner.
  • compositions of the described invention may be in the form of a sterile injectable aqueous or oleaginous suspension.
  • parenteral refers to introduction into the body by way of an injection (i.e., administration by injection), including, for example, subcutaneously (i.e., an injection beneath the skin), intramuscularly (i.e., an injection into a muscle); intravenously (i.e., an injection into a vein), intrathecally (i.e., an injection into the space around the spinal cord), intrasternal injection, or infusion techniques.
  • a parenterally administered composition of the described invention is delivered using a needle, e.g., a surgical needle.
  • surgical needle refers to any needle adapted for delivery of fluid (i.e., capable of flow) compositions of the described invention into a selected anatomical structure.
  • injectable preparations such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 , 3-butanediol.
  • a solution generally is considered as a homogeneous mixture of two or more substances; it is frequently, though not necessarily, a liquid.
  • the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent.
  • a suspension is a dispersion (mixture) in which a finely-divided species is combined with another species, with the former being so finely divided and mixed that it doesn't rapidly settle out.
  • the most common suspensions are those of solids in liquid water.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are examples of sterile, fixed oils.
  • suitable vehicles consist of solutions, for example, oily or aqueous solutions, as well as suspensions, emulsions, or implants.
  • Aqueous suspensions may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • compositions of the described invention may be in the form of a dispersible dry powder for delivery by inhalation or insufflation (either through the mouth or through the nose).
  • Dry powder compositions may be prepared by processes known in the art, such as lyophilization and jet milling, as disclosed in International Patent Publication No. WO 91/16038 and as disclosed in U.S. Pat. No. 6,921,527, the disclosures of which are incorporated by reference.
  • Spray drying for example, is a process in which a homogeneous aqueous mixture of drug and the carrier is introduced via a nozzle (e.g., a two fluid nozzle), spinning disc or an equivalent device into a hot gas stream to atomize the solution to form fine droplets.
  • the aqueous mixture may be a solution, suspension, slurry, or the like, but needs to be homogeneous to ensure uniform distribution of the components in the mixture and ultimately the powdered composition.
  • the solvent generally water, rapidly evaporates from the droplets producing a fine dry powder having particles from about 1 pm to 5 pm in diameter.
  • the spray drying is done under conditions that result in a substantially amorphous powder of homogeneous constitution having a particle size that is respirable, a low moisture content and flow characteristics that allow for ready aerosolization.
  • the particle size of the resulting powder is such that more than about 98 percent of the mass is in particles having a diameter of about 10 pm or less with about 90 percent of the mass being in particles having a diameter less than 5 pm.
  • dry powder compositions also may be prepared by lyophilization and jet milling, as disclosed in International Patent Publication No. WO 91/16038, the disclosure of which are incorporated by reference.
  • the term "dispersibility” or “dispersible” means a dry powder having a moisture content of less than about 10 percent by weight (percent w) water, according to some embodiments below about 5 percent w and in some embodiments less than about 3 percent w; a particle size of about 1.0-5.0 Am mass median diameter (MMD), according to some embodiments 1.04.0 pm MMD, and in some embodiments 1.0-3.0 pm MMD; a delivered dose of about >30 percent, according to some embodiments >40 percent, according to some embodiments >50 percent, and in some embodiments >60 percent; and an aerosol particle size distribution of about 1.0-5.0 micro m mass median aerodynamic diameter (MMAD), according to some embodiments 1.5-4.5 fun MMAD, and in some embodiments 1.5-4.0 micro m MMAD.
  • Methods and compositions for improving dispersibility are disclosed in U.S. application Ser. No. 08/423,568, filed Apr. 14, 1995, the disclosure of which is hereby incorporated by reference.
  • the term "powder” means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli.
  • the powder is said to be "respirable.”
  • the average particle size is less than about 10 microns (micro m) in diameter with a relatively uniform spheroidal shape distribution.
  • the diameter is less than about 7.5 and, according to some embodiments, less than about 5.0 micro m.
  • the particle size distribution is between about 0.1 micro m and about 5 micro m in diameter; according to some embodiments the particle size distribution is about 0.3 micro m to about 5 micro m.
  • dry means that the composition has a moisture content such that the particles are readily dispersible in an inhalation device to form an aerosol.
  • This moisture content is generally below about 10 percent by weight (percent w) water, according to some embodiments below about 5 percent w, and according to some embodiments less than about 3 percent w.
  • the amount of the pharmaceutically acceptable carrier is that amount needed to provide the necessary stability, dispersibility, consistency and bulking characteristics to ensure a uniform pulmonary delivery of the composition to a subject in need thereof.
  • the amount may be from about 0.05 percent w to about 99.95 percent w, depending on the activity of the drug being employed. According to some embodiments about 5 percent w to about 95 percent will be used.
  • the carrier may be one or a combination of two or more pharmaceutical excipients, but generally will be substantially free of any "penetration enhancers.” Penetration enhancers are surface active compounds which promote penetration of a drug through a mucosal membrane or lining and are proposed for use in intranasal, intrarectal, and intravaginal drug formulations.
  • Exemplary penetration enhancers include bile salts, e.g., taurocholate, glycocholate, and deoxycholate; fusidates, e.g., taurodehydrofusidate; and biocompatible detergents, e.g., Tweens, Laureth-9, and the like.
  • bile salts e.g., taurocholate, glycocholate, and deoxycholate
  • fusidates e.g., taurodehydrofusidate
  • biocompatible detergents e.g., Tweens, Laureth-9, and the like.
  • the dry powder compositions of the described invention are readily absorbed in the lungs without the need to employ penetration enhancers.
  • the types of pharmaceutical excipients that are useful as carriers for pulmonary delivery include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
  • HSA human serum albumin
  • bulking agents such as carbohydrates, amino acids and polypeptides
  • pH adjusters or buffers such as sodium chloride
  • salts such as sodium chloride
  • Bulking agents that are particularly valuable for pulmonary delivery include compatible carbohydrates, polypeptides, amino acids or combinations thereof.
  • Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2- hydroxypropyl-i3-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like.
  • a preferred group of carbohydrates includes lactose, trehalose, raffinose, maltodextrins, and mannitol.
  • Suitable polypeptides include aspartame.
  • Amino acids include alanine and glycine, with glycine being preferred.
  • Additives which are minor components of the composition for pulmonary delivery, may be included for conformational stability during spray drying and for improving dispersibility of the powder.
  • additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.
  • the composition of the described invention is placed within a suitable dosage receptacle in an amount sufficient to provide a subject with a unit dosage treatment.
  • the dosage receptacle is one that fits within a suitable inhalation device to allow for the aerosolization of the dry powder composition by dispersion into a gas stream to form an aerosol and then capturing the aerosol so produced in a chamber having a mouthpiece attached for subsequent inhalation by a subject in need of treatment.
  • Such a dosage receptacle includes any container enclosing the composition known in the art such as gelatin or plastic capsules with a removable portion that allows a stream of gas (e.g., air) to be directed into the container to disperse the dry powder composition.
  • a stream of gas e.g., air
  • Such containers are exemplified by those shown in U.S. Pat. Nos. 4,227,522; U.S. Pat. No. 4,192,309; and U.S. Pat. No. 4,105,027.
  • Suitable containers also include those used in conjunction with Glaxo's Ventolin(R) Rotohaler brand powder inhaler or Fison's Spinhaler(R) brand powder inhaler.
  • Another suitable unit-dose container which provides a superior moisture barrier is formed from an aluminum foil plastic laminate.
  • the pharmaceutical-based powder is filled by weight or by volume into the depression in the formable foil and hermetically sealed with a covering foil-plastic laminate.
  • a container for use with a powder inhalation device is described in U.S. Pat. No. 4,778,054 and is used with Glaxo's Diskhaler(R) (U.S. Pat. Nos. 4,627,432; 4,811,731 ; and 5,035,237). All of these references are incorporated herein by reference.
  • compositions of the invention may be used in the form of drops or sprays (e.g., a nasal spray, aerosol spray, or pump spray) or other vehicles for nasal administration
  • drops or sprays e.g., a nasal spray, aerosol spray, or pump spray
  • other vehicles for nasal administration e.g., a nasal spray, aerosol spray, or pump spray
  • Aerosol spray preparations can be contained in a pressurized container with a suitable propellant such as a hydrocarbon propellant.
  • Pump spray dispensers can dispense a metered dose or a dose having a specific particle or droplet size. Any dispensing device can be arranged to dispense only a single dose, or a multiplicity of doses.
  • compositions of the invention especially those formulated for intranasal administration, can also be provided as solutions, suspensions, or viscous compositions (e.g., gels, lotions, creams, or ointments).
  • compositions of the described invention may be in the form of suppositories for rectal administration of the composition.
  • "Rectal” or “rectally” as used herein refers to introduction into the body through the rectum where absorption occurs through the walls of the rectum.
  • These compositions can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
  • a suitable nonirritating excipient such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
  • the compositions of the invention may be formulated with traditional binders and carriers, such as triglycerides.
  • topical refers to administration of an inventive composition at, or immediately beneath, the point of application.
  • topically applying describes application onto one or more surfaces(s) including epithelial surfaces.
  • topical administration in contrast to transdermal administration, generally provides a local rather than a systemic effect, as used herein, unless otherwise stated or implied, the terms topical administration and transdermal administration are used interchangeably.
  • topical applications shall include mouthwashes and gargles.
  • Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices which are prepared according to techniques and procedures well known in the art.
  • transdermal delivery system transdermal patch or “patch” refer to an adhesive system placed on the skin to deliver a time released dose of a drug(s) by passage from the dosage form through the skin to be available for distribution via the systemic circulation.
  • Transdermal patches are a well-accepted technology used to deliver a wide variety of pharmaceuticals, including, but not limited to, scopolamine for motion sickness, nitroglycerin for treatment of angina pectoris, clonidine for
  • Patches suitable for use in the described invention include, but are not limited to, (1) the matrix patch; (2) the reservoir patch; (3) the multi-laminate drug-in- adhesive patch; and (4) the monolithic drug-in-adhesive patch; TRANSDERMAL AND TOPICAL DRUG DELIVERY SYSTEMS, 249-297 (Tapash K. Ghosh et al. eds., 1997), hereby incorporated herein by reference. These patches are well known in the art and generally available commercially.
  • compositions of the described invention may be formulated with an excipient, vehicle or carrier selected from solvents, suspending agents, binding agents, fillers, lubricants, disintegrants, and wetting agents/surfactants/solubilizing agents.
  • excipient refers to substances that facilitate the use of, but do not deleteriously react with, the active compound(s) when mixed with it.
  • active refers to the ingredient, component or constituent of the compositions of the described invention responsible for the intended therapeutic effect. Carriers must be of sufficiently high purity and of sufficiently low toxicity to render them suitable for administration to the subject being treated.
  • the carrier can be inert, or it can possess pharmaceutical benefits.
  • the carrier can be liquid or solid and is selected with the planned manner of administration in mind to provide for the desired bulk, consistency, etc., when combined with an active and the other components of a given composition.
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (including, but not limited to pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (including but not limited to lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate.); lubricants (including, but not limited to magnesium stearate, talc, silica, sollidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate); disintegrants (including but not limited to starch, sodium starch glycolate) and wetting agents (including but not limited to sodium lau
  • compositions of the described invention include, but are not limited to, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil; fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.
  • the pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • the term "pharmaceutically acceptable carrier” as used herein refers to any substantially non-toxic carrier conventionally useful for administration of pharmaceuticals in which the active component will remain stable and bioavailable.
  • the pharmaceutically acceptable carrier of the compositions of the described invention include a release agent such as a sustained release or delayed release carrier.
  • the carrier can be any material capable of sustained or delayed release of the leptin peptide active ingredient to provide a more efficient administration, resulting in less frequent and/or decreased dosage of the active ingredient, ease of handling, and extended or delayed effects.
  • Non-limiting examples of such carriers include liposomes, microsponges, microspheres, or microcapsules of natural and synthetic polymers and the like. Liposomes may be formed from a variety of phospholipids such as cholesterol, stearylamines or phosphatidylcholines.
  • the therapeutically active leptin, leptin mimic, leptin agonist, or leptin derivative peptides, as well as leptin blockers and leptin antagonists of the described invention can be formulated per se or in salt form.
  • pharmaceutically acceptable salts refers to nontoxic salts of the peptides of the described invention.
  • the peptide salts which can be used for the invention are pharmaceutically acceptable salts of organic acids or pharmaceutically acceptable salts of inorganic acids.
  • Such pharmaceutically acceptable peptide salts include, but are not limited to, those formed with free amino groups such as those derived from hydrochloric, phosphoric, sulfuric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • compositions of the described invention can be prepared readily using technology is known in the art, such as that which is described in Remington's
  • compositions of the described invention can further include one or more compatible active ingredients aimed at providing the composition with another pharmaceutical effect in addition to that provided by a leptin, leptin mimic peptide or a derivative thereof
  • “Compatible” as used herein means that the active ingredients of such a composition are capable of being combined with each other in such a manner so that there is no interaction that would substantially reduce the efficacy of each active ingredient or the composition under ordinary use conditions.
  • the composition also may be administered serially or in combination with other compositions for treating diseases, conditions or disorders resulting from accumulation of amyloid peptides.
  • compositions may include monoclonal antibodies (such as monoclonal anti- beta -Amyloids and monoclonal anti- beta -secretases); and antiinflammatory compounds (including, but not limited to nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, indomethacin, and flurbiprofen).
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • Anti-inflammatory compounds have been shown to direct A beta -lowering properties in cell cultures as well as in transgenic models of AD-like amyloidosis.
  • a composition of the described invention may be administered to a subject in a single dose or multiple doses over a period of time.
  • therapeutically effective amounts and “pharmaceutically effective amounts” are used interchangeably to refer to the amount of the composition of the invention that results in a therapeutic or beneficial effect, including a subject's perception of health or general well-being, following its administration to a subject.
  • therapeutically effective amounts and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the described invention.
  • compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition resulting from accumulation of an amyloid peptide in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications and intermediate pathological phenotypes presenting during development of the disease, disorder or condition.
  • the concentration of the active substance is selected so as to exert its therapeutic effect, but low enough to avoid significant side effects within the scope and sound judgment of the skilled artisan.
  • the effective amount of the composition may vary with the age and physical condition of the biological subject being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the specific compound, composition or other active ingredient employed, the particular carrier utilized, and like factors. Those of skill in the art can readily evaluate such factors and, based on this information, determine the particular effective concentration of a composition of the described invention to be used for an intended purpose.
  • compositions or medicants are administered to a patient suspected of, having, or already suffering from, such a disease, disorder or condition in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease, disorder or condition, including its complications and intermediate pathological phenotypes in development of the disease, disorder or condition.
  • administration of the composition of the described invention reduces or eliminates cognitive impairment in patients that have not yet developed characteristic pathology of the disease, disorder or condition.
  • an amount adequate to accomplish therapeutic or prophylactic treatment is defined herein as a therapeutically-effective dose.
  • an amount of the compositions of the described invention is usually administered in several dosages until a sufficient beneficial response has been achieved. Typically, the response is monitored and repeated dosages are given if the response starts to wane.
  • a skilled artisan can determine a pharmaceutically effective amount of the inventive compositions by determining the dose in a dosage unit (meaning unit of use) that elicits a given intensity of effect, hereinafter referred to as the "unit dose.”
  • dose-intensity relationship refers to the manner in which the intensity of effect in an individual recipient relates to dose. The intensity of effect generally designated is 50 percent of maximum intensity.
  • the corresponding dose is called the 50 percent effective dose or individual ED50.
  • the use of the term “individual” distinguishes the ED50 based on the intensity of effect as used herein from the median effective dose, also abbreviated ED50, determined from frequency of response data in a population.
  • Effectiveness refers to the property of the compositions of the described invention to achieve the desired response, and “maximum efficacy” refers to the maximum achievable effect.
  • the amount of compounds in the compositions of the described invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • phosphorylated tau accumulation modulating amount refers to a therapeutically effective amount of a leptin composition that modulates the
  • a phosphorylated tau accumulation modulating amount includes prophylactic or preventative amounts of the compositions of the described invention.
  • the dosage ranges for the administration of the compositions of the described invention are those large enough to produce the desired therapeutic effect.
  • the therapeutically effective amount of the compositions of the described invention is administered one or more times per day on a regular basis.
  • a typical dose administered to a subject is between about 0.01 mg of the composition per kg (of body weight) per day and about 0.5 mg of the composition per kg (of body weight) per day.
  • the minimum dose of the composition is contemplated as about 0.01 mg/kg/day, about 0.025 mg/kg/day, about 0.05 mg/kg/day, about 0.075 mg/kg/day, about 0.08 mg/kg/day, about 0.1 mg/kg/day, about 0.125 mg/kg/day, about 0.15 mg/kg/day, about 0.175 mg/kg/day, about 0.2 mg/kg/day, about 0.225 mg/kg/day, about 0.25 mg/kg/day, about 0.275 mg/kg/day, about 0.3 mg/kg/day, about 0.325 mg/kg/day, about 0.35 mg/kg/day, about 0.375 mg/kg/day, about 0.4 mg/kg/day, about 0.45 mg/kg/day, about 0.475 mg/kg/day, or about 0.5 mg/kg/day and the maximum dose is contemplated as about 0.5 mg/kg/day, about 0.475 mg/kg/day, about 0.45 mg/kg/day, about 0.4 mg/kg/day, or about
  • compositions in humans between 0.01 and 0.08 mg of the composition per kg (of body weight) per day.
  • compositions and methods of the described invention can be assayed by a variety of protocols.
  • the effects of increasing cognitive function in a human subject can be determined by methods routine to those skilled in the art including, but not limited to, both paper and pencil, and computer tests.
  • One of skill in the art can also directly measure amyloid peptide accumulation levels, neurofibrillary tangle formation and neurodegeneration in animal models.
  • amyloid peptide may be measured in a sample of a subject's cerebrospinal fluid (CSF) obtained by spinal tap.
  • CSF cerebrospinal fluid
  • One measure of accumulation of an amyloid peptide is an increase in levels circulating in the blood of a subject.
  • Such levels may be measured by Sandwich Enzyme-linked-Immunoabsorbent- Assays (ELISAs), using a pair of antibodies, one for capture and the other for detection.
  • variant refers to a peptide sequence that varies at one or more amino acid positions with respect to the reference peptide.
  • a variant can be a naturally- occurring variant or can be the result of spontaneous, induced, or genetically engineered mutation(s) to the nucleic acid molecule encoding the variant peptide.
  • a variant peptide can also be a chemically synthesized variant.
  • a skilled artisan likewise can produce polypeptide variants having single or multiple amino acid substitutions, deletions, additions or replacements.
  • variants may include inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids; (b) variants in which one or more amino acids are added; (c) variants in which at least one amino acid includes a substituent group; (d) variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non- conserved positions; and (d) variants in which a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, such as, for example, an epitope for an antibody.
  • the techniques for obtaining such variants including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques are known to the skilled artisan.
  • the leptin of the described invention may be altered in various ways including amino acid substitutions, deletions, truncations (e.g, fragments), and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. For example, point mutations may be introduced into the leptin coding sequence using Invitrogen's GeneTaylor Mutagenesis System, Stratagene's QuickChange System or NEB's Phusion System according to manufacturer's guidelines. The described invention is intended to encompass conservative substitutions, such as exchanging one amino acid with another amino acid having similar properties without altering peptide structure and/or function.
  • the described invention provides a recombinant leptin.
  • Methods for producing recombinant peptides are well-known in the art.
  • the nucleic acid encoding leptin of the described invention may be inserted into a replicable vector for cloning
  • the vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage.
  • plasmid vectors include, but are not limited to, pET-26+ and pCMV6-AC.
  • the appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art.
  • Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
  • a number of promoters can be used in the practice of the invention.
  • a promoter can be employed which will direct expression of a polynucleotide of the described invention in E. coli.
  • Other equivalent transcription promoters from various sources are known to those of skill in the art.
  • expression and cloning vectors usually contain a promoter operably linked to the leptin encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known.
  • Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems (Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281 :544 (1979)), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776), and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)). Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding leptin.
  • S.D. Shine-Dalgarno
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.
  • Selection genes will typically contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, (e.g., the gene encoding D-alanine racemase for Bacilli).
  • suitable selectable markers for mammalian cells include, but are not limited to, those that enable the identification of cells competent to take up the leptin-encoding nucleic acid, such as DHFR or thymidine kinase.
  • DHFR DHFR activity
  • yeast plasmid YRp7 yeast plasmid YRp7
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85: 12 (1977)).
  • the leptin of the described invention may be produced recombinantly not only directly, but also as a fusion peptide with a heterologous peptide.
  • the heterologous peptide may include, but is not limited to, an IgGl-Fc, transferrin, a signal sequence or other peptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • leptin of the described invention may be cloned into the multiple cloning site of the pFUSE-hlgGl-Fc vector.
  • the hlgGl-Fc nucleotide sequence may be excised by restriction endonuclease from the pFUSE-hlgGl-Fc vector and human transferrin nucleotide sequence inserted.
  • Host cells are transfected or transformed with expression or cloning vectors described herein for leptin production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the culture conditions such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991).
  • Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl 2 , Ca 2 P0 4 , liposome-mediated and electroporation.
  • transformation is performed using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride, as described in Sambrook et al., or electroporation is generally used for prokaryotes.
  • the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J.
  • Suitable host cells for cloning or expressing DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example,
  • Enterobacteriaceae such as E. coli.
  • E. coli Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31 ,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and KS 772 (ATCC 53,635).
  • Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E.
  • IPTG isopropyl-beta-D-thiogalactopyranoside
  • recombinant leptin may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of leptin can be disrupted by various physical or chemical means, such as freeze- thaw cycling, sonication, mechanical disruption, or cell lysing agents.
  • Various methods of protein purification may be employed, and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular peptide produced.
  • the described invention provides a route of administrating the composition.
  • the composition may be constituted into any form suitable for the mode of administration selected.
  • routes of administration include, but are not limited to, parenteral (including subcutaneous), oral, inhalation, insufflation, topical, buccal and rectal.
  • Compositions suitable for parenteral administration include sterile solutions, emulsions and suspensions.
  • Oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
  • Compositions suitable for inhalation and insufflation may take the form of an aerosolized solution.
  • compositions suitable for topical administration include creams, ointments and dermal patches.
  • Compositions suitable for buccal administration may take the form of tablets or lozenges.
  • Compositions suitable for rectal administration may take the form of suppositories.
  • Formulations for administration may be provided using any formulation known in the art and appropriate for the route of administration. Such formulations may be as provided using the guidance of such resources as REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed., Mack Publishing Co., Easton, Pa. 1990.
  • the described invention provides for a composition comprising a leptin and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers encompass any of the standard pharmaceutical carriers. For example,
  • excipients may include a solution that is isotonic with blood such as saline, Ringer's solution, or dextrose solution.
  • non-aqueous vehicles such as fixed oils and ethyl oleate may be used, as well as liposomes.
  • excipients may be included that improve the efficacy, receptor affinity, or half-life of the active ingredient.
  • the EPO-derived peptides of the methods of the described invention may be pegylated (i.e., coupled with polyethylene glycol) by means well- known in the art to prolong the half-life of the active ingredient in the circulation. (See, e.g., Kozlowski et al. J.
  • the first composition comprises a leptin derivative, exemplified by a truncated leptin protein, or a leptin with unnatural amino acid substitutions, or other modifications that may alter leptin potency.
  • the leptin may be delivered through gene-therapy, including the use of viral vectors, like adeno-associated virus, herpes simplex virus, or retroviral vectors like lentiviral vectors which can encode the leptin therapeutic wherein the viral vector can be either by systemic or stereotactically administered into the CNS, wherein the therapeutic amount of the leptin or the leptin mimic is effective to modulate accumulation of the amyloid peptide in brain, the hyperphosphorylation of tau or loss of neuronal function as it can occur in neurodegenerative disorders.
  • viral vectors like adeno-associated virus, herpes simplex virus, or retroviral vectors like lentiviral vectors which can encode the leptin therapeutic wherein the viral vector can be either by systemic or stereotactically administered into the CNS, wherein the therapeutic amount of the leptin or the leptin mimic is effective to modulate accumulation of the amyloid peptide in brain, the hyperphosphorylation of tau or loss of neuronal function
  • the method may further comprise monitoring circulating levels of the amyloid peptide or hyperphosphyrlated tau by imaging or direct measurement of these proteins in the cerebral spinal fluid (CSF).
  • the circulating levels of amyloid peptide may be detected in a sample of CSF or blood.
  • MEM Minimum essential medium
  • ATCC Manassas, Va.
  • Trypsin- EDTA and penicillin solution were purchased from MP Biomedicals (Solon, Ohio).
  • Fetal bovine serum (FBS), all-trans retinoic acid (ATRA), G418, water-soluble cholesterol, water-soluble oleic acid and recombinant human leptin were purchased from Sigma- Aldrich (St. Louis, Mo.).
  • C 2 ceramide was purchased from EMD Biosciences (San Diego, Calif.).
  • Leptin fragments (Lep 22-56; Tyr-Lep 26-39; Lep 93-105) were purchased from CPC Scientific (Sunnyvale, Calif.).
  • SY5Y cells human neuroblastoma
  • the human neuroblastoma cell line, SH- SY5Y, was purchased from ATCC. Cell culture was performed according to manufacturer's specific guidelines. Cells were propagated in MEM containing 10 percent FBS. Neuronal differentiation was performed as described previously (Greco, S. J. et al., Biochem Biophys Res Commun 376 (2008) 536-541).
  • APP 751 -Accession # NM_201413 human APP
  • FuGENE HD transfection reagent FuGENE HD transfection reagent, according to manufacturer's specific instructions (Promega Madison, Wis.). Briefly, cells were transiently transfected with APP751 or vehicle for 48 h and then switched into selection medium containing a concentration range of the antibiotic G418 (100-600 micro g/mL) to determine the optimal dose for stable selection. Selection media was changed every 3 days to remove non- viable cells.
  • Neuro2a mouse neuroblastoma stably transfected with hyg-sal34, a
  • pcDNA3.1/Hygro plasmid (Invitrogen, CA) modified to express a fusion protein of secreted alkaline phosphatase (SEAP) and a fragment of APP consisting of the C-terminal 134 amino acids (“CAPP134”) (SEQ ID NO: 10) were maintained in culture as described (Johnsingh et al., J Neurosci. 14:4769-79 (2000)) in the presence of 400 micro g/ml of hygromycin.
  • SEAP secreted alkaline phosphatase
  • CAPP134 C-terminal 134 amino acids
  • SEQ ID NO: 11 is the DNA sequence of the entire hygsal34 vector, which was derived from the pcDNA3.1/Hygro vector by genetic manipulation to insert the DNA sequences for SEAP and CAPP 134-The SEAPCAPP cDNA insert from hyg-sal 34 was also subcloned into an adenoviral vector using the Adeno Vator system (Qbiogene, CA).
  • the DNA sequence for SEAP (corresponding to nucleotides 981-2441 of hyg-sal34) (SEQ ID NO: 9) is located 5' to the DNA sequence (SEQ ID NO: 12) coding for CAPP134 (SEQ ID NO: 10).
  • SY5Y and hyg-sal 34-Neuro2a cells were treated at 80 percent confluency (see below). Primary neural cultures from mouse embryos were allowed to grow for 6-12 days following plating and prior to viral infection and treatments. [0161] About 5 micro g/ml or about 10 micro g/ml water-soluble cholesterol was added to cultures for 2 or 5 hours. Water soluble cholesterol (Sigma- Aldrich, MO) is a solution made of cholesterol balanced with cyclodextrin CDX (40 mg cholesterol per gr CDX). For comparison, cultures were treated with the equivalent amount of the resin alone, which leads to depletion of cholesterol in the cultures (Simons et al., Proc Natl Acad Sci USA 95:6460-4 (1998)).
  • leptin About 100 ng/ml or about 400 ng/ml leptin (Harbor-UCLA, CA), was added in cell culture medium for 2 or 5 h. Cells were approximately 80 percent confluent at the time of treatment. Peptide YY (3-36) (Phoenix Pharmaceuticals, Inc., CA), and CNTF (Sigma- Aldrich, MO) were added at about 2511M or 150 l.IM for the same incubation periods. TOFA, etomoxir (Research Biochemicals International, MA) and cerulenin (Sigma-Aldrich, MO) were used as described below.
  • SY5Y cells in culture were metabolically labeled with 35 S-[Met] as described (Figueiredo-Pereira et al. J Neurochem. 72: 1417-22 (1999)), followed by immunoprecipitation, resolution of the immunoprecipitates on SDS-PAGE, autoradiography, and densitometric analysis of the autoradiogram.
  • Neuro2a cells were stably transfected with hyg-sal34 (K. Sambamurti, S.
  • Leptin was detected using a rabbit polyclonal antibody raised against mouse leptin, cross-reacting with human leptin (obtained from Dr. A. F. Parlow, Harbor-UCLA, CA). Immunofluorescent confocal microscopy was performed on 2 percent paraformaldehyde - fixed primary neural cells. Filipin staining was performed as described (Feng et al., Nat Cell Biol. 5:781-92 (2003)).
  • ApoE was isolated from the conditioned media of human embryonic kidney (HEK- 293) cells stably-transfected with human apoE (having the E3 allele or the E4 allele) cDNA (Tezapsidis et al., FASEB J. 17: 1322-1324 (2003)). These preparations, while usually poor in
  • RAP is an antagonist of a number of lipoprotein receptors (LaDu et al., Neurochem Int. 39:427-34 (2001)). After 24 h, the media were collected and subjected to scintillation counting for eradiation (Kang et al., 2000. J Clin Invest. 106: 1159-66 (2000)). The amount of radioactivity was measured in both the trichloroacetic acid (10 percent) TCA pellets (representing intact A beta) and the
  • the amount of TCA-precipitable radioactivity in the soluble fraction of cell lysates was compared to that in the total lysates, the ratio of which was typically about 0.8 to about 0.9 (not shown), to further verify that radioactivity was reduced in the media as a reflection of AP uptake by the cells, rather than due to non-specific binding to the extracellular surface of membranes or oligomerization/aggregation of A beta.
  • Human SREBP-1 (SEQ ID NO: 17) and SREBP-2 (SEQ ID NO: 19) cDNAs were obtained by polymerase chain reaction ("PCR") using a human brain cDNA expression library as a template. Briefly, 5'-gagaggatccaacagggcaggacacgaa-3' (linker italicized, BamHI site underlined) (SEQ ID NO: 20) was used as forward primer and 5'- gagagaattcggctgctgccaagggaca-3' (linker italicized, EcoRI site underlined) (SEQ ID NO: 21) was used as a reverse primer, generating a 1461 nt fragment of human SREBP-1 (GenBank Accession No.
  • Genlnfo Identifier (GI):409404) predicted to encode for SREBP-1 (1445 amino acids) (SEQ ID NO: 24).
  • SREBP-1 1445 amino acids
  • 5'- gagaggatecaaggttgtcgggtgtcatg-3' (linker italicized, BamHI site underlined) (SEQ ID NO: 22) was used as a forward primer and 5'-gagagaattcggctggctcatctttgacctt-3' (linker italicized, EcoRI site underlined) (SEQ ID NO: 23) as a reverse primer, generating a 1492 nt fragment of human SREBP-2 (GenBank Accession No. UO2031 , 01:451329), predicted to encode for SREBP-2 (1-467 amino acids) (SEQ ID NO: 25). The resulting 1.5-kb fragment was cloned into the BamHI and EcoRI sites of the pcDNA3.1 vector.
  • SEQ ID NO: 14 is the amino acid sequence of PS 1 in humans.
  • SEQ ID NO: 15 is the amino acid sequence of PS1MI46V- A single mutation at codon 146 co-segregates with the disease in members of early-onset Alzheimer's disease families. A base pair change from the normal sequence predicts M to V amino acid substitution at codon 146.
  • Plasma leptin concentrations were determined by a radioimmunoassay (RIA) (Chung et al., Am. J. Physiol. 274:R985-R990 (1998)), using a kit from LINCO Research, Inc. (Missouri).
  • RIA radioimmunoassay
  • mice Tg2576 or wild-type littermates were maintained in pathogen- free environment at 25 degrees centigrade on a 12-12 h light-dark cycle. Mice were euthanized between the ages of 31 and 40 weeks. They were provided ad libidum access for up to 9 weeks (i.e., 1 week prior to leptin treatments and 8 weeks during such treatments) to a high fat diet (D 12451) containing about 45 percent of the total calories from fat (Research Diets, NJ) or to a low fat diet (D 12450B) containing about 10 percent of the total calories from fat.
  • D 12451 high fat diet
  • D 12450B low fat diet
  • mice under each diet were administered leptin or a placebo (PBS) from the age of 32 wks to up to 40 wks of age.
  • PBS placebo
  • mice were anaesthetized with intraperitoneal injection of ketamine (55 mg/ml) and xylazine (7-10 mg/ml) and surgically fitted with an Alzet miniosmotic pump (model 2004, Durect Co, CA) placed subcutaneously.
  • Local subcutaneous injection of 0.5 ml of 0.5 percent lidocaine insured postoperative relief.
  • mice received daily about 20 pg leptin in PBS (0.25 micro 1/h of 3.33 mg/ml recombinant murine leptin) and the other half were infused with PBS.
  • PBS phosphatidylcholine
  • Four from each group two males and two females were euthanized after 4 weeks treatment.
  • Osmotic pumps were replaced in the rest and the mice then treated for a total period of 8 weeks. Wild- type littermates were also treated with leptin under high or low diet regimens.
  • the animal protocol was reviewed and approved by the Institutional Animal Core and Use Committee (IACUC) at the Columbia University Medical Center.
  • Leptin includes, but is not limited to, recombinant human leptin (PEG-OB, Hoffman La Roche) and recombinant methionyl human leptin (Amgen).
  • Leptin derivatives e.g., truncated forms of leptin (see para. 33 above), useful in the described invention include: U.S. Pat. Nos.
  • leptin fusion products useful in the described invention include, but are not limited to, fc-leptin, which is a fusion peptide derived from leptin and the Fc
  • fusion protein or “fusion product” as used herein refers to a protein created through genetic engineering from two or more proteins/peptides by creating a fusion gene (i.e., removing the stop codon from the DNA sequence of the first protein and appending the DNA sequence of the second protein in frame) so that the DNA sequence encoding the two or more
  • proteins/peptides is expressed by a cell as a single protein.
  • RA-SY5Y at 2x10 4 cells/well, were seeded in 96- well microplates and treated for 18 h with a range of concentrations of cholesterol to determine effective doses for 50-70 percent viability (data not shown), or in the presence of a range of concentrations of full length leptin or leptin fragments to determine effective doses to prevent cell death.
  • Viability was assessed using the Cell-Titer Blue Viability Assay (Promega) by adding 20 micro L of the reagent to each well for 4 h, and plates read by a microplate reader with fluorescence capabilities at EX53o-570nm EM58o-620nm- Viability was determined using a standard curve of known cell number and plotted as a percent of non-treated or vehicle control.
  • RA-SY5Y were treated with leptin (100 ng/ml) or leptin fragments (25 and 100 nM) in the presence of ceramide (25 micro M), or linoleic acid (30 micro g/mL) for 6 h, and then harvested by scraping. Preparation of whole cell lysates from cell pellets was performed as described previously (Greco, S. J. et al., Biochem Biophys Res Comm 376 (2008) 536-541).
  • a beta (i_ 4 o ) levels in cell culture media from SY5YAPP75I treated for 18 h with the aforementioned metabolic insults in the presence or absence of leptin were determined using the human amyloid beta 1-40 ELISA kit (Invitrogen; Carlsbad, Calif.), and phospho- and total tau levels in RA-SY5Y lysates were determined using the human Tau pSer , pThr and total tau ELISA kits (Invitrogen) according to manufacturer's specific instructions.
  • a beta ( 1-40), phosho- and total tau levels were calculated from a standard curve developed with OD at 450 nm using 8 serial dilutions of known concentration.
  • FIG. 4 shows that leptin affects Af3 production through BACE in rafts.
  • panel (a) Neuro2a cells stably transfected with hyg-sal34 were treated for about 2 h or about 5 h with about 100 ng/ml leptin, Ob (black); about 125 mg/ml cyclodextrin, CDX (striped gray); about 5 mg/ml cholesterol, Ch (pale gray); and leptin plus cholesterol, Ob+Ch (medium gray). Media were collected and assayed for total Ala by ELISAs (Figueiredo-Pereira et al., J Neurochem. 72: 1417-22 (1999)).
  • Results are expressed as a percentage of the corresponding controls that did not receive drug treatment, measured at about 2 h and about 5 h respectively.
  • Water soluble cholesterol (Sigma-Aldrich, MO) is a solution made of cholesterol balanced with CDX (40 mg cholesterol per gr CDX).
  • panel (b) Neuro2a cells stably transfected with hyg-sal34 were treated for about 2 h or 5 h with about 400 ng/ml leptin, Ob (black); about 250 mg/ml cyclodextrin, CDX (striped gray), about 10 mg/ml cholesterol, Ch (pale gray) and leptin plus cholesterol, Ob+Ch (medium gray) were used.
  • SY5Y cells in culture were treated with about 400 ng/ml leptin or about 10 tg/ml cholesterol, or both, in the presence of the y secretase inhibitors L-685,458 (100 nM) or Z-VL-CHO (10011M) for about 5 h.
  • Extracts prepared from harvested cells were analysed by SDS-PAGE and Western blotting using an antibody directed against the C-terminal fraction of APP (C-APP, lanes 1-4) or actin (top lanes 5-8) or full-length APP (bottom lanes 5-8).
  • Immunoreactive bands C99 and C83 correspond to beta - and gamma secretase-generated fragments.
  • results are expressed as the percent distribution of BACE activity within the gradient derived from cell cultures in the absence (black) or presence (gray) of leptin in the medium. Asterisks indicate that value is significantly different to that of the corresponding control (set at p ⁇ 0.05).
  • Leptin caused a dose- and time-dependent decrease in the levels of A beta detected in the media of transfected Neuro2a cells (56 plus or minus 5 percent following 5 h treatment with about 400 ng/ml leptin, Fig. 4 b). Leptin was almost as efficient as methyl-P- cyclodextrin in lowering A beta (Fig. 4 a, 4b). In agreement with published data (Refolo et al., Neurobiol Dis. 7:321-31 (2000)), inclusion of water-soluble cholesterol in the culture media increased AP production (205 plus or minus 6 percent after 5 h with 10 1.1 , M, Fig. 4 b).
  • Leptin partially reduced the amyloidogenic potency of cholesterol when co-administered with cholesterol (150 plus or minus 4 percent after 5 h with the highest concentrations of leptin, Fig. 4 b).
  • 125 I-A beta was included in the media during treatments in the presence or absence of 1 mM 1,10-phenanthroline, a general metalloprotease inhibitor which effectively inhibits degradation of secreted AP in vitro (Qiu et al., J Biol Chem. 272:6641- 6646 (1997)), none of these treatments caused any significant differences in the degradation
  • LRs were prepared from a Triton X-100-insoluble membrane fraction further resolved by separation on a discontinuous sucrose gradient. All steps were carried out at 4 degrees centigrade Confluent cells were scraped into 2 ml Mes-buffered saline (MBS, 25 mM Mes, 0.15M NaCl, pH 6.5) containing 1 percent (vol/vol) Triton X-100 and resuspended by passing them 5 times through a 25-gauge needle. An equal volume of 90 percent (wt/vol) sucrose in MBS then was added.
  • MBS Mes-buffered saline
  • BACE activity in extracts from control cells was detected in a low density fraction also containing flotillin (Fig. 4 d), an integral membrane protein known to be a marker for neuronal LRs (Bickel et al., J Biol Chem. 272: 13793-802 (1997)). Noticeably, the bulk of BACE activity was detected outside LRs, at higher density fractions. In addition, the distribution of APP immunoreactivity, as detected by Western blotting, was very similar to that of BACE activity in gradient fractions. Only a small fraction co-migrated with the flotillin peak (Fig. 4 d).
  • Leptin treatment resulted in a subtle change of the composition and/or density of LRs, as determined by the distribution of BACE activity, APP and flotillin on sucrose gradient fractions. Flotillin migrated at heavier subcellular fractions as compared to controls, and the activity of BACE in the low density fractions was almost absent. A similar shift in the elution position for both flotillin and BACE was observed when cells were treated with CDX (not shown).
  • Fig. 6 shows that leptin can modulate free cholesterol-rich membrane domains and surplus cholesterol may trigger local leptin production.
  • Neural cultures from E15 rat cerebral cortex were processed for enrichment of neurons (a-d) or astrocytes (e-h) as described (Takeshima et al., /. Neurosci. Methods 67: 27-41 (1996)). After about 7 days to about 10 days in culture, cultures were treated for about 5 h with about 10 pg/ml cholesterol (b, f) or about 400 ng/ml leptin plus cholesterol (c, g) or leptin alone (d, h). Controls (a, e) were treated with culture media alone.
  • etomoxir ethyl-2-[6-4-chlorophenoxy)hexyl)oxirane-2-carboxylate
  • CPTl carinitine palmitoyl transferase 1
  • Fig. 5 shows that leptin affects apoE-dependent An-uptake and the possible involvement of SREBPs.
  • a beta uptake was measured in SY5Y cells following their treatment at 0 ng/ml, 100 ng/ml or 400 ng/ml leptin. Uptake also was measured in cells previously transfected with antisense DNA for PS 1 as described (Tezapsidis et al., FASEB 117:1322-1324 (2003)) (black). Uptake is expressed as the percentage of that observed with A beta pre-incubated with apoE3 (medium gray) in the absence of leptin (first set of columns).
  • Asterisks indicate that the value is significantly different to that set as 100 percent (set at p ⁇ 0.05).
  • SY5Y cells were transiently transfected with SREBP-1 or SREBP-2 cDNA or an empty vector (Control). Then A beta was measured in the medium by ELISAs (Figueiredo-Pereira et al., J Neurochem. 72: 1417-22 (1999)) following treatment with (+) or without (-) leptin. Results are expressed as the percentage of the A beta produced in cells transfected with empty vector that did not receive leptin treatment, set at 100 percent (grey bar).
  • a beta uptake was measured in SY5Y cells prepared as in panel (c). Uptake was performed using A beta /apoE3 complexes. Results are expressed as the percentage of the A beta taken-up by cells transfected with empty vector that did not receive leptin treatment, set at 100 percent (black bar).
  • Leptin increased the uptake of apoE-A beta in a dose-dependent fashion (Fig. 5 a, striped and white bars for apoE3 and apoE4, respectively).
  • the E3 allele of apoE was more efficient in delivering A beta to the cell than the E4 allele. This indicates that the apoE isoform associated with increased risk for AD may be more resistant to the beneficial action of leptin in promoting lipid delivery to neurons and degradation of A beta.
  • SY5Y cells were preloaded with cholesterol by introducing a preincubation step with cholesterol/CDX, and compared to controls preincubated with medium.
  • Results are expressed: as mean ⁇ SEM from 4 experiments, each with 3 determinations. Values are expressed as a percentage of total ⁇ found in the conditioned media of cells not receiving treatment.
  • S Y5Y cells produced 252 ⁇ 50 pM
  • Neuro2a-SEAP-APP produced 820 ⁇ 210 pM
  • Neurons/SEAPP-APP produced 131 ⁇ 83 pM.
  • Student's t test was used and statistical significance was set at p50.05.
  • NS statistically non-significant
  • TOF A 5- (tetradecyloxy)-2-furancarboxylic acid
  • ACC Acetyl CoA carboxylase
  • F AS Fatty acid synthase
  • CPT -1 carnitine palmitoyl transferase-1
  • SREBP-la SEQ ID NO: 17
  • SREBP-lc SEQ ID NO: 18
  • SREBP-2 SEQ ID NO: 19
  • SREBP-la Two isoforms, SREBP-la and SREBP lc, are transcribed from the SREBP- 1 gene by alternative (or multiple) promoter usage for the same gene.
  • the acidic transactivation domain that mediates interactions with chromatin modifying coactivators is shorter in SREBP-lc.
  • SREBP-lc is a weaker transcriptional activator than SREBP-la (Shimano et al. J. Cli. Inv. 99 (1997) 846-854).
  • SREBP-1 refers to the a isoform of SREBP-1.
  • SREBP-2 (SEQ ID NO: 19) is more selective in activating the transcription of cholesterol biosynthetic genes, whereas SREBP 1 (SEQ ID NO: 17) and SREBP-lc (SEQ ID NO: 18) preferentially regulate fatty acid synthesis, however there is considerable overlap in their transcriptional activity.
  • transactivation refers to a technique used in molecular biology to control gene expression by stimulating transcription. It can be used to turn genes on and off. During transactivation, the transactivation gene and special promoters of DNA are inserted into the genome at areas of interest. The transactivator gene expresses a transcription factor that binds to specific promoter region(s) of DNA, causing that gene to be expressed. The expression of one transactivator gene can activate multiple genes, as long as they have the specific promoter region attached.
  • coactivators as used herein refers to a diverse array of gene regulatory proteins that do not themselves bind DNA but assemble on DNA-bound gene regulatory protein. They connect sequence- specific DNA binding activators to the general
  • Coactivator functions can be broadly divide into two classes: (a) adaptors that direct activator recruitment of the transcriptional apparatus, (b) chromatin-remodeling or -modifying enzymes.
  • SREBP-lc SEQ ID NO: 18
  • SY5Y cells were transfected with modified pcDNA3.1 vectors to drive the expression of SREBP-1 (SEQ ID NO: 17) or SREBP-2 (SEQ ID NO: 19) under the CMV promoter, and some of the experiments of A beta production or uptake in the presence or absence of leptin as already described were repeated.
  • SREBP-2 (SEQ ID NO: 19) transfected cells were more resistant to the inhibition of AP production by leptin as compared to SREBP-1 (SEQ ID NO: 17) transfected cells (Fig. 5 c).
  • SREBP-2 (SEQ ID NO: 19) cells were resistant to the increase of apoE/A beta uptake by leptin (Fig. 5 d).
  • transient expression of SREBP-1 (SEQ ID NO: 17) increased the production of A beta to 138 plus or minus 22 percent as compared to controls (Fig. 5 c) and reduced the uptake of apoE/A beta to 41 plus or minus 5 percent as compared to controls (Fig. 5 d).
  • SREBP-2 (SEQ ID NO: 19) expression increased production of A beta to 166 plus or minus 25 percent and inhibited uptake of apoE/A beta to 25 plus or minus 8 percent.
  • leptin limits the availability of a common precursor for fatty acids and cholesterol (i.e. acetyl-CoA) or b) post-leptin receptor signaling events somehow turn-off SREBP-1 (SEQ ID NO: 17), causing a reduction in cholesterol, which is important for A beta turnover. While the minor changes observed in SREBP- 1 (SEQ ID NO: 17) transfected cells in the presence of leptin support the second possibility, both may be working in cohort.
  • leptin appears to serve as a local feedback signal to inhibit further cholesterol synthesis and uptake, which in turn has an impact on Ap production and uptake. Consequently, deficiencies in either leptin or transduction of its signal in neural cells could be contributory to AD-related pathways.
  • glia are the cell group prominently synthesizing apoE, cholesterol and phospholipid rich HDL-like lipoprotein particles (Fagan et al., J Biol Chem. 274:30001-7 (1999)).
  • glia or "glial cell” are used interchangeably to refer to the connective tissue cells of the CNS that serve as the supportive structure that holds together and protects neurons).
  • Lipids are required by neurons during plasticity-related neuritic arborization/outgrowth or during neural progenitor cell proliferation.
  • Neuroplasticity refers to the ability of neural circuits to undergo changes in function or organization due to previous activity). Nonetheless, excess cholesterol and AP can be harmful.
  • bi-directional communication between neurons and glia may serve to balance local lipid requirement. It has been demonstrated previously that leptin can modulate hippocampal excitability via activation of large conductance calcium-activated potassium ion channels (Shanley et al., Nat Neurosci. 5:299-300 (2002)), supporting a link between endocrine factors and AD.
  • Plasma leptin levels were measured in transgenic mice engineered to express mutations linked to familial AD: APP with the Swedish mutation (APP swe ) (SEQ ID NO: 13), PS1 with the M146V substitution (PSl M i46v) (SEQ ID NO: 15), and both APP swe (SEQ ID NO: 13) and PS1MI46V (SEQ ID NO: 15).
  • APP Swedish mutation
  • PS1 PS1 with the M146V substitution
  • PS1MI46V SEQ ID NO: 13
  • PS1MI46V SEQ ID NO: 15
  • Fig. 7 shows a deficiency of leptin in AD transgenic mice and the effect of leptin supplementation on amyloid load.
  • plasma leptin was quantified in one year old mice with the following genotypes: i) double mutant APP swe /PS lMi46v ii) single mutant PS1MI46V and wild-type (a cross between C57BL/6Ntac and B6SJLF1). Asterisk indicates that value is significantly different to that of non-transgenic controls (set at p ⁇ 0.05). Plasma Ail was also measured in these mice prior to treatment.
  • Panel (b) shows Tg2576 mice under high fat (HFD) and low fat (LFD) diets one week prior to the implantation of the Alzet pump subcutaneously (s.c) for constant delivery of leptin (+) or vehicle PBS (-) at 8 months of age. The pump was replaced after 4 weeks.
  • Formic acid extracts of brains obtained as described previously were used to determine the A beta 40 (SEQ ID NO: 4) and A beta 42 (SEQ ID NO: 5) content by commercially available ELISA kits (KMI Diagnostics, MN), as described by the manufacturer.
  • mice Only APP swe - expressing mice (Tg2576) contained detectable amounts of A13 species. At 8 months of age the Tg2576 mouse has very low levels of A beta.
  • plasma leptin was determined by radioimmunoassay ("RIA") (LINCO Research, Inc.) in 8 month old Tg2576 and WT littermate mice and then again following treatments as described in Fig. 4 b. Leptin also was measured in WT but not Tg2576 mice prior to treatment.
  • plasma insulin was determined by RIA (LINCO Research, Inc.) in 8 month old WT and Tg2576 mice and then again following a 2 month LFD or HFD with (+) or without (-) leptin infusion.
  • plasma total A beta (A beta 40 (SEQ ID NO: 4) plus A beta 42/43 (SEQ ID NO: 5/SEQ ID NO: 6) was measured in 8 month Tg2576 mice and then again following a 2 month LFD or HFD with (+) or without (-) leptin infusion.
  • APPs we expressing transgenic Tg2576 mice under the high fat diet had higher levels of both A beta 40 and A beta 42 in formic acid extracts of brain homogenates when compared to those under the low fat diet (Fig. 7 c), in agreement with others (Refolo et al., Neurobiol Dis. 7:321-31 (2000)).
  • RA-SY5Y were treated for 18 h with full length or fragments of leptin ranging from 5 nM (white bars) to 500 nM (black bars), or no leptin control (checkered bars) in the presence (Fig. 9B) or absence (Fig. 9A) of cholesterol, and cell viability measured.
  • RA- SY5Y were treated for 6 h with full length leptin or leptin fragments (25 or 100 nM) or no leptin control (checkered bars) in the presence (Fig. 10B) or absence (Fig. 10A) of linoleic acid, and phosphorylation of tau at pTau 396 was measured by ELISA. Challenging cells with linoleic acid resulted in an approximately 50 percent increase in tau phosphorylation (Fig. 10B, checkered bar). Simultaneous treatment with full length or fragments of leptin showed limited effects on tau phosphorylation in the absence of linoleic acid (Fig.
  • leptin receptors are present throughout the brain including the hippocampus and olfactory bulb, domains affected early during the course of the disease. Because dysregulation of pathways associated with leptin may play a critical role in the pathogenesis of AD, leptin treatment may be beneficial in some AD cases, specifically those experiencing weight loss and/or have low circulating leptin levels.

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Abstract

La présente invention concerne des méthodes de traitement, de prévention ou de diagnostic de la pathologie de troubles cognitifs progressifs provenant de l'accumulation d'un peptide amyloïde, en particulier, la maladie d'Alzheimer, le syndrome de Down et l'angiopathie amyloïde cérébrale, chez des sujets mammifères à l'aide d'une composition comprenant une quantité thérapeutiquement efficace d'une leptine, d'un mimétique de leptine, d'un dérivé de leptine, d'un agoniste de leptine ou d'un activateur de protéine kinase dépendante de l'AMP, seul ou en combinaison avec, un ou plusieurs composés lipolitique/antilipogénique. L'invention concerne en outre des procédés pour améliorer la fonction cognitive à l'aide d'une composition comprenant une quantité thérapeutiquement efficace de leptine, d'un mimétique de leptine, d'un dérivé de leptine, d'un activateur de protéine kinase dépendante de l'AMP, d'un agoniste de leptine, d'un bloqueur de leptine, d'un mimétique d'un bloqueur de leptine, d'un antagoniste de leptine, d'un inhibiteur de protéine kinase dépendante de l'AMP ; ou d'un sel pharmaceutiquement acceptable de ceux-ci.
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