WO2008098371A1 - Compositions et procédé de réduction de la bêta-amyloïde dans un mammifère - Google Patents

Compositions et procédé de réduction de la bêta-amyloïde dans un mammifère Download PDF

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WO2008098371A1
WO2008098371A1 PCT/CA2008/000297 CA2008000297W WO2008098371A1 WO 2008098371 A1 WO2008098371 A1 WO 2008098371A1 CA 2008000297 W CA2008000297 W CA 2008000297W WO 2008098371 A1 WO2008098371 A1 WO 2008098371A1
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inositol
compound
disease
alzheimer
combination
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PCT/CA2008/000297
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English (en)
Inventor
Claudio A. Cuello
Martin Bruno
Adriana Ducatenzeiler
Vanessa Partridge
Wanda Leon
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The Royal Institution For The Advancement Of Learning/Mcgill University
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Publication of WO2008098371A1 publication Critical patent/WO2008098371A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/65—Tetracyclines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to combinations, compositions and methods for reducing amyloid beta (A ⁇ ) intracellular accumulation and/or reducing extracellular A ⁇ oligomers or A ⁇ aggregates in a mammal. More particularly, the present invention provides combinations, compositions and methods for treating Alzheimer's disease.
  • a ⁇ amyloid beta
  • AD Alzheimer's disease
  • a ⁇ aggregated ⁇ -amyloid protein
  • tau protein ⁇ -amyloid protein
  • Experimental work in AD transgenic models indicates that this cholinergic depletion is driven by the progressive amyloid pathology. It is well established that the CNS cholinergic system participates in higher CNS functions such as learning and memory. It is generally accepted that cholinergic hypofunction contributes to dementia-related cognitive decline.
  • proNGF the NGF precursor protein
  • a ⁇ is involved in the activation of poly(ADP-ribose) polymerase isoform 1 (PARP-1) in the adult hippocampus 4 and its activity is enhanced in AD 5 .
  • PARP-1 is known to promote microglia activation, proliferation and MMP-9-mediated neurotoxic effects 6 .
  • PARP-1 activation also plays an important role in up-regulating inflammatory cascades 7 ' 8 and its inhibition has been recently shown to protect the brain against systemic inflammation 9 and reduce MMP-9 brain expression 10 .
  • NO participates prominently in inflammation and activates MMP-9 by S-nitrosylation in response to oxidative stress 11 .
  • McLaurin further proposes in US2004/0204387A1 and in international application No. PCT/CA2005/001744 published under No. WO2006/053428A1 , methods and compositions comprising scyllo-inositol or substituted scyllo-inositol compounds for treating disorders involving amyloid formation, deposition, accumulation or persistence.
  • McLaurin indicates that scyllo-inositol not only inhibit de novo amyloid aggregation but also inhibits already established cerebral amyloid deposition and improved cognitive outcomes.
  • Minocycline a semisynthetic tetracycline with anti-inflammatory properties, is a small highly lipophilic molecule which is able to cross the blood-brain barrier, is readily absorbed after oral ingestion, and has a good clinical safety record 16 .
  • Minocycline has also demonstrated some neuroprotective properties in certain disease and lesion models. It effectively inhibits PARP-1 14 and iNOS 15 displaying anti-inflammatory properties. It is thought that microglial cells are key in the cellular cascade resulting in neuronal cell death. These cells are associated with amyloid plaques and produce a number of pro-inflammatory cytokines that can results in neuronal death.
  • Minocycline has been shown to attenuate glial cell activation and transcription of downstream pro- inflammatory mediators in mice injected with a specific immunotoxin able to mimic the selective loss of basal forebrain cholinergic neurons and cognitive impairment in mice.
  • Alzheimer's disease by administration of anti-microbial agents having aut ⁇ -Chlamydia pneumoniae activity including for example, minocycline, doxycycline, etc.
  • anti-microbial agents having aut ⁇ -Chlamydia pneumoniae activity including for example, minocycline, doxycycline, etc.
  • the methods of Balin et al. clearly demand for the individual to be or to have been infected with Chlamydia pneumoniae.
  • Alzheimer's disease none of them targets multiple aspects of Alzheimer's disease including inflammation, NGF dysmetabolism and A ⁇ accumulation.
  • Alzheimer's disease These are: A) the removal-inhibition of highly toxic A ⁇ oligomers, B) the inhibition of the A ⁇ -induced inflammatory cascade and C) the correction of the NGF dysmetabolism.
  • the present invention may thus be used to simultaneously target these three main aspects of the AD pathology.
  • the present invention therefore provides a combined therapy which may concurrently target the NGF metabolism pathway and the A ⁇ accumulation and aggregation.
  • the present invention relates in one aspect to the combination of an inositol compound and a tetracycline compound.
  • the combination of compounds surprisingly work together to significantly reduce A ⁇ accumulation in the brain of a mammal. As there is no cure to Alzheimer's disease nor any effective treatment, this new combination represents a potential solution of a long awaited therapeutic treatment of this and related diseases.
  • the present invention therefore provides in another aspect, a pharmaceutical composition which may comprise or consists in a) a tetracycline compound b) an inositol compound and; c) a pharmaceutically acceptable carrier.
  • a pharmaceutical composition which may comprise or consists in a) a tetracycline compound b) an inositol compound and; c) a pharmaceutically acceptable carrier.
  • a ⁇ oligomers have been observed to be the main cause of loss of synaptic function and for loss of cognitive function and as such, the pharmaceutical composition of the present invention may improve the clinical outcome of patients having or susceptible of having Alzheimer's disease.
  • the tetracycline compound and the inositol compound may be mixed together or may be provided separately (e.g., in a kit) with indications on how the drugs are to be administered or on how it may be mixed or combined.
  • the combination of both compounds may reduce A ⁇ intracellular accumulation and/or extracellular accumulation of A ⁇ oligomers or aggregates in the brain of a mammal in a more efficient manner than scyllo-inositol.
  • the compounds may be used to reduce A ⁇ accumulation extracellularly and/or intracellular ⁇ in neurons such as for example, in neurons of the cerebral cortex (neocortex and entorhinal) and/or in neurons of the hippocampal complex, etc.
  • the combinations and compositions of the present invention may be advantageously administered prior to disease onset or in the early stage of the disease.
  • individuals who are found to be susceptible of developping Alzheimer's disease are individuals who are found to be susceptible of developping Alzheimer's disease.
  • Such individuals may be those having a family member diagnosed with Alzheimer's disease, those having a genetic predisposition (e.g., at least one mutation in the amyloid precursor protein (APP) gene, carrier of Apoe allele associated with risk factor, etc.) associated with AD and/or having a family member having a genetic predisposition associated with AD.
  • Such predispositions may also be associated with the over-expression of APP and/or increased proteolytic processing of APP.
  • Other individuals who may benefit from the treatment are those with an early diagnosis of AD.
  • Table 1 is from John Hardy, National Institute on Aging, Bethesda, MD, and Richard Crook, MD., Mayo Clinic, Jacksonville, FL. (http://www.alzforum.org/res/com/mut/app/table1.asp)
  • the tetracycline compound of the present invention is one which, may be able to inhibit (partially or totally) PARP-1 and/or iNOS activity or expression.
  • the tetracycline compound and/or the inositol compound may be able to cross the blood brain barrier.
  • the present invention also relates in another aspect thereof to the use of a tetracycline compound and an inositol compound in the making of a pharmaceutical composition, drug or medicament for inhibiting amyloid beta (A ⁇ ) intracellular accumulation and/or reducing A ⁇ extracellular oligomers or aggregates in the brain of a mammal (an individual in need).
  • a ⁇ amyloid beta
  • the present therapy may thus be useful in the treatment or prevention of Alzheimer's disease.
  • the present therapy may also be useful in the treatment or prevention of Down's syndrome.
  • the present invention provides a kit which may comprise a vial or vials (e.g., without limitation, container(s), package(s), etc.) containing a tetracycline compound and an inositol compound (separately or together).
  • the kit may comprise instructions as to the method of administration of the drugs.
  • the present invention provides an article of manufacture which may comprise; a) a vial containing a tetracycline compound and an inositol compound; or b) packaged together; a first vial containing a tetracycline compound and a second vial containing an inositol compound.
  • the present invention relates in an additional aspect to a method for reducing or inhibiting amyloid beta (A ⁇ ) intracellular accumulation and/or reducing A ⁇ extracellular oligomers or aggregates in an individual in need thereof (mammal) (for example, for reducing the A ⁇ oligomer burden in the brain of an individual).
  • the method may comprise administering to the individual in need a tetracycline compound and an inositol compound in combination.
  • An exemplary embodiment of the method of the present invention may comprise first identifying an individual as being susceptible of developping AD and then administering the combinations or compositions of the present invention.
  • Another exemplary embodiment of the method of the present invention may comprise first identifying an individual as being at an early stage of Alzheimer's disease and then administering the combinations or compositions of the present invention.
  • the tetracycline compound and inositol compound may be administered, for example, prior to a substantial A ⁇ intracellular accumulation occurrence or prior to the extracellular accumulation of A ⁇ oligomers or before to the formation of A ⁇ plaques or aggregates.
  • the compounds of the present invention may be administered to prevent or to treat Alzheimer's disease and more particularly the administration may be performed at an early stage of Alzheimer's disease in prodromic stages such as in the mild cognitive impairment phase or even before the appearance of a clinical sign in patients at risk
  • the tetracycline compound and inositol compound may be administered after a substantial A ⁇ intracellular accumulation has occurred or after the formation of A ⁇ plaques or aggregates.
  • the compounds of the present invention may be administered to prevent or to treat Alzheimer's disease and more particularly the administration may be performed at a mild, moderate or severe phase of Alzheimer's disease.
  • the present invention provides the use of a tetracycline compound and (e.g., in combination with) an inositol compound for the treatment of a mammal which may have or may be susceptible of having A ⁇ intracellular accumulation or extracellular A ⁇ oligomers or aggregates.
  • Alzheimer's disease having or susceptible of having or developing Alzheimer's disease.
  • individuals susceptible of having Alzheimer's disease include individuals having a family member diagnosed with Alzheimer's disease, those having a genetic predisposition associated with AD and/or having a family member having such predisposition.
  • the mammal may be at a stage of
  • Alzheimer's disease characterized as being mild, moderate or severe.
  • composition of the present invention may be used to improve the behavioral and pathological outcomes of the AD-like amyloid pathology.
  • Pharmaceutical composition of the present invention may thus be used at a preclinical phase or at a phase characterized as being mild, moderate or severe
  • Figure 1 is a schematic representation of the cascade responsible for the processing of the activity-dependent released of proNGF, its conversion to mature NGF (mNGF) and its degradation;
  • Figure 2A Upper panel is a gelatin zymograph depicting the increased activity of MMP-9 and MMP-2 in Alzheimer's disease brain; and lower panel are Western blots illustrating the level of expression of pro-MMP9 and active MMP-9 in in AD brain in comparison with non-demented age-matched control;
  • FIG. 2B Upper panels are Western blots illustrating the increase level of expression of proNGF in AD brain in comparison with non-demented age-matched control, lower panel is a quantitative histogram illustrating the results of the upper panel. ; Expression levels of proNGF have been normalized to those of ⁇ -tubulin;
  • FIG. 2C Upper panels are Western blots illustrating the decreased expression levels of plasminogen and plasmin in AD brain in comparison with non- demented age-matched control, lower panel are quantitative histograms illustrating the results of the upper panel. Expression levels of plasminogen and plasmin have been normalized to those of ⁇ -tubulin;
  • Figure 2D presents Western blots illustrating the increased expression levels of PARP-1 and inducible nitric oxide synthase (iNOS) in AD brain in comparison to non-demented age-matched control. Expression levels of PARP-1 and iNOS have been normalized to those of ⁇ -tubulin,
  • Figure 2E is a quantitative histogram illustrating the increase in PARP-1 expression level in AD brain in comparison with non-demented age-matched control of Figure 2D. Expression levels of PARP-1 have been normalized to those of ⁇ -tubulin;
  • Figure 2F is a quantitative histogram illustrating the increase in iNOS-1 expression level in AD brain in comparison to non-demented age-matched control of Figure 2D . Expression levels of iNOS have been normalized to those of ⁇ -tubulin;
  • Figure 3A is a Dot-blot assay measuring soluble monomers (Mono) and soluble A ⁇ oligomers (Oligo) preparations using an oligomer-specific antibody A11 or the 6E10 antibody able to detect A ⁇ mono and oligomeric forms;
  • Figure 3B are Western blots illustrating the elevated level of pro-NGF expression and the activity of MMP-9 and MMP-2 in four months old Fischer-344 rats infused for 72 hours with soluble monomeric or soluble oligomeric forms of A ⁇ in the hippocampal region of the brain;
  • Figure 3C are quantitative histograms representing the relative pro-
  • Figure 3D are Western blots measuring the levels of pro-NGF, PARP-1 , iNOS expression and activity of MMP-9 in the presence of A ⁇ oligomers (Oligo) alone or after treatment with either 3-aminobenzamide or minocycline;
  • Figure 3E are quantitative histograms representing the ratio of pro-
  • NGF/ ⁇ -tubulin expression the ratio of PARP-1/ ⁇ -tubulin expression, the ratio of iNOS/ ⁇ - tubulin expression and the MMP-9 activity in the presence of A ⁇ oligomers (Oligo) alone or treatment with either 3-aminobenzamide or minocycline as obtained in Figure 3D.
  • Expression levels have been normalized to those of ⁇ -tubulin;
  • Figure 4 is a schematic representation of the observed dysregulation of the cascade responsible for the processing and degradation of NGF observed in AD;
  • Figure 5A comprises a graphic illustration of the transgene over- expression from 3, 6 and 9 months transgenic (Tg) mice (the A ⁇ peptide values correspond to A ⁇ 1-42) and immunohistochemical data on the distribution and intensity of the intracellular A ⁇ immunoreactivity in neurons of the cerebral cortex (neocortex and entorhinal) and in neurons of the hippocampal complex at 3 months of age. At later stage (4 to 6 months) these mice develop A ⁇ amyloid plaques as revealed by Thioflavin S in the cerebral cortex ;
  • Figure 5B presents Western blots illustrating the level of pro-NGF expression of APP and a zymograph illustrating increased pro-MMP-9, MMP-9 and MMP-2 activity in Tg mice. Expression levels of APP have been normalized to those of ⁇ - tubulin. On the right side are graphs illustrating the early behavioral impairment of this transgenic model in the learning of the escape behavior in the Morris Water Maze task at 3 months and 9 months of age;
  • Figure 6B represents immunohistochemical data on the distribution and intensity of the intracellular A ⁇ immunoreactivity in neurons of the cerebral cortex (neocortex and entorhinal) and in neurons of the hippocampal complex (dentate granular and pyramidal CA1 to CA3 and hylum).
  • Scale bar in e) applies to images in a) and c) and scale bar in f) to b) and d).
  • tetracycline compound refers to a family of compounds sharing a similar structure with tetracycline (e.g., four hydrocarbon rings derivation).
  • tetracycline compound refers to naturally- occurring molecule, semi-synthetic molecules, synthetic molecules, stereoisomers and enantiomers or salts thereof having the biological activity described herein.
  • tetracycline compound include without limitation, tetracycline, chlortetracycline, oxytetracycline, demeclocycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, as well as tetracycline analogues, tetracycline derivatives, tetracycline prodrugs or substituted tetracycline such as those disclosed or referred to, for example in US patent Nos.
  • tetracycline compound may be for example, minocycline and/or doxycycline or salt thereof. It is to be understood that the pharmaceutical composition may comprise at least one tetracycline compound but it may comprise combination of different tetracycline compounds.
  • inositol compound refers to a family of compounds sharing a similar structure with inositol and includes for example and without limitation naturally-occurring molecule, semi-synthetic molecules, synthetic molecules, stereoisomers, and enantiomers or salts thereof having the biological activity described herein.
  • exemplary embodiments of "inositol compound” include without limitation, scyllo-inositol, epi-inositol, myo-inositol, as well as inositol analogues, inositol derivatives, inositol prodrugs or substituted inositol and more particularly substituted scyllo-inositol such as those disclosed or referred to, for example in US publication No. US2004/0204387A1 and in international application No. PCT/CA2005/001744 or salts thereof.
  • inositol compound may be for example, scyllo-inositol or a substituted scyllo-inositol. It is to be understood that the pharmaceutical composition may comprise at least one inositol compound but it may also comprise combination of different inositol compounds.
  • the tetracycline compound and/or the inositol compound may be in a pure or substantially pure form.
  • Staging of Alzheimer's disease may be determined by methods known in the art. Method for the diagnosis of Alzheimer's disease may be found for example at http://www.alz.org/alzheimers disease diagnosis. asp.
  • Symptoms associated with the different stages of Alzheimer's disease are discussed at, e.g., http://www.alzheimersdisease.com/info/about/stages- alzheimers.jsp.
  • Administration of the compounds may be performed using methods known in the art.
  • the compounds of the present invention may be administered separately, via different routes or sites of administration, as a single formulation, etc.
  • the compounds may be administered separately, the compounds may be administered at the same time or at different (and/or defined) time intervals (e.g., sequentially, consecutively). Administration may be performed simultaneously (e.g., concurrently).
  • administering and “administration” is intended to mean a mode of delivery which may include, without limitation, intra-arterially, intra-nasally, intra- peritoneally, intravenously, intramuscularly, sub-cutaneously, transdermal ⁇ , per os etc.
  • the term "therapeutically effective” or “effective amount” is intended to mean an amount of a compound sufficient to substantially improve some symptom associated with a disease or a medical condition.
  • an agent or compound which decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be therapeutically effective.
  • a therapeutically effective amount of an agent or compound is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in an individual.
  • compositions means therapeutically effective amounts of the compound(s) together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers.
  • Such compositions may be in the form of liquids or lyophilized or otherwise dried formulations and may include diluents such as water or of various buffer content (e.g., Tris-HCI., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts).
  • buffer content e.g., Tris-HCI., acetate, phosphate
  • additives such as albumin or gelatin to prevent absorption to surfaces
  • detergents e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts.
  • Solubilizing agents e.g., glycerol, polyethylene glycerol
  • anti-oxidants e.g., ascorbic acid, sodium metabisulfite
  • preservatives e.g., thimerosal, benzyl alcohol, parabens
  • bulking substances or tonicity modifiers e.g., lactose, mannitol
  • covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
  • Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils).
  • particulate compositions coated with polymers e.g., poloxamers or poloxamines.
  • Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes.
  • the pharmaceutical composition may be administered parenterally, transmucosally, transdermal ⁇ , intramuscularly, intravenously, intradermal ⁇ , subcutaneously, intraperitonealy, intraventricular ⁇ , intracranial ⁇ or intratumorally.
  • pharmaceutical composition may also comprise composition suitable for ingestion, i.e., in the form of food supplement.
  • the formulations of the present invention include those suitable for oral, rectal, ophthalmic, (including intravitreal or intracameral) nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into associate the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • treatment for purposes of the present invention may refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • treating means also to inhibit (partially or totally), to delay the apparition or progression of the disease or to reverse the disease and/or its symptoms.
  • Figure 1 is a schematic representation of the cascade responsible for the processing of the activity-dependent release of proNGF, its conversion to mature NGF (mNGF) and its degradation by the activated matrix metallo-protease 9 (MMP-9) based on the work of Bruno and Cuello (PNAS, 2006). It was hypothesized that the activation of this pathway may favor cholinergic function, which is known to switch the APP towards a non-amyloidogenic ( ⁇ -secretase) pathway via the activation of muscarinic receptors M1 and M3.
  • MMP-9 matrix metallo-protease 9
  • a ⁇ has been shown to be involved in the activation of poly(ADP-ribose) polymerase isoform 1 (PARP-1) in the adult hippocampus 4 and its activity is enhanced in AD 5 .
  • PARP-1 is known to promote microglia activation, proliferation and MMP-9-mediated neurotoxic effects 6 .
  • PARP-1 activation also plays an important role in up-regulating inflammatory cascades 7 8 and its inhibition has been recently shown to protect the brain against systemic inflammation 9 and reduce MMP-9 brain expression 10 .
  • NO participates prominently in inflammation and activates MMP-9 by S-nitrosylation in response to oxidative stress 11 . It is demonstrated herein that A ⁇ oligomers enhance both PARP-1 and iNOS activity.
  • MMP-9, NGF and pro-NGF, iNOS and PARP-1 in AD brain results are illustrated in Figure 2A-F.
  • the volume of the immunoreactive bands was determined by densitometry using a computer-assisted image analysis system. Values were normalized to ⁇ -tubulin.
  • a Dot-blot assay is presented in Figure 3A for corroborating the presence of soluble monomers (Mono) and the prepared soluble A ⁇ oligomers (Oligo) following established protocols.
  • the presence of soluble oligomers was detected using oligomer-specific antibody A11 (Gift from Dr Glabe, Irvine, Ca).
  • oligomer-specific antibody A11 As a positive control, the detection of A ⁇ mono and oligomeric forms were detected by re-blotting using 6E10 antibody (Chemicon).
  • 6E10 antibody 6E10 antibody
  • the volume of the immunoreactive bands was determined by densitometry using a computer-assisted image analysis system. Values were normalized to ⁇ -tubulin. Statistical significance between pixel values for control and treated groups was calculated using Mest. Bars represent mean intensity ⁇ SEM.
  • FIG. 4 is a schematic representation of the dysregulation of the cascade responsible for the processing and degradation of NGF observed in AD (based on the results of Figure 2 and Figure 3).
  • Tg mice A transgenic (Tg) mouse model of AD was developed. These Tg mice carry a human APP transgene bearing the Swedish double mutation and the 717 Indiana mutations under the control of the Thy1 promoter. These mice were characterized for transgene expression and AD-like pathology as indicated above ( Figure 5).
  • Figure 5A is a graphic illustration of the transgene over-expression from 3 to 9 months of age.
  • a ⁇ peptide values correspond to A ⁇ 1-42.
  • Figure 5B is a Western blot and Zymography illustration showing the early (3 months of age) up-regulation of proNGF (upper panel) and of MMP-9 (zymography, lower panel) in this transgenic model of the AD-like A ⁇ pathology.
  • mice express the APP transgene 12 fold higher than endogenous levels; display a clear cut phase of intracellular A ⁇ accumulation in neurons of the neocortex, entorhinal cortex, amygdala and hippocampus from 1 to 3 months of age, diffuse extracellular amyloid plaques starting at 4 months of age, and mature amyloid (thioflavin-S positive) starting at 5 months of age.
  • Tg mice display a dysregulation of the NGF metabolic protease cascade with an AD-like elevation of cortical and hippocampal proNGF levels.
  • the animals have progressive behavioral impairments detected as early as 3 months and total inability to learn the Morris water maze task at 9 months.
  • the pathology of these Tg mice is accompanied by progressive dysregulation of key kinases related to synaptic plasticity and also by the involvement of the NGF- convertase cascade described by Bruno and Cuello 11 which are akin to the observations made in human AD brain (see Figure 2).
  • This transgenic model is fairly similar to that reported by Westaway and collaborators (coded CRND8).
  • the effectiveness of a combination therapy comprising minocycine and scyllo-inositiol is assessed in our Tg mouse model.
  • the initial dosage used is 50mg/kg/day, through subcutaneous release pellets of minocycline as this dosage has been shown to protect basal forebrain cholinergic neurons 18 and inhibit PARP-1 14 and iNOS 15 .
  • the initial dosage was 30mg/kg/day, as it has been shown to be effective in preventing A ⁇ amyloid aggregation when orally administered 13 .
  • Both drugs are water soluble and are not toxic in human or animals at these doses. In our pilot experiment, no sign of toxicity or side effects were observed in treated animals.
  • the combined therapy is evaluated to assess the added benefits for the prevention and/or reversal of the pathological and behavioral effects of the AD-like A ⁇ amyloid overexpression.
  • Tg and non-Tg littermate mice are segregated in gender and age-matched cohorts and treated separately or simultaneously for 3 months with scyllo-inositol/minocycline at two different stages of the AD-like pathology (early stage : Aim 1 and late stage: Aim 2).
  • These treatment-initiation stages are for Aim 1 at the intracellular AB, pre-plaque, pathology stage and for Aim 2 starting at A ⁇ amyloid mature plaque pathology stage.
  • Aim 1 The effectiveness of the combination minocycline/scyllo-inositol treatment is tested when administered at "early" stages of the AD-like pathology, beginning at 8 weeks of age and continuing until 5 months of age.
  • Group 1 non-Tg mice without treatment
  • Group 2 non-Tg mice treated with minocycline
  • Group 3 non-Tg mice treated with scyllo-inositol
  • Group 4 non-Tg mice treated with minocycline/scyllo-inositol
  • Group 5 Tg mice without treatment
  • Group 6 Tg mice treated with minocycline
  • Group 7 Tg mice treated with scyllo-inositol
  • Group 8 Tg mice treated with minocycline/scyllo-inositol. Each group includes 12 mice.
  • Aim 2 The effectiveness of the combination minocycline/scyllo-inositol treatment is tested when administered at "late" stages of the AD-like pathology, beginning at 5 months of age and continuing until 9 months of age.
  • Tg mice and non-Tg littermates are separated in Groups 9 to 16, analogous to Aim 1 above.
  • the behavioral outcome will be most important as this Tg mice model at this age displays a complete impairment in acquiring the escape behavior in the Morris water maze task.
  • this Tg animal displays advanced inflammation and NGF dysmetabolism.
  • Behavioral test at the end of the treatments, animals are evaluated cognitively, measured by spatial reference learning in the Morris water maze test, as previously described.
  • Tissue processing following the behavioral evaluation. Anesthetized mice are intracardially perfused and brains separated in hemispheres for immunocytochemical (immersion aldehyde-fixed) and neurochemical analysis (frozen).
  • Immunohistochemistrv 35-50 ⁇ m sections are stained with McSAI and 6E10 antibodies for identification and quantification of intracellular A ⁇ and plaque burden (using MCID program, as described previously by us). Tissue sections are stained for the vesicular acetylcholine transporter (VAChT) after which their cortical presynaptic cholinergic boutons are quantified. This result is compared with the quantification of the total synaptophysin presynaptic population, as previously described.
  • VAChT vesicular acetylcholine transporter
  • Neurochemistry cerebral A ⁇ 40 and 42 are investigated in homogenized brain samples using the Biosource kit, following the manufacturer's instructions. Soluble A ⁇ oligomers are investigated by Western blotting, using 6E10 antibody (Chemicon). PARP-1 , iNOS and proNGF levels are resolved by Western blots using commercial antibodies. ProMMP-9 and MMP-9 levels and activity are examined by Western blot and gelatin zymography, respectively.
  • ChAT Choline acetyl transferase
  • Figure 6B represents immunohistochemical data on the distribution and intensity of the intracellular A ⁇ immunoreactivity in neurons of the cerebral cortex (neocortex and entorhinal) and in neurons of the hippocampal complex (dentate granular and pyramidal CA1 to CA3 and hylum).
  • c) and d) illustrate the effects of the combined minocycline/scyllo-inositol treatment as compared with scyllo-inositol alone, e) and T).
  • Scale bar in e) applies to images in a) and c) and scale bar in f) to b) and d).
  • Representative images are from the contralateral brain hemispheres that correspond to the brain hemispheres analyzed by Western blotting in A).
  • the activity of the combined therapy or other combined therapy may, for example, be assessed by observing a decrease in the level of A ⁇ oligomers and of intracellular A ⁇ accumulation following treatment of Tg mice. An improvement in the cognitive function following treatment of Tg mice is also indicative of the composition's activity.
  • Fahnestock M Michalski B, Xu B 1 Coughlin MD.
  • the precursor pro-nerve growth factor is the predominant form of nerve growth factor in brain and is increased in Alzheimer's disease.
  • Kauppinen TM Swanson RA. Poly(ADP-ribose) polymerase-1 promotes microglial activation, proliferation, and matrix metalloproteinase-9-mediated neuron death. J Immunol 2005; 174(4):2288-2296.
  • Mattson MP Akbari Y, LaFerla FM., Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron. 2003 JuI 31 ;39(3):409-21 ;

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Abstract

La présente invention concerne des combinaisons, des compositions et des procédés pour réduire l'accumulation intracellulaire de bêta-amyloïde (Aß) et/ou pour réduire les oligomères d'Aß ou agrégats d'Aß extracellulaires dans un mammifère. Plus particulièrement, la présente invention concerne des combinaisons, des compositions et des procédés pour traiter ou prévenir la maladie d'Alzheimer.
PCT/CA2008/000297 2007-02-16 2008-02-15 Compositions et procédé de réduction de la bêta-amyloïde dans un mammifère WO2008098371A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7618944B2 (en) 2007-03-01 2009-11-17 Intezyne Technologies, Inc. Encapsulated amyloid-beta peptides
US8188046B2 (en) 2006-10-16 2012-05-29 University Of South Florida Amyloid beta peptides and methods of use
US8859628B2 (en) 2003-02-27 2014-10-14 JoAnne McLaurin Method for preventing, treating and diagnosing disorders of protein aggregation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB817181A (en) * 1956-10-24 1959-07-29 Bristol Lab Inc Therapeutic products containing tetracycline
GB819652A (en) * 1955-02-14 1959-09-09 Merck & Co Inc Antibiotic composition

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB819652A (en) * 1955-02-14 1959-09-09 Merck & Co Inc Antibiotic composition
GB817181A (en) * 1956-10-24 1959-07-29 Bristol Lab Inc Therapeutic products containing tetracycline

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8859628B2 (en) 2003-02-27 2014-10-14 JoAnne McLaurin Method for preventing, treating and diagnosing disorders of protein aggregation
US9833420B2 (en) 2003-02-27 2017-12-05 JoAnne McLaurin Methods of preventing, treating, and diagnosing disorders of protein aggregation
US8188046B2 (en) 2006-10-16 2012-05-29 University Of South Florida Amyloid beta peptides and methods of use
US7618944B2 (en) 2007-03-01 2009-11-17 Intezyne Technologies, Inc. Encapsulated amyloid-beta peptides

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