WO2008098371A1 - Compositions and method for reducing amyloid beta in a mammal - Google Patents

Abstract

The present invention relates to combinations, compositions and methods for reducing amyloid beta (Aβ) intracellular accumulation and/or reducing extracellular Aβ oligomers or Aβ aggregates in a mammal. More particularly, the present invention provides combinations, compositions and methods for treating or preventing Alzheimer's disease.

Description

COMPOSITIONS AND METHOD FOR REDUCING AMYLOID BETA IN A MAMMAL

FIELD OF THE INVENTION

[0001] The present invention relates to combinations, compositions and methods for reducing amyloid beta (Aβ) intracellular accumulation and/or reducing extracellular Aβ oligomers or Aβ aggregates in a mammal. More particularly, the present invention provides combinations, compositions and methods for treating Alzheimer's disease.

BACKGROUND OF THE INVENTION

[0002] Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by a progressive loss of memory and deterioration of higher cognitive functions. The brain of an individual with AD exhibits extracellular plaques of aggregated β-amyloid protein (Aβ), intracellular neurofibrillary tangles containing hyperphosphorylated tau protein accompanied by an important loss of cholinergic markers in the hippocampus and the cerebral cortex. 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. The fact that the boosting of the cholinergic system by the application of anticholinesterases improves cognitive outcomes for periods of 6 to 18 months in fairly advances stages of AD strongly supports the involvement of this transmitter-system in the AD-related cognitive decline. Indeed, the cholinergic system has been shown to be particularly vulnerable to the amyloid pathology. The forebrain cholinergic neurons which project to the cerebral cortex and the hippocampus are known to be highly dependent on the supply of endogenous NGF for the maintenance of the biochemical-morphological phenotype, as well as for the steady state number of cholinergic synapses in the cerebral cortex⁹. However, there is no evidence for the diminished synthesis of NGF levels in AD. On the contrary and paradoxically, an up-regulation in the levels of proNGF (the NGF precursor protein) has been well documented¹. This apparent paradox can now be explained by our recent discovery that it is proNGF, and not mature NGF, which is released from the cerebral cortex in a activity-dependent manner and that the maturation of proNGF as well as the degradation of the mature NGF is dependent upon a protease cascade² (See simplified scheme in Figure 1). The validity of this NGF metabolic pathway has been established in in vivo pharmacological experimentations². We have further observed a marked dysregulation of this NGF metabolic pathway in the AD cerebral cortex³. In brief, a failure in the conversion of proNGF to NGF (lower plasminogen/plasmin activity) is exacerbated and augmented by NGF degradation (a rise in MMP-9 activity). These finding would explain the preferential cholinergic vulnerability in AD.

[0003] Previous studies also reinforced this rationale. It has been shown that Aβ is involved in the activation of poly(ADP-ribose) polymerase isoform 1 (PARP-1) in the adult hippocampus⁴ and its activity is enhanced in AD⁵. PARP-1 is known to promote microglia activation, proliferation and MMP-9-mediated neurotoxic effects⁶. PARP-1 activation also plays an important role in up-regulating inflammatory cascades⁷'⁸ and its inhibition has been recently shown to protect the brain against systemic inflammation⁹ and reduce MMP-9 brain expression¹⁰. NO participates prominently in inflammation and activates MMP-9 by S-nitrosylation in response to oxidative stress¹¹.

[0004] Previous reports in transgenic mouse models have demonstrated beneficial effects of single therapy. For example, the deposition of oligomers may be prevented by immunoneutralization with anti-Aβ antibodies or the application of scyllo-inositol (McLaurin et al.¹³). 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. In this work McLaurin indicates that scyllo-inositol not only inhibit de novo amyloid aggregation but also inhibits already established cerebral amyloid deposition and improved cognitive outcomes.

[0005] 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 record¹⁶. Minocycline has also demonstrated some neuroprotective properties in certain disease and lesion models. It effectively inhibits PARP-1¹⁴ and iNOS¹⁵ 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.

[0006] In US2004/0063674A1 and US2006/0194773A1 , Levy, S.B. et al., propose to use tetracycline compounds for treating neurological diseases. However, Levy et al., specifically disclaim the use of some tetracycline compounds described in US 2001/0014670 including minocycline for the treatment of Alzheimer's disease.

[0007] In US2001/0014670A1 , Balin et al. proposes methods of treating

Alzheimer's disease by administration of 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.

[0008] In US2003/0203881 A1 , Duncan suggests the use of tetracycline compounds for the treatment of Alzheimer's disease. Duncan does not provide a rationale nor experimental evidence in support of this suggestion.

[0009] In US2006/148766A1 , Suh et al., discuss the use of minocycline alone for preventing and treating dementia and learning and memory impairment. In Seabrook TJ et al., administration of minocycline to trangenic mice of less than 12 months of age is associated with an increase in amyloid plaques, thereby teaching that minocycline may have a deleterious effect when administered to individuals considered at the pre-plaque stage (Tg mice at 5 months of age) or at the early-stage (Tg mice at 9 months of age) of Alzheimer's disease.

[0010] Although significant efforts have been made to develop therapeutics against

Alzheimer's disease, none of them targets multiple aspects of Alzheimer's disease including inflammation, NGF dysmetabolism and Aβ accumulation.

[0011] The present invention seeks to meet these needs and other needs. SUMMARY OF THE INVENTION

[0012] We thus propose herein to concurrently address three main aspects of

Alzheimer's disease. These are: A) the removal-inhibition of highly toxic Aβ oligomers, B) the inhibition of the A^β-induced inflammatory cascade and C) the correction of the NGF dysmetabolism.

[0013] The present invention may thus be used to simultaneously target these three main aspects of the AD pathology.

[0014] The present invention therefore provides a combined therapy which may concurrently target the NGF metabolism pathway and the Aβ accumulation and aggregation.

[0015] We believe that by addressing the NGF metabolism pathway at an early stage with agents having little or no toxicity targeting MMP-9 it may prevent the Aβ-driven initial inflammation and concomitant dysregulation of the NGF metabolism, thus rescuing the cholinergic phenotype in AD-like models of the amyloid pathology. This may be a positive outcome as it is well established that muscarinic cholinergic stimulation favors a non-amyloidogenic pathway of the Amyloid Precursor Protein (APP metabolism)²⁰²¹.

[0016] 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 Aβ 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.

[0017] 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. We came to the unexpected discovery that the combination of both compounds may reduce Aβ oligomers brain burden and Aβ intracellular accumulation in a more efficient manner than scyllo-inositol alone. Aβ 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.

[0018] In accordance with the present invention the combination of both compounds may reduce Aβ intracellular accumulation and/or extracellular accumulation of Aβ oligomers or aggregates in the brain of a mammal in a more efficient manner than scyllo-inositol. For example, the compounds may be used to reduce Aβ 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.

[0019] The combinations and compositions of the present invention may be advantageously administered prior to disease onset or in the early stage of the disease. Among those who sould benefit from a prophylactic treatment using the combinations and compositions of the present invention 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.

[0020] Examplary embodiments of mutations in the APP gene associated with AD are listed in Table 1.

Table t

Mutation ^! Phenotype i References

I Glu665Asp AD but may not be _{Peacock et aL 1994} pathogenic. '

' ,^Lf ^O/Asn/Leu | | _{Mu|lan et aL 1992}

(Swedish) I ^l '

His677Arg ¹ AD Janssen et al., 2003 D678N I FAD Wakutani et al., 2004

I j AD, but may not be i Kamino et al., 1992 I pathogenic.

Glu693Gly (Artie)

Reproduced plasma Nilsbeth et a!.. 2001 |Aβ40 and 42, high protfibril levels.

IAD or Cerebral

Asp694Asn (Iowa) J Grabowski et al., 2001 ! hemorrhage i AD, but may not be

Ala713Thr i Carter et al., 1992 pathogenic

Thr714Ala (Iranian) ι ^AD Pasaler et al., 2002 Val715Met (French) I AD Ancolio et al., 1999

^! De Jonqhe et al., 2001 ; Cruts et

Val715AIa (German) , AD al., 2003

• He716VaI (Florida) ^AD Eckman et al., 1997 lle716Thr AD Terrini et al.. 2002

Val717Phe (Indiana) AD Murrell et al , 1991

Val717Gly AD Chartier-Harlin et al , 1991

I Val717lle (London) sAD Goate et al., 1991

CSF tau and A-β consistent with AD;

! Val717Leu Murrell et al.. 2000 earlier onset than other 717 mutations.

^• Leu723Pro

AD I Kwok. 2000 (Australian)

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)

[0021] In an exemplary embodiment, 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.

[0022] In an exemplary embodiment of the present invention, the tetracycline compound and/or the inositol compound may be able to cross the blood brain barrier.

[0023] 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 (Aβ) intracellular accumulation and/or reducing Aβ extracellular oligomers or aggregates in the brain of a mammal (an individual in need).

[0024] 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.

[0025] In a further aspect, 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.

[0026] In yet a further aspect, 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.

[0027] The present invention relates in an additional aspect to a method for reducing or inhibiting amyloid beta (Aβ) intracellular accumulation and/or reducing Aβ extracellular oligomers or aggregates in an individual in need thereof (mammal) (for example, for reducing the Aβ 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.

[0028] 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.

[0029] 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.

[0030] In accordance with an embodiment of the invention, the tetracycline compound and inositol compound may be administered, for example, prior to a substantial Aβ intracellular accumulation occurrence or prior to the extracellular accumulation of Aβ oligomers or before to the formation of Aβ 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

[0031] In accordance with an additional embodiment of the invention, the tetracycline compound and inositol compound may be administered after a substantial Aβ intracellular accumulation has occurred or after the formation of Aβ 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.

[0032] In another aspect, 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 Aβ intracellular accumulation or extracellular Aβ oligomers or aggregates.

[0033] As used herein "an individual in need" is to be understood as an individual

(patient) having or susceptible of having or developing Alzheimer's disease. Those 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.

[0034] In accordance with the present invention, the mammal may be at a stage of

Alzheimer's disease characterized as being mild, moderate or severe.

[0035] Pharmaceutical 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

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In the appended drawings: [0037] 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;

[0038] 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;

[0039] Figure 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 β-tubulin;

[0040] Figure 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 β-tubulin;

[0041] 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 β-tubulin,

[0042] 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 β-tubulin;

[0043] 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 β-tubulin; [0044] Figure 3A is a Dot-blot assay measuring soluble monomers (Mono) and soluble Aβ oligomers (Oligo) preparations using an oligomer-specific antibody A11 or the 6E10 antibody able to detect Aβ mono and oligomeric forms;

[0045] 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 Aβ in the hippocampal region of the brain;

[0046] Figure 3C are quantitative histograms representing the relative pro-

NGF/ β-tubulin ratio and the MMP-9 activity in the monomeric or Aβ oligomer-infused rats of Figure 3B;

[0047] Figure 3D are Western blots measuring the levels of pro-NGF, PARP-1 , iNOS expression and activity of MMP-9 in the presence of Aβ oligomers (Oligo) alone or after treatment with either 3-aminobenzamide or minocycline;

[0048] Figure 3E are quantitative histograms representing the ratio of pro-

NGF/ β-tubulin expression, the ratio of PARP-1/ β-tubulin expression, the ratio of iNOS/β- tubulin expression and the MMP-9 activity in the presence of Aβ oligomers (Oligo) alone or treatment with either 3-aminobenzamide or minocycline as obtained in Figure 3D. Expression levels have been normalized to those of β-tubulin;

[0049] Figure 4 is a schematic representation of the observed dysregulation of the cascade responsible for the processing and degradation of NGF observed in AD;

[0050] Figure 5A comprises a graphic illustration of the transgene over- expression from 3, 6 and 9 months transgenic (Tg) mice (the Aβ peptide values correspond to Aβ 1-42) and immunohistochemical data on the distribution and intensity of the intracellular Aβ 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 Aβ amyloid plaques as revealed by Thioflavin S in the cerebral cortex ;

[0051] 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 β- 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;

[0052] Figure 6A illustrates a Western blot analysis of Aβ oligomers (6E10 antibody) in soluble fractions from 3 month old transgenic mice untreated (n=2), minocycline/scyllo-inositol treated (n=3) and scyllo-inositol treated (n=2) and;

[0053] Figure 6B represents immunohistochemical data on the distribution and intensity of the intracellular Aβ 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). a) Low and b) High magnification (neocortex) micrographs in untreated transgenic mice, c) Low and d) High magnification (neocortex) of Tg mice treated with the combined minocycline/scyllo-inositol composition and e) Low and f) High magnification of Tg mice treated with scyllo-inositol alone. Scale bar in e) applies to images in a) and c) and scale bar in f) to b) and d).

DETAILED DESCRIPTION

[0054] As used herein the term "tetracycline compound" refers to a family of compounds sharing a similar structure with tetracycline (e.g., four hydrocarbon rings derivation). The term "tetracycline compound" as used herein, refers to naturally- occurring molecule, semi-synthetic molecules, synthetic molecules, stereoisomers and enantiomers or salts thereof having the biological activity described herein.

[0055] Exemplary embodiments of "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. 2,980,584; 2,990,331 ; 3,062,717, 3,165,531 ; 3,454,697; 3,557,280; 3,674,859; 3,957,980; 4,018,889; 4,024,272; 4,126,680; 6,624,168; 6,846,939; 7,056,902; in US publication Nos. US2003/0203881A1 ; US2004/0063674A1 ; US2006/0194773A1 , and in international application Nos. PCT/US2006/004233 and PCT/US02/07856 and salts thereof. More specific exemplary and specific embodiment of the 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.

[0056] As used herein the term "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.

[0057] 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. More specific exemplary embodiment of the 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.

[0058] In an exemplary embodiment of the present invention, the tetracycline compound and/or the inositol compound may be in a pure or substantially pure form.

[0059] 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.

[0060] Symptoms associated with the different stages of Alzheimer's disease are discussed at, e.g., http://www.alzheimersdisease.com/info/about/stages- alzheimers.jsp.

[0061] Administration of the compounds may be performed using methods known in the art. For example, the compounds of the present invention may be administered separately, via different routes or sites of administration, as a single formulation, etc. Although 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).

[0062] The term "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.

[0063] 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. For example, in the treatment of cancer, 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.

[0064] As used herein, "pharmaceutical composition" 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). 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. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are 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. In one embodiment the pharmaceutical composition may be administered parenterally, transmucosally, transdermal^, intramuscularly, intravenously, intradermal^, subcutaneously, intraperitonealy, intraventricular^, intracranial^ or intratumorally. The term "pharmaceutical composition" may also comprise composition suitable for ingestion, i.e., in the form of food supplement.

[0065] 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.

[0066] The term "treatment" or "treating" 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. As used herein the term "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.

EXAMPLE 1 Maturation and degradation of NGF in normal mice

[0067] 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 (α-secretase) pathway via the activation of muscarinic receptors M1 and M3.

[0068] As indicated above, Aβ has been shown to be involved in the activation of poly(ADP-ribose) polymerase isoform 1 (PARP-1) in the adult hippocampus⁴ and its activity is enhanced in AD⁵. PARP-1 is known to promote microglia activation, proliferation and MMP-9-mediated neurotoxic effects⁶. 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 inflammation⁹ and reduce MMP-9 brain expression¹⁰. NO participates prominently in inflammation and activates MMP-9 by S-nitrosylation in response to oxidative stress¹¹. It is demonstrated herein that Aβ oligomers enhance both PARP-1 and iNOS activity.

[0069] We therefore determined the level of expression of proMMP-9, active

MMP-9, NGF and pro-NGF, iNOS and PARP-1 in AD brain. These 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 β-tubulin.

[0070] In order to determine the level of each of these proteins, frozen tissue

(middle frontal gyrus) from non-demented (n=6) and from Alzheimer's disease (n=6) patients (Netherlands Brain Bank) were homogenized and then centrifuged at 14,00Og for 30 min at 4⁰C, and the supernatants containing solubilized protein were analyzed following standard protocols. The band density was determined using a computer- assisted image analysis system. Values were normalized to β-tubulin. Statistical significance between pixel values for control and AD groups was calculated using a Mest, and bars represent mean intensities ±SEM for each group.

[0071] More particularly, we show in Figure 2A an increased levels of proMMP-

9 and active MMP-9 in AD brains as demonstrated by gelatin zymography and by Western blot. In Figure 2B, a marked increase in proNGF levels is also observed in AD brains as compared with age-matched controls (n=6 for each group, ^** P < 0.01 , t-test). [0072] In Figure 2C, lower levels of plasminogen (80KDa) and plasmin (30KDa) bands in Alzheimer's brain (n=6 for each group, ^*P < 0.05 and **P < 0.001 , t-test) are also observed.

[0073] In Figures 2D-2F the increased levels of the inflammation regulator

PARP-1 and of inducible nitric oxide synthase (iNOS) in AD brain samples (n=6 each group, ^*P < 0.05; ^**P < 0.001 , t-test) compared to age-matched controls are clearly observed.

[0074] Results of these experiments indicate that besides increased levels and activity of MMP-9, elevated levels of PARP-1 and inducible nitric oxide synthase (iNOS) was observed in AD brain. Therefore, PARP-1 and iNOS represent attractive candidates for regulating NGF metabolism and/or degradation.

EXAMPLE 2 Minocycline prevents soluble Aβ oligomer-induced up- regulation of PARP-1, iNOS, MMP-9 and proNGF

[0075] Lee CZ et a/.⁴⁴ showed that minocycline and doxycycline are potent inhibitors of cerebral MMP-9 expression and activity, offering tetracycline compounds great potential for therapeutic use.

[0076] In addition, Alano CC et al ™ report that PARP-1 enzymatic activity is directly inhibited by minocycline and other tetracycline derivatives that have previously been shown to have neuroprotective and anti-inflammatory actions. PARP-1 inhibitors significantly reduced MMP-9 level and activity¹⁰.

[0077] The effect of minocycline on NGF metabolism was thus assessed herein.

Results of these experiments are illustrated in Figure 3.

[0078] A Dot-blot assay is presented in Figure 3A for corroborating the presence of soluble monomers (Mono) and the prepared soluble Aβ oligomers (Oligo) following established protocols. The presence of soluble oligomers was detected using oligomer-specific antibody A11 (Gift from Dr Glabe, Irvine, Ca). As a positive control, the detection of Aβ mono and oligomeric forms were detected by re-blotting using 6E10 antibody (Chemicon). [0079] To test the in vivo impact of monomeric vs. oligomeric forms of Aβ on proNGF and MMP-9 levels, four month old Fischer-344 rats were bilaterally implanted with a canula into the hippocampus (CA1 region) and continuously infused (ALZET pump) for 72hrs (method as described by Bruno and Cuello, PNAS 2006) with soluble monomeric (3μg total) and soluble oligomeric (3μg total) Aβ preparations. These results are presented in Figure 3B and Figure 3C. Unilateral application of oligomeric Aβ resulted in increased levels of proNGF (n=4, **P < 0.001 , t-test) and of gelatinolytic activity of MMP-9 (n=4, **P < 0.001 , t-test), as compared with the soluble monomeric Aβ-infused site.

[0080] In Figures 3D-E, the PARP-1 inhibitor 3-aminobenzamide (3-AB,

10mg/kg/day) or Minocycline (Mc 50mg/kg/day ) was shown to significantly prevent the up-regulation of proNGF (n=4 each group, ^**P < 0.001 , t-test), MMP-9 (n=4 each group, ^**P < 0.001 , t-test), PARP-1 (n=4 each group, ^**P < 0.001 , t-test) and iNOS (n=4 each group, *P < 0.05, t-test). The Western blot analyses were made using anti-NGF, PARP-1 and anti-NOS2 antibodies. The levels of MMP-9 were detected by gelatin zymography. The volume of the immunoreactive bands was determined by densitometry using a computer-assisted image analysis system. Values were normalized to β-tubulin. Statistical significance between pixel values for control and treated groups was calculated using Mest. Bars represent mean intensity ±SEM.

[0081] We have thus demonstrated that pre-treatment with minocycline prevented the up-regulation of MMP-9 and proNGF elicited by the infusion of Aβ oligomers in the rat cerebral cortex.

[0082] We thus propose an early therapeutic intervention for preventing proNGF accumulation and dysregulation of NGF metabolism by the application of therapeutic agents targeting MMP-9 by inhibiting PARP-1 up-regulation and excessive NO production though iNOS.

[0083] By establishing an experimental therapy which would prevent the Aβ- driven initial inflammation and concomitant dysregulation of the NGF metabolism, we may rescue the cholinergic phenotype in AD-like models of the amyloid pathology in Alzheimer's patients. This would be a positive outcome as it is well established that muscarinic cholinergic stimulation favors a non-amyloidogenic pathway of the APP metabolism¹². [0084] Figure 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). Note that in AD the failure of NGF maturation leads to increased proNGF levels (as observed in AD) and the activation of MMP-9, which exacerbates degradation of NGF (double trophic factor support jeopardy). This situation will deprive forebrain cholinergic neurons of trophic support, and the diminished release of acetylcholine would favor the switch towards an amyloidogenic processing of APP. In this scenario, Aβ will increase the synthesis of nitric oxide (NO) and also stimulate the synthesis of proMMP-9 shown here in Figures 3A to 3E. Again, these results indicate that PARP-1 and iNOS represent attractive targets for AD therapeutics.

EXAMPLE 3 Transgenic mice characterization

[0085] 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).

[0086] More particularly, Figure 5A is a graphic illustration of the transgene over-expression from 3 to 9 months of age. Aβ peptide values correspond to Aβ 1-42. Note the early occurrence of abnormal accumulation of intracellular Aβ in AD vulnerable areas (e.g. hippocampus and cortex) akin to early stages of AD and Down's syndrome, and the presence of mature amyloid plaques (Thioflavin S positive at 6 months of age). 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 Aβ pathology. Brain homogenates were resolved by Western blot using an anti-NGF antibody. Note higher levels of proNGF compared to age- matched littermates (n=6 each group, *P < 0.05, t-test). Increased proMMP-9 and active MMP-9 gelatinolytic activity was found in the transgenic mice cortical brain tissue compared to non-transgenic littermates (n=6 each group, ^**P < 0.001 , t-test). Illustrated below the Western blots of Figure 5B are graphs representing the early behavioral impairment of this transgenic model in the learning of the escape behavior in the Morris Water Maze task. Six months values, not shown, are intermediate. The swim-speed, distances and latencies to locate the escape platform were recorded using a video tracking system (HVS Image, Buckingham, UK). This model is therefore suitable to assess therapeutics for AD. [0087] Results indicate that Tg mice express the APP transgene 12 fold higher than endogenous levels; display a clear cut phase of intracellular Aβ 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. These 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 Cuello¹¹ 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). These Tg mice survive well beyond the 12-18 month period with maximal pathology.

EXAMPLE 4 Assessment of new therapeutic combination in AD-Tg mice

[0088] In an exemplary embodiment, the effectiveness of a combination therapy comprising minocycine and scyllo-inositiol is assessed in our Tg mouse model.

[0089] Methodology, sampling size and data analysis

[0090] Doses: 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 neurons¹⁸ and inhibit PARP-1¹⁴ and iNOS¹⁵. For scyllo-inositol, the initial dosage was 30mg/kg/day, as it has been shown to be effective in preventing Aβ amyloid aggregation when orally administered¹³. 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.

[0091] 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 Aβ amyloid overexpression. For this, 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 Aβ amyloid mature plaque pathology stage.

[0092] Animals: The effectiveness of minocycline/scyllo-inositol treatment is tested by using 6 to 12 animals per group in order to minimize the impact of intraexperimental variations and to secure sufficient statistic power to determine the biological significance of the results.

[0093] 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.

[0094] For this purpose, 8 week-old Tg mice and non-Tg littermates are separated in four different groups based on treatment, as follows:

[0095] Group 1: non-Tg mice without treatment;

[0096] Group 2: non-Tg mice treated with minocycline;

[0097] Group 3: non-Tg mice treated with scyllo-inositol;

[0098] Group 4: non-Tg mice treated with minocycline/scyllo-inositol,

[0099] Group 5: Tg mice without treatment;

[00100] Group 6: Tg mice treated with minocycline,

[00101] Group 7: Tg mice treated with scyllo-inositol;

[00102] Group 8:Tg mice treated with minocycline/scyllo-inositol. Each group includes 12 mice.

[00103] 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.

[00104] For this purpose, six month-old Tg mice and non-Tg littermates are separated in Groups 9 to 16, analogous to Aim 1 above. In this animal cohort 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. Also, at the treatment initiation stage, this Tg animal displays advanced inflammation and NGF dysmetabolism. [00105] 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.

[00106] 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).

[00107] Immunohistochemistrv: 35-50μm sections are stained with McSAI and 6E10 antibodies for identification and quantification of intracellular Aβ 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.

[00108] Neurochemistry: cerebral Aβ 40 and 42 are investigated in homogenized brain samples using the Biosource kit, following the manufacturer's instructions. Soluble Aβ 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.

[00109] Measurement of Choline acetyl transferase (ChAT) activity: are investigated in homogenized brain tissue applying the Fonnum's method, as described.

[00110] A pilot experiment was carried out. Two month-old Tg and non-Tg mice were treated for 28 days (n=3 per group) with minocycline plus scyllo-inositol or scyllo- inositol alone. Results of this experimentation are illustrated in Figure 6.

[00111] Figure 6A illustrates a Western blot analysis (6E10 antibody) of soluble fractions from 3 month old transgenic mice untreated (n=2), minocycline/scyllo-inositol treated (n=3) and scyllo-inositol treated (n=2). Treatment with scyllo-inositol diminished the levels of Aβ trimers and large oligomers in transgenic mice, when compared with non- treated Tg mice. Note that the combined application of scyllo-inositol with minocycline resulted in a further depletion of the two molecular forms of Aβ, the high molecular weight and also the Aβ trimers, the latter known to impair LTP formation²⁰. Similar additive effects of minocycline/scyllo-inositol combined treatment were observed in preventing accumulation of proNGF and increased activity of MMP-9 (not shown).

[00112] Figure 6B represents immunohistochemical data on the distribution and intensity of the intracellular Aβ 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). a) Low and b) High magnification (neocortex) micrographs illustrating the maximal intracellular Aβ accumulation preceding Aβ-plaque formation in untreated transgenic mice. Some small, initial, diffuse Aβ-plaques can be noticed in the hippocampal formation, 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).

[00113] This experiment shows encouraging results regarding Aβ mobilization which is significantly reduced in the combined therapy (see Figure 6). In contrast, scyllo-inositol alone did not prevent the up-regulation of proNGF and MMP-9 observed in non treated transgenic mice group (see Figure 6).

EXAMPLE 5 Effect of combined therapy on cognitive functions and NGF metabolism

[00114] The effects of the separate vs. combined minocycline/scyllo-inositol treatments on cognitive functions (using the Morris water maze), the phenotype of the basal forebrain cholinergic system and their impact on the cortical and hippocampal levels of PARP-1 , iNOS, MMP-9 and proNGF is investigated. The ratio in Aβ40 and Aβ42 is determined by Elisa and the relative proportion of soluble Aβ oligomers is examined by Western blotting and also by dot-blot assay using oligomeric-specific antibodies. As there is recent evidence indicating that intracellular A^β also plays a phatophysiological role in AD, the two compartments (intra- and extra-cellular) is immunocytochemically analyzed utilizing quantitative computer assisted image analysis.

[00115] The activity of the combined therapy or other combined therapy may, for example, be assessed by observing a decrease in the level of Aβ oligomers and of intracellular Aβ 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.

[00116] It has been shown that the earliest aspect of the Aβ amyloid pathology in Down Syndrome is the intraneuronal accumulation of Aβ peptide²¹' ²², preceding plaque formation. As the pathological accumulation of intracellular Aβ provokes synaptic dysfunction²³ and cognitive impairments²⁴' ²⁵ it is reasonable to assume that a therapeutic approach as the combination therapy described here, clearing most of the intracellular Aβ peptide in a transgenic animal model, may thus be effective as a therapeutic approach in early phases of Down Syndrome.

[00117] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

[00118] Although the present invention has been described hereinabove by way of exemplary embodiments, it can be modified without departing from the spirit, scope and the nature of the invention.

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Claims

WE CLAIM:

1. A combination comprising a. a tetracycline compound and b. an inositol compound.

2. The combination of claim 1 , wherein said combination is administered simultaneously.

3. The combination of claim 1 , wherein said combination is administered separately.

4. The combination of any one of claims 1 to 3, for use in a method for reducing amyloid beta accumulation in the brain of an individual.

5. The combination of any one of claims 1 to 4, for use in a method of prophylactic treatment of Alzheimer's disease.

6. The combination of any one of claims 1 to 5, wherein said combination is administered to an individual identified as being susceptible of developping Alzheimer's disease.

7. The combination of any one of claims 1 to 5, wherein said combination is administered to an individual identified as being at an early stage of Alzheimer's disease.

8. The combination of any one of claims 1 to 7, wherein the tetracycline compound is minocycline or doxycycline.

9. The combination of any one of claims 1 to 8, wherein the inositol compound is scyllo- inositol or a substituted scyllo-inositol.

10. The combination of claim 4, 6 or 7, wherein said individual is a carrier of an APP gene associated with Alzheimer's disease susceptibility.

11. A pharmaceutical composition comprising a. a tetracycline compound; b. an inositol compound and; c. a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claim 11 , wherein said tetracycline compound is able to inhibit PARP-1 and/or iNOS activity or expression.

13. The pharmaceutical composition of claims 11 or 12, wherein the tetracycline compound is able to cross the blood brain barrier.

14. The pharmaceutical composition of any one of claims 11 to 13, wherein said tetracycline compound is minocycline.

15. The pharmaceutical composition of any one of claims 11 to 13, wherein said tetracycline compound is doxycycline.

16. The pharmaceutical composition of any one of claims 11 to 15, wherein said inositol compound is scyllo-inositol or a substituted scyllo-inositol.

17. The pharmaceutical composition of any one of claims 11 to 16, wherein said inositol compound is myo-inositol.

18. The pharmaceutical composition of any one of claims 11 to 16, wherein said inositol compound is epi-inositol.

19. The use of a tetracycline compound and an inositol compound in the making of a pharmaceutical composition, drug or medicament for reducing Aβ intracellular accumulation or for reducing extracellular Aβ oligomers or aggregates in the brain of a mammal in need.

20. The use as defined in claim 19, wherein the mammal in need has or is susceptible of having Alzheimer's disease.

21. A kit comprising a vial or vials containing a tetracycline compound and an inositol compound.

22. An article of manufacture comprising; 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.

23. A method for reducing Aβ intracellular accumulation or extracellular Aβ oligomers or aggregates in a mammal in need, the method comprising administering to said mammal, a tetracycline compound and an inositol compound.

24. The method of claim 23, wherein the mammal in need has or is susceptible of having Alzheimer's disease,

25. The method of any one of claims 23 or 24, wherein the tetracycline compound and inositol compound are administered prior to the apparition of clinical symptoms of Alzheimer's disease.

26. The method of any one of claims 23 or 24, wherein the tetracycline compound and inositol compound are administered during the mild cognitive impairment phase of Alzheimer's disease.

27. The method of any one of claims 23 or 24, wherein the tetracycline compound and inositol compound are administered at a mild, moderate or severe phase of Alzheimer's disease.

28. The use of a tetracycline compound and an inositol compound for the treatment of a mammal having or susceptible of having Aβ intracellular accumulation or extracellular Aβ oligomers or aggregates.

29. The use as defined in claim 28, wherein the mammal has or is susceptible of having Alzheimer's disease.

30. A method for treating or preventing Alzheimer's disease, the method comprising administering to a mammal in need, a tetracycline compound and an inositol compound.

31. The method of claim 30, wherein the mammal in need has or is susceptible of having Alzheimer's disease.

32. A method for treating Down Syndrome the method comprising administering to a mammal in need, a tetracycline compound and an inositol compound.

33. The method of claim 32, wherein the mammal in need has or is susceptible of having Down Syndrome.